G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
calmodulin 1 (phosphorylase kinase, delta)
G00001122 (Mus musculus)

Databases (7)

ENSG00000143933 (Ensembl human gene)
801 (Entrez Gene)
5 (G2Cdb plasticity & disease)
CALM1 (GeneCards)
114180 (OMIM)
Marker Symbol
HGNC:1442 (HGNC)
Protein Sequence
P62158 (UniProt)

Synonyms (3)

  • CAMI
  • DD132
  • PHKD

Diseases (4)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000103: Leukaemia N Y (10221337) Translocation (T) Y
D00000259: Osteoporosis N Y (10323246) Unknown (?) ?
D00000193: Alzheimer's disease Y Y (9237482) Single nucleotide polymorphism (SNP) N
D00000103: Leukaemia N Y (16956826) Translocation fusion (with another gene) (TF) Y
D00000103: Leukaemia N Y (16921363) Translocation fusion (with another gene) (TF) Y
D00000091: T-cell acute lymphoblastic leukaemia N Y (16873670) Deletion (D) Y
D00000091: T-cell acute lymphoblastic leukaemia N Y (15774621) Translocation fusion (with another gene) (TF) Y


  • The cryptic chromosomal deletion del(11)(p12p13) as a new activation mechanism of LMO2 in pediatric T-cell acute lymphoblastic leukemia.

    Van Vlierberghe P, van Grotel M, Beverloo HB, Lee C, Helgason T, Buijs-Gladdines J, Passier M, van Wering ER, Veerman AJ, Kamps WA, Meijerink JP and Pieters R

    Erasmus MC/Sophia Children's Hospital, Department of Pediatric Oncology/Hematology, 3000 CB Rotterdam, The Netherlands.

    To identify new cytogenetic abnormalities associated with leukemogenesis or disease outcome, T-cell acute lymphoblastic leukemia (T-ALL) patient samples were analyzed by means of the array-comparative genome hybridization technique (array-CGH). Here, we report the identification of a new recurrent and cryptic deletion on chromosome 11 (del(11)(p12p13)) in about 4% (6/138) of pediatric T-ALL patients. Detailed molecular-cytogenetic analysis revealed that this deletion activates the LMO2 oncogene in 4 of 6 del(11)(p12p13)-positive T-ALL patients, in the same manner as in patients with an LMO2 translocation (9/138). The LMO2 activation mechanism of this deletion is loss of a negative regulatory region upstream of LMO2, causing activation of the proximal LMO2 promoter. LMO2 rearrangements, including this del(11)(p12p13) and t(11;14) (p13;q11) or t(7;11)(q35;p13), were found in the absence of other recurrent cytogenetic abnormalities involving HOX11L2, HOX11, CALM-AF10, TAL1, MLL, or MYC. LMO2 abnormalities represent about 9% (13/138) of pediatric T-ALL cases and are more frequent in pediatric T-ALL than appreciated until now.

    Blood 2006;108;10;3520-9

  • Dual-color split signal fluorescence in situ hybridization assays for the detection of CALM/AF10 in t(10;11)(p13;q14-q21)-positive acute leukemia.

    La Starza R, Crescenzi B, Krause A, Pierini V, Specchia G, Bardi A, Nieddu R, Ariola C, Nanni M, Diverio D, Aventin A, Sborgia M, Martelli MF, Bohlander SK and Mecucci C

    Hematology and Bone Marrow Transplantation Unit, Policlinico Monteluce, via Brunamonti, 06122 Perugia, Italy.

    We developed dual-color split fluorescence in situ hybridization (FISH) assays to detect AF10 and/or CALM rearrangements. Among nine cases of acute leukemia with translocation breakpoints at 10p13 and 11q14-21, a CALM/AF10 rearrangement was found in seven and was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) in all. In 2/7 cases, FISH detected CALM/AF10 in extramedullary leukemic infiltrations in the mediastinum and breast. As expected, FISH was less sensitive than RT-PCR for disease monitoring of CALM-AF10 positive cases. This new FISH assay reliably discriminates between MLL/AF10 and CALM/AF10 genomic rearrangements, identifies variant and complex CALM/AF10 translocations and detects the CALM/AF10 rearrangement in extramedullary leukemic infiltrations.

    Haematologica 2006;91;9;1248-51

  • Leukaemic transformation by CALM-AF10 involves upregulation of Hoxa5 by hDOT1L.

    Okada Y, Jiang Q, Lemieux M, Jeannotte L, Su L and Zhang Y

    Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA.

    Chromosomal translocation is a common cause of leukaemia and the most common chromosome translocations found in leukaemia patients involve the mixed lineage leukaemia (MLL) gene. AF10 is one of more than 30 MLL fusion partners in leukaemia. We have recently demonstrated that the H3K79 methyltransferase hDOT1L contributes to MLL-AF10-mediated leukaemogenesis through its interaction with AF10 (ref. 5). In addition to MLL, AF10 has also been reported to fuse to CALM (clathrin-assembly protein-like lymphoid-myeloid) in patients with T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML). Here, we analysed the molecular mechanism of leukaemogenesis by CALM-AF10. We demonstrate that CALM-AF10 fusion is both necessary and sufficient for leukaemic transformation. Additionally, we provide evidence that hDOT1L has an important role in the transformation process. hDOT1L contributes to CALM-AF10-mediated leukaemic transformation by preventing nuclear export of CALM-AF10 and by upregulating the Hoxa5 gene through H3K79 methylation. Thus, our study establishes CALM-AF10 fusion as a cause of leukaemia and reveals that mistargeting of hDOT1L and upregulation of Hoxa5 through H3K79 methylation is the underlying mechanism behind leukaemia caused by CALM-AF10 fusion.

    Funded by: NHLBI NIH HHS: HL72240, R21 HL072240; NIAID NIH HHS: AI48407, R01 AI048407, R01 AI077454, R01 AI080432, R56 AI048407; NIGMS NIH HHS: GM68804, R01 GM068804

    Nature cell biology 2006;8;9;1017-24

  • HOXA genes are included in genetic and biologic networks defining human acute T-cell leukemia (T-ALL).

    Soulier J, Clappier E, Cayuela JM, Regnault A, García-Peydró M, Dombret H, Baruchel A, Toribio ML and Sigaux F

    Institut National de la Santé et de la Recherche Médicale Lymphocyte et Cancer, and Molecular Hematology Laboratory, Hôpital Saint Louis, Paris, France.

    Using a combination of molecular cytogenetic and large-scale expression analysis in human T-cell acute lymphoblastic leukemias (T-ALLs), we identified and characterized a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA and the TCRB locus. Real-time quantitative polymerase chain reaction (RQ-PCR) analysis showed that the expression of the whole HOXA gene cluster was dramatically dysregulated in the HOXA-rearranged cases, and also in MLL and CALM-AF10-related T-ALL cases, strongly suggesting that HOXA genes are oncogenic in these leukemias. Inclusion of HOXA-translocated cases in a general molecular portrait of 92 T-ALLs based on large-scale expression analysis shows that this rearrangement defines a new homogeneous subgroup, which shares common biologic networks with the TLX1- and TLX3-related cases. Because T-ALLs derive from T-cell progenitors, expression profiles of the distinct T-ALL subgroups were analyzed with respect to those of normal human thymic subpopulations. Inappropriate use or perturbation of specific molecular networks involved in thymic differentiation was detected. Moreover, we found a significant association between T-ALL oncogenic subgroups and ectopic expression of a limited set of genes, including several developmental genes, namely HOXA, TLX1, TLX3, NKX3-1, SIX6, and TFAP2C. These data strongly support the view that the abnormal expression of developmental genes, including the prototypical homeobox genes HOXA, is critical in T-ALL oncogenesis.

    Blood 2005;106;1;274-86

  • Mixed-lineage leukemia with t(10;11)(p13;q21): an analysis of AF10-CALM and CALM-AF10 fusion mRNAs and clinical features.

    Kumon K, Kobayashi H, Maseki N, Sakashita A, Sakurai M, Tanizawa A, Imashuku S and Kaneko Y

    Department of Cancer Chemotherapy, Saitama Cancer Center Hospital, Ina, Japan.

    A fusion transcript of AF10 and CALM was isolated recently from the U937 cell line with t(10;11)(p13;q21). We performed reverse transcription-polymerase chain reaction and sequencing analysis on the t(10;11) leukemia samples obtained from four patients and one cell line, and we identified reciprocal fusion transcripts of AF10 and CALM in all the samples. The fusion transcripts in the five samples showed four different breakpoints in AF10 and three different breakpoints in CALM. In addition, the fusion transcripts in one sample showed a nucleotide sequence deletion in AF10, and those in two samples showed a nucleotide sequence deletion in CALM; the deletions were thought to be caused by alternative splicing. The variety of breakpoints and splice sites in the two genes resulted in five different-sized AF10-CALM mRNAs and in four different-sized CALM-AF10 mRNAs. Clinical features of 11 patients, including 6 of our own and 5 reported by others, in whom the fusion of AF10 and CALM was identified, are characterized by young age of the patients, mixed-lineage immunophenotype with coexpression of T-cell and myeloid antigens, frequent occurrence of a mediastinal mass, and poor clinical outcome.

    Genes, chromosomes & cancer 1999;25;1;33-9

  • A genome-wide scan for loci linked to forearm bone mineral density.

    Niu T, Chen C, Cordell H, Yang J, Wang B, Wang Z, Fang Z, Schork NJ, Rosen CJ and Xu X

    Program for Population Genetics, Harvard School of Public Health, FXB-101, Boston, MA 02115-6096, USA.

    Osteoporosis is a chronic disorder characterized by low bone mass and fragility fractures. It affects more than 25 million men and women in the United States alone. Although several candidate genes, such as the vitamin-D-receptor gene or the estrogen-receptor gene, have been suggested in the pathogenesis of osteoporosis, the genetic dissection of this disorder remains a daunting task. To search systematically for chromosomal regions containing genes that regulate bone mineral density (BMD), we scanned the entire autosomal genome by using 367 polymorphic markers among 218 individuals (153 sibpairs) from 96 nuclear families collected from three townships of Anqing, China. In these 96 families, DNA samples from both parents were available for 82 (85.4%) families. By using age- and gender-adjusted forearm BMD measurements, a peak on chromosome 2 near D2S2141, D2S1400, and D2S405, a region previously linked to spinal BMD, showed evidence of linkage to both proximal and distal forearm BMD (multipoint LOD=2.15 and 2.14 for proximal and distal forearm BMD, respectively). One region on chromosome 13 (multipoint LOD=1.67) in the proximity of D13S788 and D13S800 showed evidence of linkage to distal forearm BMD only. Possible candidate genes included CALM2 (calmodulin 2) at 2p21.3-p21.1, a putative STK (serine/threonine kinase) at 2p23-24, POMC (pro-opiomelanocortin) at 2p23.3, and COL4A1 and COL4A2 (collagen IV alpha-1 and alpha-2 subunits) at 13q34. Because of the limited sample size, the suggestive evidence of linkage of this study should be considered as tentative and needs to be replicated in other larger populations.

    Funded by: NCRR NIH HHS: RR03655-11; NHLBI NIH HHS: HL 94-011, HL54998-01; ...

    Human genetics 1999;104;3;226-33

  • Mutation analysis of chromosome 19 calmodulin (CALM3) gene in Alzheimer's disease patients.

    Ibarreta D, Tao J, Parrilla R and Ayuso MS

    Department of Physiopathology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain.

    The calcium buffering capacity of lymphoblasts from patients suffering of late onset Alzheimer's disease (AD) has been reported to be diminished. Calmodulin is a calcium binding protein codified by three genes, one of them (CALM3) maps to chromosome 19, nearby a gene, apoE, associated with late onset AD. In this study we screened for structural changes in the CALM3 gene from AD patients by PCR-SSCP analysis. We observed several point mutations in the intronic flanking regions of exons 3 and 4 of CALM 3 gene. However, we failed to detect any structural changes in the regions encoding the calcium binding domains of this gene. Similar results were obtained by RT-PCR analysis of CALM3 transcripts from AD patients carrying apoE epsilon4 allele. It is concluded that structural alterations in the CALM3 gene are not associated with the altered Ca2+ homeostasis shown by lymphoblasts from these patients.

    Neuroscience letters 1997;229;3;157-60

Literature (197)

Pubmed - human_disease

  • The cryptic chromosomal deletion del(11)(p12p13) as a new activation mechanism of LMO2 in pediatric T-cell acute lymphoblastic leukemia.

    Van Vlierberghe P, van Grotel M, Beverloo HB, Lee C, Helgason T, Buijs-Gladdines J, Passier M, van Wering ER, Veerman AJ, Kamps WA, Meijerink JP and Pieters R

    Erasmus MC/Sophia Children's Hospital, Department of Pediatric Oncology/Hematology, 3000 CB Rotterdam, The Netherlands.

    To identify new cytogenetic abnormalities associated with leukemogenesis or disease outcome, T-cell acute lymphoblastic leukemia (T-ALL) patient samples were analyzed by means of the array-comparative genome hybridization technique (array-CGH). Here, we report the identification of a new recurrent and cryptic deletion on chromosome 11 (del(11)(p12p13)) in about 4% (6/138) of pediatric T-ALL patients. Detailed molecular-cytogenetic analysis revealed that this deletion activates the LMO2 oncogene in 4 of 6 del(11)(p12p13)-positive T-ALL patients, in the same manner as in patients with an LMO2 translocation (9/138). The LMO2 activation mechanism of this deletion is loss of a negative regulatory region upstream of LMO2, causing activation of the proximal LMO2 promoter. LMO2 rearrangements, including this del(11)(p12p13) and t(11;14) (p13;q11) or t(7;11)(q35;p13), were found in the absence of other recurrent cytogenetic abnormalities involving HOX11L2, HOX11, CALM-AF10, TAL1, MLL, or MYC. LMO2 abnormalities represent about 9% (13/138) of pediatric T-ALL cases and are more frequent in pediatric T-ALL than appreciated until now.

    Blood 2006;108;10;3520-9

  • Dual-color split signal fluorescence in situ hybridization assays for the detection of CALM/AF10 in t(10;11)(p13;q14-q21)-positive acute leukemia.

    La Starza R, Crescenzi B, Krause A, Pierini V, Specchia G, Bardi A, Nieddu R, Ariola C, Nanni M, Diverio D, Aventin A, Sborgia M, Martelli MF, Bohlander SK and Mecucci C

    Hematology and Bone Marrow Transplantation Unit, Policlinico Monteluce, via Brunamonti, 06122 Perugia, Italy.

    We developed dual-color split fluorescence in situ hybridization (FISH) assays to detect AF10 and/or CALM rearrangements. Among nine cases of acute leukemia with translocation breakpoints at 10p13 and 11q14-21, a CALM/AF10 rearrangement was found in seven and was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) in all. In 2/7 cases, FISH detected CALM/AF10 in extramedullary leukemic infiltrations in the mediastinum and breast. As expected, FISH was less sensitive than RT-PCR for disease monitoring of CALM-AF10 positive cases. This new FISH assay reliably discriminates between MLL/AF10 and CALM/AF10 genomic rearrangements, identifies variant and complex CALM/AF10 translocations and detects the CALM/AF10 rearrangement in extramedullary leukemic infiltrations.

    Haematologica 2006;91;9;1248-51

  • Leukaemic transformation by CALM-AF10 involves upregulation of Hoxa5 by hDOT1L.

    Okada Y, Jiang Q, Lemieux M, Jeannotte L, Su L and Zhang Y

    Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA.

    Chromosomal translocation is a common cause of leukaemia and the most common chromosome translocations found in leukaemia patients involve the mixed lineage leukaemia (MLL) gene. AF10 is one of more than 30 MLL fusion partners in leukaemia. We have recently demonstrated that the H3K79 methyltransferase hDOT1L contributes to MLL-AF10-mediated leukaemogenesis through its interaction with AF10 (ref. 5). In addition to MLL, AF10 has also been reported to fuse to CALM (clathrin-assembly protein-like lymphoid-myeloid) in patients with T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML). Here, we analysed the molecular mechanism of leukaemogenesis by CALM-AF10. We demonstrate that CALM-AF10 fusion is both necessary and sufficient for leukaemic transformation. Additionally, we provide evidence that hDOT1L has an important role in the transformation process. hDOT1L contributes to CALM-AF10-mediated leukaemic transformation by preventing nuclear export of CALM-AF10 and by upregulating the Hoxa5 gene through H3K79 methylation. Thus, our study establishes CALM-AF10 fusion as a cause of leukaemia and reveals that mistargeting of hDOT1L and upregulation of Hoxa5 through H3K79 methylation is the underlying mechanism behind leukaemia caused by CALM-AF10 fusion.

    Funded by: NHLBI NIH HHS: HL72240, R21 HL072240; NIAID NIH HHS: AI48407, R01 AI048407, R01 AI077454, R01 AI080432, R56 AI048407; NIGMS NIH HHS: GM68804, R01 GM068804

    Nature cell biology 2006;8;9;1017-24

  • HOXA genes are included in genetic and biologic networks defining human acute T-cell leukemia (T-ALL).

    Soulier J, Clappier E, Cayuela JM, Regnault A, García-Peydró M, Dombret H, Baruchel A, Toribio ML and Sigaux F

    Institut National de la Santé et de la Recherche Médicale Lymphocyte et Cancer, and Molecular Hematology Laboratory, Hôpital Saint Louis, Paris, France.

    Using a combination of molecular cytogenetic and large-scale expression analysis in human T-cell acute lymphoblastic leukemias (T-ALLs), we identified and characterized a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA and the TCRB locus. Real-time quantitative polymerase chain reaction (RQ-PCR) analysis showed that the expression of the whole HOXA gene cluster was dramatically dysregulated in the HOXA-rearranged cases, and also in MLL and CALM-AF10-related T-ALL cases, strongly suggesting that HOXA genes are oncogenic in these leukemias. Inclusion of HOXA-translocated cases in a general molecular portrait of 92 T-ALLs based on large-scale expression analysis shows that this rearrangement defines a new homogeneous subgroup, which shares common biologic networks with the TLX1- and TLX3-related cases. Because T-ALLs derive from T-cell progenitors, expression profiles of the distinct T-ALL subgroups were analyzed with respect to those of normal human thymic subpopulations. Inappropriate use or perturbation of specific molecular networks involved in thymic differentiation was detected. Moreover, we found a significant association between T-ALL oncogenic subgroups and ectopic expression of a limited set of genes, including several developmental genes, namely HOXA, TLX1, TLX3, NKX3-1, SIX6, and TFAP2C. These data strongly support the view that the abnormal expression of developmental genes, including the prototypical homeobox genes HOXA, is critical in T-ALL oncogenesis.

    Blood 2005;106;1;274-86

  • Mixed-lineage leukemia with t(10;11)(p13;q21): an analysis of AF10-CALM and CALM-AF10 fusion mRNAs and clinical features.

    Kumon K, Kobayashi H, Maseki N, Sakashita A, Sakurai M, Tanizawa A, Imashuku S and Kaneko Y

    Department of Cancer Chemotherapy, Saitama Cancer Center Hospital, Ina, Japan.

    A fusion transcript of AF10 and CALM was isolated recently from the U937 cell line with t(10;11)(p13;q21). We performed reverse transcription-polymerase chain reaction and sequencing analysis on the t(10;11) leukemia samples obtained from four patients and one cell line, and we identified reciprocal fusion transcripts of AF10 and CALM in all the samples. The fusion transcripts in the five samples showed four different breakpoints in AF10 and three different breakpoints in CALM. In addition, the fusion transcripts in one sample showed a nucleotide sequence deletion in AF10, and those in two samples showed a nucleotide sequence deletion in CALM; the deletions were thought to be caused by alternative splicing. The variety of breakpoints and splice sites in the two genes resulted in five different-sized AF10-CALM mRNAs and in four different-sized CALM-AF10 mRNAs. Clinical features of 11 patients, including 6 of our own and 5 reported by others, in whom the fusion of AF10 and CALM was identified, are characterized by young age of the patients, mixed-lineage immunophenotype with coexpression of T-cell and myeloid antigens, frequent occurrence of a mediastinal mass, and poor clinical outcome.

    Genes, chromosomes & cancer 1999;25;1;33-9

  • A genome-wide scan for loci linked to forearm bone mineral density.

    Niu T, Chen C, Cordell H, Yang J, Wang B, Wang Z, Fang Z, Schork NJ, Rosen CJ and Xu X

    Program for Population Genetics, Harvard School of Public Health, FXB-101, Boston, MA 02115-6096, USA.

    Osteoporosis is a chronic disorder characterized by low bone mass and fragility fractures. It affects more than 25 million men and women in the United States alone. Although several candidate genes, such as the vitamin-D-receptor gene or the estrogen-receptor gene, have been suggested in the pathogenesis of osteoporosis, the genetic dissection of this disorder remains a daunting task. To search systematically for chromosomal regions containing genes that regulate bone mineral density (BMD), we scanned the entire autosomal genome by using 367 polymorphic markers among 218 individuals (153 sibpairs) from 96 nuclear families collected from three townships of Anqing, China. In these 96 families, DNA samples from both parents were available for 82 (85.4%) families. By using age- and gender-adjusted forearm BMD measurements, a peak on chromosome 2 near D2S2141, D2S1400, and D2S405, a region previously linked to spinal BMD, showed evidence of linkage to both proximal and distal forearm BMD (multipoint LOD=2.15 and 2.14 for proximal and distal forearm BMD, respectively). One region on chromosome 13 (multipoint LOD=1.67) in the proximity of D13S788 and D13S800 showed evidence of linkage to distal forearm BMD only. Possible candidate genes included CALM2 (calmodulin 2) at 2p21.3-p21.1, a putative STK (serine/threonine kinase) at 2p23-24, POMC (pro-opiomelanocortin) at 2p23.3, and COL4A1 and COL4A2 (collagen IV alpha-1 and alpha-2 subunits) at 13q34. Because of the limited sample size, the suggestive evidence of linkage of this study should be considered as tentative and needs to be replicated in other larger populations.

    Funded by: NCRR NIH HHS: RR03655-11; NHLBI NIH HHS: HL 94-011, HL54998-01; ...

    Human genetics 1999;104;3;226-33

  • Mutation analysis of chromosome 19 calmodulin (CALM3) gene in Alzheimer's disease patients.

    Ibarreta D, Tao J, Parrilla R and Ayuso MS

    Department of Physiopathology and Human Molecular Genetics, Centro de Investigaciones Biológicas (CSIC), Velázquez, Madrid, Spain.

    The calcium buffering capacity of lymphoblasts from patients suffering of late onset Alzheimer's disease (AD) has been reported to be diminished. Calmodulin is a calcium binding protein codified by three genes, one of them (CALM3) maps to chromosome 19, nearby a gene, apoE, associated with late onset AD. In this study we screened for structural changes in the CALM3 gene from AD patients by PCR-SSCP analysis. We observed several point mutations in the intronic flanking regions of exons 3 and 4 of CALM 3 gene. However, we failed to detect any structural changes in the regions encoding the calcium binding domains of this gene. Similar results were obtained by RT-PCR analysis of CALM3 transcripts from AD patients carrying apoE epsilon4 allele. It is concluded that structural alterations in the CALM3 gene are not associated with the altered Ca2+ homeostasis shown by lymphoblasts from these patients.

    Neuroscience letters 1997;229;3;157-60

Pubmed - other

  • Single-molecule force spectroscopy distinguishes target binding modes of calmodulin.

    Junker JP and Rief M

    Physik-Department E22, Technische Universität München, James-Franck-Strasse, 85748 Garching, Germany.

    The eukaryotic signaling protein calmodulin (CaM) can bind to more than 300 known target proteins to regulate numerous functions in our body in a calcium-dependent manner. How CaM distinguishes between these various targets is still largely unknown. Here, we investigate fluctuations of the complex formation of CaM and its target peptide sequences using single-molecule force spectroscopy by AFM. By applying mechanical force, we can steer a single CaM molecule through its folding energy landscape from the fully unfolded state to the native target-bound state revealing equilibrium fluctuations between numerous intermediate states. We find that the prototypical CaM target sequence skMLCK, a fragment from skeletal muscle myosin light chain kinase, binds to CaM in a highly cooperative way, while only a lower degree of interdomain binding cooperativity emerges for CaMKK, a target peptide from CaM-dependent kinase kinase. We identify minimal binding motifs for both of these peptides, confirming that affinities of target peptides are not exclusively determined by their pattern of hydrophobic anchor residues. Our results reveal an association mode for CaMKK in which the peptide binds strongly to only partially Ca(2+)-saturated CaM. This binding mode might allow for a fine-tuning of the intracellular response to changes in Ca(2+) concentration.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;34;14361-6

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.

    Svendsen JM, Smogorzewska A, Sowa ME, O'Connell BC, Gygi SP, Elledge SJ and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Structure-specific endonucleases mediate cleavage of DNA structures formed during repair of collapsed replication forks and double-strand breaks (DSBs). Here, we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312. Human SLX4 forms a multiprotein complex with the ERCC4(XPF)-ERCC1, MUS81-EME1, and SLX1 endonucleases and also associates with MSH2/MSH3 mismatch repair complex, telomere binding complex TERF2(TRF2)-TERF2IP(RAP1), the protein kinase PLK1 and the uncharacterized protein C20orf94. Depletion of SLX4 causes sensitivity to mitomycin C and camptothecin and reduces the efficiency of DSB repair in vivo. SLX4 complexes cleave 3' flap, 5' flap, and replication fork structures; yet unlike other endonucleases associated with SLX4, the SLX1-SLX4 module promotes symmetrical cleavage of static and migrating Holliday junctions (HJs), identifying SLX1-SLX4 as a HJ resolvase. Thus, SLX4 assembles a modular toolkit for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure.

    Funded by: Howard Hughes Medical Institute; NCI NIH HHS: T32 CA009216, T32CA09216; NIA NIH HHS: R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: R01 GM070565, R01 GM070565-04

    Cell 2009;138;1;63-77

  • The association of CaM and Hsp70 regulates S-phase arrest and apoptosis in a spatially and temporally dependent manner in human cells.

    Huang M, Wei JN, Peng WX, Liang J, Zhao C, Qian Y, Dai G, Yuan J, Pan FY, Xue B, Sha JH and Li CJ

    The Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China.

    The cell cycle is controlled by regulators functioning at the right time and at the right place. We have found that calmodulin (CaM) has specific distribution patterns during different cell-cycle stages. Here, we identify cell-cycle-specific binding proteins of CaM and examine their function during cell-cycle progression. We first applied immunoprecipitation methods to isolate CaM-binding proteins from cell lysates obtained at different cell-cycle phases and then identified these proteins using mass spectrometry methods. A total of 41 proteins were identified including zinc finger proteins, ribosomal proteins, and heat shock proteins operating in a Ca(2+)-dependent or independent manner. Fifteen proteins were shown to interact with CaM in a cell-phase-specific manner. The association of the selected proteins and CaM were confirmed with in vitro immunoprecipitation and immunostaining methods. One of the identified proteins, heat shock protein 70 (Hsp70), was further studied with respect to its cell-cycle-related function. In vivo fluorescence resonance energy transfer (FRET) analysis showed that the interaction of CaM and Hsp70 was found in the nucleus during the S phase. Overexpression of Hsp70 is shown to arrest cells at S phase and, thus, induce cell apoptosis. When we disrupted the CaM-Hsp70 association with HSP70 truncation without the CaM-binding domain, we found that S-phase arrest and apoptosis could be rescued. The results suggest that the spatial and temporal association of CaM and Hsp70 can regulate cell-cycle progression and cell apoptosis.

    Cell stress & chaperones 2009;14;4;343-53

  • Calcium fingerprints induced by calmodulin interactors in eukaryotic cells.

    Dagher R, Brière C, Fève M, Zeniou M, Pigault C, Mazars C, Chneiweiss H, Ranjeva R, Kilhoffer MC and Haiech J

    UMR CNRS 7200, Université de Strasbourg, Faculté de Pharmacie 74, route du Rhin, F-67401 Illkirch, France.

    Calcium (Ca2+) is a ubiquitous second messenger which promotes cell responses through transient changes in intracellular concentrations. The prominent role of Ca2+ in cell physiology is mediated by a whole set of proteins constituting a Ca2+-signalling toolkit involved in Ca2+-signal generation, deciphering and arrest. The different Ca2+-signalosomes deliver Ca2+-signals with spatial and temporal dynamics to control the function of specific cell types. Among the intracellular proteins involved in Ca2+-signal deciphering, calmodulin (CaM) plays a pivotal role in controlling Ca2+-homeostasis and downstream Ca2+-based signalling events. Due to its ubiquitous expression in eukaryotic cells and the variety of proteins it interacts with, CaM is central in Ca2+-signalling networks. For these reasons, it is expected that disrupting or modifying CaM interactions with its target proteins will affect Ca2+-homeostasis and cellular responses. The resulting calcium response will vary depending on which interactions between CaM and target proteins are altered by the molecules and on the specific Ca2+-toolkit expressed in a given cell, even in the resting state. In the present paper, the effect of six classical CaM interactors (W5, W7, W12, W13, bifonazole and calmidazolium) was studied on Ca2+-signalling in tumor initiating cells isolated from human glioblastoma (TG1) and tobacco cells (BY-2) using the fluorescent Ca2+-sensitive Indo-1 dye and aequorin, respectively. Various Ca2+-fingerprints were obtained depending both on the CaM interactor used and the cell type investigated. These data demonstrate that interaction between the antagonists and CaM results in a differential inhibition of CaM-dependent proteins involved in Ca2+-signal regulation. In addition, the distinct Ca2+-fingerprints in tobacco and human tumor initiating glioblastoma cells induced by a given CaM interactor highlight the specificity of the Ca2+-signalosome in eukaryotic cells.

    Biochimica et biophysica acta 2009;1793;6;1068-77

  • Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis.

    Martins-de-Souza D, Gattaz WF, Schmitt A, Rewerts C, Marangoni S, Novello JC, Maccarrone G, Turck CW and Dias-Neto E

    Laboratório de Neurociências, Faculdade de Medicina da USP, Instituto de Psiquiatria, Universidade de São Paulo, Rua Dr. Ovídio Pires de Campos, No 785, s/n Consolação, São Paulo, SP, CEP 05403-010, Brazil. danms90@gmail.com

    Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

    Journal of neural transmission (Vienna, Austria : 1996) 2009;116;3;275-89

  • Gene variants associated with ischemic stroke: the cardiovascular health study.

    Luke MM, O'Meara ES, Rowland CM, Shiffman D, Bare LA, Arellano AR, Longstreth WT, Lumley T, Rice K, Tracy RP, Devlin JJ and Psaty BM

    Celera, Alameda, California 94502, USA. may.luke@celera.com

    The purpose of this study was to determine whether 74 single nucleotide polymorphisms (SNPs), which had been associated with coronary heart disease, are associated with incident ischemic stroke.

    Methods: Based on antecedent studies of coronary heart disease, we prespecified the risk allele for each of the 74 SNPs. We used Cox proportional hazards models that adjusted for traditional risk factors to estimate the associations of these SNPs with incident ischemic stroke during 14 years of follow-up in a population-based study of older adults: the Cardiovascular Health Study (CHS).

    Results: In white CHS participants, the prespecified risk alleles of 7 of the 74 SNPs (in HPS1, ITGAE, ABCG2, MYH15, FSTL4, CALM1, and BAT2) were nominally associated with increased risk of stroke (one-sided P<0.05, false discovery rate=0.42). In black participants, the prespecified risk alleles of 5 SNPs (in KRT4, LY6G5B, EDG1, DMXL2, and ABCG2) were nominally associated with stroke (one-sided P<0.05, false discovery rate=0.55). The Val12Met SNP in ABCG2 was associated with stroke in both white (hazard ratio, 1.46; 90% CI, 1.05 to 2.03) and black (hazard ratio, 3.59; 90% CI, 1.11 to 11.6) participants of CHS. Kaplan-Meier estimates of the 10-year cumulative incidence of stroke were greater among Val allele homozygotes than among Met allele carriers in both white (10% versus 6%) and black (12% versus 3%) participants of CHS.

    Conclusions: The Val12Met SNP in ABCG2 (encoding a transporter of sterols and xenobiotics) was associated with incident ischemic stroke in white and black participants of CHS.

    Funded by: NHLBI NIH HHS: N01 HC-55222, N01 HC015103, N01 HC035129, N01 HC045133, N01-HC-75150, N01-HC-85079, N01-HC-85086, N01HC55222, N01HC75150, N01HC85079, N01HC85086, U01 HL080295, U01 HL080295-01

    Stroke 2009;40;2;363-8

  • Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling.

    Labasque M, Reiter E, Becamel C, Bockaert J and Marin P

    Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Montpellier F-34094, France.

    The serotonin (5-hydroxytryptamine; 5-HT)(2C) receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT(2C) receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca(2+)-dependent "1-10" CaM-binding motif located in the proximal region of the 5-HT(2C) receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both beta-arrestin recruitment by 5-HT(2C) receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on beta-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT(2C) receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT(2C) receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT(2C) receptor agonists and their antidepressant-like activity.

    Molecular biology of the cell 2008;19;11;4640-50

  • Lack of association between the CALM1 core promoter polymorphism (-16C/T) and susceptibility to knee osteoarthritis in a Chinese Han population.

    Shi D, Ni H, Dai J, Qin J, Xu Y, Zhu L, Yao C, Shao Z, Chen D, Xu Z, Yi L, Ikegawa S and Jiang Q

    The Center of Diagnosis and Treatment for Joint Disease, Drum Tower Hospital Affiliated to Medical School of Nanjing University, Nanjing 210008, Jiangsu, PR China. tim801215@hotmail.com

    Background: CALM1 gene encodes calmodulin (CaM), an important and ubiquitous eukaryotic Ca2+-binding protein. Several studies have indicated that a deficient CaM function is likely to be involved in the pathogenesis of osteoarthritis (OA). Using a convincing genome-wide association study, a Japanese group has recently demonstrated a genetic association between the CALM1 core promoter polymorphism (-16C/T transition SNP, rs12885713) and OA susceptibility. However, the subsequent association studies failed to provide consistent results in OA patients of differently selected populations. The present study is to evaluate the association of the -16C/T polymorphism with knee OA in a Chinese Han population.

    Methods: A case-control association study was conducted. The polymorphism was genotyped in 183 patients who had primary symptomatic knee OA with radiographic confirmation and in 210 matched controls. Allelic and genotypic frequencies were compared between patients and control subjects.

    Results: No significant difference was detected in genotype or allele distribution between knee OA and control groups (all P > 0.05). The association was also negative even after stratification by sex. Furthermore, no association between the -16C/T SNP genotype and the clinical variables age, sex, BMI (body mass index) and K/L (Kellgren/Lawrence) score was observed in OA patients.

    Conclusion: The present study suggests that the CALM1 core promoter polymorphism -16C/T is not a risk factor for knee OA susceptibility in the Chinese Han population. Further studies are needed to give a global view of this polymorphism in pathogenesis of OA.

    BMC medical genetics 2008;9;91

  • [Is calmodulin 1 gene/estrogen receptor-alpha gene polymorphisms correlated with double curve pattern of adolescent idiopathic scoliosis?].

    Zhao D, Qiu GX and Wang YP

    Department of Orthopedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China.

    Objective: To investigate the association between calmodulin1 (CALM1) gene or estrogen receptor-alpha (ESR1) polymorphisms and double curve of adolescent idiopathic scoliosis (AIS).

    Methods: 67 double curve patients (30 degrees < Cobb angle < 90 degrees ), 100 controls. There were 4 polymorphic loci, rs12885713 (-16C > T) and rs5871 locus in CALM1 gene, rs2234693 (PvuII) and rs9340799 (XbaI) in ESR1 gene analyzed sequence by ABI3730 genetic analyzer.

    Results: There were 60 patients with Cobb angle > or = 40 degrees . According to the apical location of major curve, there were 40 thoracic curve patients. Furthermore, 1) there are statistical differences on the polymorphic distribution of ESR1 gene rs2234693 site between the double curve, Cobb angle > or = 40 degrees or thoracic curve patients and the controls, respectively (chi(2) = 6.081, 5.554, 6.1935; P = 0.014, 0.0128, 0.0184); 2) between the double curve cases and the controls, there is difference on the polymorphic distribution of rs12885713 site in CALM1 gene (chi(2) = 4.478; P = 0.034); 3) Between the thoracic curve patients and the controls, there is difference on the distribution of rs5871 allele polymorphism in CALM1 gene (chi(2) = 6.6061; P = 0.0102).

    Conclusion: Double curve patterns might be related to ESR1 gene rs2234693 (PvuII) site polymorphism. It is necessary to clarify the association between the polymorphisms of ESR1 gene and CALM1 gene and different subtypes of adolescent idiopathic scoliosis in the further study.

    Zhonghua yi xue za zhi 2008;88;35;2452-6

  • Association of the CALM1 core promoter polymorphism with knee osteoarthritis in patients of Greek origin.

    Poulou M, Kaliakatsos M, Tsezou A, Kanavakis E, Malizos KN and Tzetis M

    Department of Medical Genetics, Athens University, Aghia Sophia Children's Hospital, Athens, Greece.

    Purpose: Osteoarthritis (OA) is characterized by focal areas of loss of articular cartilage in synovial joints, associated with varying degrees of osteophyte formation, subchondral bone change, and synovitis. The Calmodulin 1 gene (CALM1) encodes for a ubiquitous eukaryotic Ca2+ binding protein and is the principal mediator of the calcium signal. The protein thus affects the chondrocyte's response to mechanical load. A functional core promoter single nucleotide polymorphism (SNP) -16C/T (rs12885713) was recently associated with hip OA (HOA) in the Japanese population, while no association was found in a British group of patients with HOA. Our objective was to assess whether this SNP was also associated with knee OA (KOA) in a Greek Caucasian population sample.

    Methods: The -16T/C SNP was genotyped in a 351 case-control cohort--158 patients with idiopathic KOA and 193 controls.

    Results: No significant differences were found in genotype frequencies for the -16T/C SNP of CALM1 gene between cases and controls (p = 0.581).

    Conclusion: Our data implied that the -16TT (rs12885713) CALM1 core promoter genotype is not a risk factor for OA etiology in Greek Caucasians on its own, but associated with the Asporin (ASPN) D14 or D15 risk allele, it could influence KOA susceptibility.

    Genetic testing 2008;12;2;263-5

  • Calmodulin binding to the Fas-mediated death-inducing signaling complex in cholangiocarcinoma cells.

    Chen Y, Pawar P, Pan G, Ma L, Liu H and McDonald JM

    Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. ybchen@path.uab.edu

    We have previously demonstrated that the antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells partially through Fas-mediated apoptosis pathways. Recently, CaM has been shown to bind to Fas, which is regulated during Fas or CaM antagonist-mediated apoptosis in Jurkat cells and osteoclasts. Accordingly, the present studies were designed to determine whether Fas interacts with CaM in cholangiocarcinoma cells and to elucidate its role in regulating Fas-mediated apoptosis. CaM bound to Fas in cholangiocarcinoma cells. CaM was identified in the Fas-mediated death inducing signaling complex (DISC). The amount of CaM recruited into the DISC was increased after Fas-stimulation, a finding confirmed by immunofluorescent analysis that demonstrated increased membrane co-localization of CaM and Fas upon Fas-stimulation. Consistently, increased Fas microaggregates in response to Fas-stimulation were found to bind to CaM. Fas-induced recruitment of CaM into the DISC was inhibited by the Ca(2+) chelator, EGTA, and the CaM antagonist, trifluoperazine (TFP). TFP decreased DISC-induced cleavage of caspase-8. Further, inhibition of actin polymerization, which has been demonstrated to abolish DISC formation, inhibited the recruitment of CaM into the DISC. These results suggest an important role of CaM in mediating DISC formation, thus regulating Fas-mediated apoptosis in cholangiocarcinoma cells. Characterization of the role of CaM in Fas-mediated DISC formation and apoptosis signaling may provide important insights in the development of novel therapeutic targets for cholangiocarcinoma.

    Journal of cellular biochemistry 2008;103;3;788-99

  • Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer.

    Park HY, Kim SA, Korlach J, Rhoades E, Kwok LW, Zipfel WR, Waxham MN, Webb WW and Pollack L

    School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.

    A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are approximately 490 micros for the transitions in the C-terminal domain and approximately 20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.

    Funded by: NIBIB NIH HHS: 9 P41 EB001976, P41 EB001976; NIGMS NIH HHS: P01 GM066275, P01-GM066275

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;2;542-7

  • Association of gene variants with incident myocardial infarction in the Cardiovascular Health Study.

    Shiffman D, O'Meara ES, Bare LA, Rowland CM, Louie JZ, Arellano AR, Lumley T, Rice K, Iakoubova O, Luke MM, Young BA, Malloy MJ, Kane JP, Ellis SG, Tracy RP, Devlin JJ and Psaty BM

    Celera, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA. dov.shiffman@celera.com

    Objective: We asked whether single nucleotide polymorphisms (SNPs) that had been nominally associated with cardiovascular disease in antecedent studies were also associated with cardiovascular disease in a population-based prospective study of 4522 individuals aged 65 or older.

    Based on antecedent studies, we prespecified a risk allele and an inheritance model for each of 74 SNPs. We then tested the association of these SNPs with myocardial infarction (MI) in the Cardiovascular Health Study (CHS). The prespecified risk alleles of 8 SNPs were nominally associated (1-sided P<0.05) with increased risk of MI in White CHS participants. The false discovery rate for these 8 was 0.43, suggesting that about 4 of these 8 are likely to be true positives. The 4 of these 8 SNPs that had the strongest evidence for association with cardiovascular disease before testing in CHS (association in 3 antecedent studies) were in KIF6 (CHS HR=1.29; 90%CI 1.1 to 1.52), VAMP8 (HR=1.2; 90%CI 1.02 to 1.41), TAS2R50 (HR=1.13; 90%CI 1 to 1.27), and LPA (HR=1.62; 90%CI 1.09 to 2.42).

    Conclusions: Although most of the SNPs investigated were not associated with MI in CHS, evidence from this investigation combined with previous studies suggests that 4 of these SNPs are likely associated with MI.

    Funded by: NHLBI NIH HHS: K08 HL077499, N01 HC015103, N01 HC035129, N01 HC045133, N01-HC-55222, N01-HC-75150, N01-HC-85079, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC-85084, N01-HC-85085, N01-HC-85086, N01HC55222, N01HC75150, N01HC85079, N01HC85086, R01 HL077499, U01 HL080295, U01 HL080295-01, U01 HL080295-02, U01 HL080295-03, U01 HL080295-04

    Arteriosclerosis, thrombosis, and vascular biology 2008;28;1;173-9

  • Calmodulin bound to stress fibers but not microtubules involves regulation of cell morphology and motility.

    Yuan J, Shi GX, Shao Y, Dai G, Wei JN, Chang DC and Li CJ

    Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Nanjing Normal University, #1 Wen Yuan Road, Nanjing, China.

    Calmodulin (CaM) is a major cytoplasmic calcium receptor that performs multiple functions including cell motility. To investigate the mechanism of the regulation of CaM on cell morphology and motility, first we checked the distribution of CaM in the living cells using GFP-CaM as an indicator. We found that GFP-CaM showed a fiber-like distribution pattern in the cytosol of living Potorous tridactylis kidney (PtK2) cells but not in living HeLa cells. The endogenous CaM in heavily permeabilized HeLa was also found to display a fiber-like distribution pattern. Further examination showed that the distribution pattern of GFP-CaM was same as that of stress fibers, but not microtubules. Co-immunoprecipitation also showed that CaM can interact with actin directly or indirectly. The microinjection of trp peptide, a specific inhibitor of CaM, attenuated the polymerization of stress fibers and induced the alteration of cell morphology. A wound-healing assay and a single cell tracking experiment showed that CaM in PtK2 cells could increase cell motility. The data we have got from living cells suggested that CaM affect cell morphology and motility through binding to stress fibers and regulate f-actin polymerization.

    The international journal of biochemistry & cell biology 2008;40;2;284-93

  • A polymorphism in the protease-like domain of apolipoprotein(a) is associated with severe coronary artery disease.

    Luke MM, Kane JP, Liu DM, Rowland CM, Shiffman D, Cassano J, Catanese JJ, Pullinger CR, Leong DU, Arellano AR, Tong CH, Movsesyan I, Naya-Vigne J, Noordhof C, Feric NT, Malloy MJ, Topol EJ, Koschinsky ML, Devlin JJ and Ellis SG

    Celera, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA. may.luke@celera.com

    Objectives: The purpose of this study was to identify genetic variants associated with severe coronary artery disease (CAD).

    We used 3 case-control studies of white subjects whose severity of CAD was assessed by angiography. The first 2 studies were used to generate hypotheses that were then tested in the third study. We tested 12,077 putative functional single nucleotide polymorphisms (SNPs) in Study 1 (781 cases, 603 controls) and identified 302 SNPs nominally associated with severe CAD. Testing these 302 SNPs in Study 2 (471 cases, 298 controls), we found 5 (in LPA, CALM1, HAP1, AP3B1, and ABCG2) were nominally associated with severe CAD and had the same risk alleles in both studies. We then tested these 5 SNPs in Study 3 (554 cases, 373 controls). We found 1 SNP that was associated with severe CAD: LPA I4399M (rs3798220). LPA encodes apolipoprotein(a), a component of lipoprotein(a). I4399M is located in the protease-like domain of apolipoprotein(a). Compared with noncarriers, carriers of the 4399M risk allele (2.7% of controls) had an adjusted odds ratio for severe CAD of 3.14 (confidence interval 1.51 to 6.56), and had 5-fold higher median plasma lipoprotein(a) levels (P=0.003).

    Conclusions: The LPA I4399M SNP is associated with severe CAD and plasma lipoprotein(a) levels.

    Arteriosclerosis, thrombosis, and vascular biology 2007;27;9;2030-6

  • Ca(2+)/calmodulin directly interacts with the pleckstrin homology domain of AKT1.

    Dong B, Valencia CA and Liu R

    School of Pharmacy and Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca(2+).calmodulin (Ca(2+).CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903-38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca(2+).CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca(2+)-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969-5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca(2+).CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca(2+).CaM. In addition, Ca(2+).CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca(2+).CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane.

    Funded by: NIDDK NIH HHS: R21DK067480

    The Journal of biological chemistry 2007;282;34;25131-40

  • [Is polymorphism of CALM1 gene or growth hormone receptor gene associated with susceptibility to adolescent idiopathic scoliosis?].

    Zhuang QY, Wu ZH and Qiu GX

    Department of Orthopedics, Peking Union Medical College Hospital, Peking Union Medical College, Beijing, China.

    Objective: To investigate the major effect of the candidate genes, calmodulin 1 (CALM1) and growth hormone receptor (GHR) gene, in mechanism of adolescent idiopathic scoliosis (AIS) and to evaluate the cross-influence between the polymorphism of the candidates genes and risk factors of AIS.

    Methods: Peripheral blood samples were collected from 30 AIS patients, 6 boys and 24 girls, aged 15.7, and 30 gender and age-matched controls. Genomic DNA was extracted. PCR amplification and sequencing of the segments containing SNPs chosen from candidate genes were conducted. The SNPs were genotyped then. Statistical analysis was conducted. Questionnaire survey was conducted in terms of the risk factors.

    Results: (1) The AIS patients had higher corrected standing height and an earlier growth spurt than the controls. (2) The frequencies of CC genotype of CALM1 gene at -16C > T locus and homozygous genotype of GHR gene at exon 10 I526L were significantly higher in the AIS patients than in the controls. (3) The frequencies of SNP03G-05A and SNP03G-05C haplotypes of GHR gene were significantly higher in the AIS patients than in the controls. (4) The frequency of PPGG (PP = homozygous genotype of GHR gene at I526L, GG = homozygous genotype of MTNR1B gene at rs1562444 locus) of the AIS patients was significantly higher than that of the controls. (5) The AIS patients who had homozygous genotype of GHR gene at I526L had an earlier growth spurt and higher standing height than those who had heterozygous genotype.

    Conclusion: The -16C > T polymorphism at the promoter region of CALM1 gene and the homozygous genotype of GHR gene at I526L may be associated with high susceptibility to AIS.

    Zhonghua yi xue za zhi 2007;87;31;2198-202

  • HIV-1 Nef upregulates CCL2/MCP-1 expression in astrocytes in a myristoylation- and calmodulin-dependent manner.

    Lehmann MH, Masanetz S, Kramer S and Erfle V

    Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. orlataler@web.de

    HIV-associated dementia (HAD) correlates with infiltration of monocytes into the brain. The accessory HIV-1 negative factor (Nef) protein, which modulates several signaling pathways, is constitutively present in persistently infected astroctyes. We demonstrated that monocytes responded with chemotaxis when subjected to cell culture supernatants of nef-expressing astrocytic U251MG cells. Using a protein array, we identified CC chemokine ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1) as a potential chemotactic factor mediating this phenomenon. CCL2/MCP-1 upregulation by Nef was further confirmed by ribonuclease protection assay, RT-PCR and ELISA. By applying neutralizing antibodies against CCL2/MCP-1 and using CCR2-deficient monocytes, we confirmed CCL2/MCP-1 as the exclusive factor secreted by nef-expressing astrocytes capable of attracting monocytes. Additionally, we showed that Nef-induced CCL2/MCP-1 expression depends on the myristoylation moiety of Nef and requires functional calmodulin. In summary, we suggest that Nef-induced CCL2/MCP-1 expression in astrocytes contributes to infiltration of monocytes into the brain, and thereby to progression of HAD.

    Journal of cell science 2006;119;Pt 21;4520-30

  • The role of calmodulin recruitment in Ca2+ stimulation of adenylyl cyclase type 8.

    Simpson RE, Ciruela A and Cooper DM

    The Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.

    Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.

    Funded by: NIGMS NIH HHS: GM 32483; Wellcome Trust

    The Journal of biological chemistry 2006;281;25;17379-89

  • A calmodulin-binding site on cyclin E mediates Ca2+-sensitive G1/s transitions in vascular smooth muscle cells.

    Choi J, Chiang A, Taulier N, Gros R, Pirani A and Husain M

    Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Faculty of Medicine, Department of Medicine, University of Toronto, Ontario, Canada.

    Calcium transients are known to control several transition points in the eukaryotic cell cycle. For example, we have previously shown that a coordinate elevation in the intracellular free calcium ion concentration is required for G1- to S-phase cell cycle progression in vascular smooth muscle cells (VSMC). However, the molecular basis for this Ca2+ sensitivity was not known. Using buffers with differing [Ca2+], we found that the kinase activity of mouse and human cyclin E/CDK2, but not other G1/S-associated cell cycle complexes, was responsive to physiological changes in [Ca2+]. We next determined that this Ca2+-responsive kinase activity was dependent on a direct interaction between calmodulin (CaM), one of the major Ca2+-signal transducers of eukaryotic cells, and cyclin E. Pharmacological inhibition of CaM abrogated the Ca2+ sensitivity of cyclin E/CDK2 and retarded mouse VSMC proliferation by causing G1 arrest. We next defined the presence of a highly conserved 22 amino acid N-terminal CaM-binding motif in mammalian cyclin E genes (dissociation constant, 1.5+/-0.1 micromol/L) and showed its essential role in mediating Ca2+-sensitive kinase activity of cyclin E/CDK2. Mutant human cyclin E protein, lacking this CaM-binding motif, was incapable of binding CaM or responding to [Ca2+]. Taken together, these findings reveal CaM-dependent cyclin E/CDK2 activity as a mediator of the known Ca2+ sensitivity of the G1/S transition of VSMC.

    Circulation research 2006;98;10;1273-81

  • The CALM1 core promoter polymorphism is not associated with hip osteoarthritis in a United Kingdom Caucasian population.

    Loughlin J, Sinsheimer JS, Carr A and Chapman K

    Institute of Musculoskeletal Sciences, University of Oxford, Botnar Research Centre, Oxford, UK. john.loughlin@ndcls.ox.ac.uk

    Objective: A convincing genetic association with hip osteoarthritis (OA) of a functional single nucleotide polymorphism (SNP) in the core promoter of the calmodulin 1 gene CALM1 was recently reported in a Japanese population. The T-allele of the SNP encoded OA susceptibility and this was mediated by a reduced expression of CALM1. Our objective was to assess whether the SNP was also associated with hip OA in UK Caucasians.

    Methods: The SNP was genotyped in 920 cases that had undergone elective joint replacement of the hip due to end-stage primary OA and in 752 age-matched controls.

    Results: Our study had greater than 97% power to observe an effect comparable to that seen in the Japanese study. However, there was no significant difference (P< or =0.05) in genotype or allele frequencies between our cases and our controls. There was also no significant difference when the cases were stratified by sex.

    Conclusion: Our data on a cohort of 1672 individuals implies that the CALM1 core promoter polymorphism is not a risk factor for OA etiology in Caucasians. Our study does not call in to question the veracity of the Japanese report. Instead it highlights the heterogeneous nature of OA genetic susceptibility.

    Osteoarthritis and cartilage 2006;14;3;295-8

  • A direct interaction between the N terminus of adenylyl cyclase AC8 and the catalytic subunit of protein phosphatase 2A.

    Crossthwaite AJ, Ciruela A, Rayner TF and Cooper DM

    Department of Pharmacology, University of Cambridge, UK.

    Although protein scaffolding complexes compartmentalize protein kinase A (PKA) and phosphodiesterases to optimize cAMP signaling, adenylyl cyclases, the sources of cAMP, have been implicated in very few direct protein interactions. The N termini of adenylyl cyclases are highly divergent, which hints at isoform-specific interactions. Indeed, the Ca(2+)-sensitive adenylyl cyclase 8 (AC8) contains a Ca(2+)/calmodulin binding site on the N terminus that is essential for stimulation of activity by the capacitative entry of Ca(2+) in the intact cell. Here, we have used the N terminus of AC8 as a bait in a yeast two-hybrid screen of a human embryonic kidney (HEK) 293 cell cDNA library and identified the catalytic subunit of the serine/threonine protein phosphatase 2A (PP2A(C)) as a binding partner. Confirming the highly specific nature of this novel interaction, glutathione-S-transferase fusion proteins containing the full-length N terminus of AC8 affinity precipitated catalytically active PP2A(C) from both HEK293 and mouse forebrain membranes-the latter a normal source of AC8. The scaffolding subunit of PP2A (PP2A(A); 65 kDa) was also precipitated by the N terminus of AC8, indicating that AC8 may occur in a complex with the PP2A core dimer. The interaction between the N terminus of AC8 and PP2A(C) was antagonized by Ca(2+)/calmodulin. However, PP2A(C) and Ca(2+)/calmodulin did not share identical binding specificities in the N terminus of AC8. PKA-mediated phosphorylation did not influence either calmodulin or PP2A(C) association with AC8. In addition, both PP2A(C) and AC8 occurred in lipid rafts. These findings are the first demonstration of an association between adenylyl cyclase and any downstream element of cAMP signaling.

    Funded by: NIGMS NIH HHS: GM32483; Wellcome Trust

    Molecular pharmacology 2006;69;2;608-17

  • Point mutations in the C-terminus of HIV-1 gp160 reduce apoptosis and calmodulin binding without affecting viral replication.

    Micoli KJ, Mamaeva O, Piller SC, Barker JL, Pan G, Hunter E and McDonald JM

    Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

    One hallmark of AIDS progression is a decline in CD4+ T lymphocytes, though the mechanism is poorly defined. There is ample evidence that increased apoptosis is responsible for some, if not all, of the decline. Prior studies have shown that binding of cellular calmodulin to the envelope glycoprotein (Env) of HIV-1 increases sensitivity to fas-mediated apoptosis and that calmodulin antagonists can block this effect. We show that individual mutation of five residues in the C-terminal calmodulin-binding domain of Env is sufficient to significantly reduce fas-mediated apoptosis in transfected cells. The A835W mutation in the cytoplasmic domain of gp41 eliminated co-immunoprecipitation of Env with calmodulin in studies with stably transfected cells. Four point mutations (A835W, A838W, A838I, and I842R) and the corresponding region of HIV-1 HXB2 were cloned into the HIV-1 proviral vector pNL4-3 with no significant effect on viral production or envelope expression, although co-immunoprecipitation of calmodulin and Env was decreased in three of these mutant viruses. Only wild-type envelope-containing virus induced significantly elevated levels of spontaneous apoptosis by day 5 post-infection. Fas-mediated apoptosis levels positively correlated with the degree of calmodulin co-immunoprecipitation, with the lowest apoptosis levels occurring in cells infected with the A835W envelope mutation. While spontaneous apoptosis appears to be at least partially calmodulin-independent, the effects of HIV-1 Env on fas-mediated apoptosis are directly related to calmodulin binding.

    Funded by: NCI NIH HHS: CA/72823, R01 CA072823; NIAID NIH HHS: AI33319, AI49090, R01 AI033319, R01 AI049090, R37 AI033319

    Virology 2006;344;2;468-79

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Isotope-labeled vibrational circular dichroism studies of calmodulin and its interactions with ligands.

    Pandyra AA, Yamniuk AP, Andrushchenko VV, Wieser H and Vogel HJ

    Department of Chemistry, University of Calgary, Alberta, Canada.

    In this work we have studied ligand-induced secondary structure changes in the small calcium regulatory protein calmodulin (CaM) using vibrational circular dichroism (VCD) spectroscopy. We find that, due to its chiral sensitivity, VCD spectroscopy has increased ability over IR spectroscopy to detect changes in the structure and flexibility of secondary structure elements upon ligand binding. Moreover, we demonstrate that the uniform isotope labeling of CaM with (13)C shifts its amide I' VCD band by about approximately 43 cm(-1) to lower wavenumbers, which opens up a spectral window to simultaneously visualize a bound target protein. Therefore this study also provides the first example of how isotope labeling enables protein-protein interactions to be studied by VCD with good separation of the signals for both isotope-labeled and unlabeled proteins.

    Biopolymers 2005;79;5;231-7

  • Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex.

    Van Petegem F, Chatelain FC and Minor DL

    Cardiovascular Research Institute, Department of Biochemistry and Biophysics, California Institute for Quantitative Biomedical Research, University of California, San Francisco, 1700 4th St., Box 2532, San Francisco, California 94143-2532, USA.

    Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca(V)s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca(2+)/calmodulin (Ca(2+)/CaM) and a Ca(V) channel consensus isoleucine-glutamine (IQ) motif: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the high-resolution structure of the Ca(2+)/CaM-Ca(V)1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca(2+)/CaM lobes through sets of conserved 'aromatic anchors.' Ca(2+)/N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca(2+)/N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca(2+)/N lobe aromatic anchors uncovers their role in Ca(V)1.2 CDF. Together, our data suggest that Ca(V) subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca(V)s.

    Funded by: NHLBI NIH HHS: R01 HL080050, R01 HL080050-02

    Nature structural & molecular biology 2005;12;12;1108-15

  • Calmodulin modulates hepatic membrane polarity by protein kinase C-sensitive steps in the basolateral endocytic pathway.

    Tyteca D, van Ijzendoorn SC and Hoekstra D

    Department of Cell Biology/Section Membrane Cell Biology, University Medical Center Groningen, A.Deusinglaan 1, 9713 AV Groningen, The Netherlands.

    Membrane polarity is maintained by a complex intermingling of various trafficking pathways, including basolateral and apical endocytosis. The present work was undertaken to better define the role of basolateral endocytic transport in apical membrane homeostasis. When polarized HepG2 hepatoma cells were incubated with calmodulin antagonists, the cells lost their polarity, as reflected by an inhibition of lipid transport of a fluorescent sphingomyelin to the apical membrane and an impediment of its recycling to the basolateral membrane. Instead, an accumulation of the lipid in dilated early endosomal compartments was observed, presumably due to a frustration of vesiculation. Interestingly, lipid transport to the apical pole, lipid recycling to the basolateral membrane and cell polarity were reestablished, while dilated compartments disappeared, when the cells were simultaneously treated with specific inhibitors of protein kinase C (PKC). Consistently, following activation of PKC, extensive dilation/vacuolation of early sorting endosomes was observed, very similar as seen upon treatment with calmodulin antagonists. Thus, the results indicate that membrane trafficking at early steps of the basolateral endocytic pathway in HepG2 cells is regulated by an intricate interplay between calmodulin and PKC. This interference, although not affecting endocytosis as such, compromises cell polarity by impeding membrane trafficking from early endosomes to the apical membrane.

    Experimental cell research 2005;310;2;293-302

  • Calcium/calmodulin regulates ubiquitination of the ubiquitin-specific protease TRE17/USP6.

    Shen C, Ye Y, Robertson SE, Lau AW, Mak DO and Chou MM

    Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.

    The TRE17 (USP6/TRE-2) oncogene induces tumorigenesis in both humans and mice. However, little is known regarding its regulation or mechanism of transformation. TRE17 encodes a TBC (Tre-2/Bub2/Cdc16)/Rab GTPase-activating protein homology domain at its N terminus and a ubiquitin-specific protease at its C terminus. In the current study, we identified the ubiquitous calcium (Ca2+)-binding protein calmodulin (CaM) as a novel binding partner for TRE17. CaM bound directly to TRE17 in a Ca2+-dependent manner both in vitro and in vivo. The CaM-binding site was mapped to two hydrophobic motifs near the C terminus of the TBC domain. Point mutations within these motifs significantly reduced the interaction of TRE17 with CaM. We further found that TRE17 is monoubiquitinated and promotes its own deubiquitination in vivo. CaM binding-deficient mutants of TRE17 exhibited significantly reduced monoubiquitination, suggesting that binding of Ca2+/CaM to TRE17 promotes this modification. Consistent with this notion, treatment of cells with the CaM inhibitor W7 reduced levels of TRE17 monoubiquitination. Interestingly, the calcium ionophore A23187 induced accumulation of a polyubiquitinated TRE17 species. The effect of A23187 was attenuated in CaM binding-deficient mutants of TRE17. Taken together, these studies indicate a role for Ca2+/CaM in regulating ubiquitination through direct interaction with TRE17.

    Funded by: NCI NIH HHS: CA-81415

    The Journal of biological chemistry 2005;280;43;35967-73

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Calmodulin physically interacts with the erythropoietin receptor and enhances Jak2-mediated signaling.

    Kakihana K, Yamamoto M, Iiyama M and Miura O

    Department of Hematology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.

    Stimulation of the erythropoietin receptor (EpoR) induces a transient increase in intracellular Ca2+ level as well as activation of the Jak2 tyrosine kinase to stimulate various downstream signaling pathways. Here, we demonstrate that the universal Ca2+ receptor calmodulin (CaM) binds EpoR in a Ca2+-dependent manner in vitro. Binding studies using various EpoR mutants in hematopoietic cells showed that CaM binds the membrane-proximal 65-amino-acid cytoplasmic region (amino acids 258-312) of EpoR that is critical for activation of Jak2-mediated EpoR signaling. Structurally unrelated CaM antagonists, W-13 and CMZ, inhibited activation of Jak2-mediated EpoR signaling pathways, whereas W-12, a W-13 analog, did not show any significant inhibitory effect. Moreover, overexpression of CaM augmented Epo-induced tyrosine phosphorylation of the EpoR. W-13, but not W-12, also inhibited Epo-induced proliferation and survival. Together, these results indicate that CaM binds to the membrane-proximal EpoR cytoplasmic region and plays an essential role in activation of Jak2-mediated EpoR signaling.

    Biochemical and biophysical research communications 2005;335;2;424-31

  • Ca2+ and calmodulin regulate the binding of filamin A to actin filaments.

    Nakamura F, Hartwig JH, Stossel TP and Szymanski PT

    Hematology Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Filamin A (FLNa) cross-links actin filaments (F-actin) into three-dimensional gels in cells, attaches F-actin to membrane proteins, and is a scaffold that collects numerous and diverse proteins. We report that Ca(2+)-calmodulin binds the actin-binding domain (ABD) of FLNa and dissociates FLNa from F-actin, thereby dissolving FLNa.F-actin gels. The FLNa ABD has two calponin homology domains (CH1 and CH2) separated by a linker. Recombinant CH1 but neither FLNa nor its ABD binds Ca(2+)-calmodulin in the absence of F-actin. Extending recombinant CH1 to include the negatively charged region linker domain makes it, like full-length FLNa, unable to bind Ca(2+)-calmodulin. Ca(2+)-calmodulin does, however, dissociate the FLNa ABD from F-actin provided that the CH2 domain is present. These findings identify the first evidence for direct regulation of FLNa, implicating a mechanism whereby Ca(2+)-calmodulin selectively targets the FLNa.F-actin complex.

    Funded by: NHLBI NIH HHS: HL19429, HL56252

    The Journal of biological chemistry 2005;280;37;32426-33

  • Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal.

    Sim H, Rimmer K, Kelly S, Ludbrook LM, Clayton AH and Harley VR

    Prince Henry's Institute of Medical Research, Level 4 Block E, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.

    The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

    Molecular endocrinology (Baltimore, Md.) 2005;19;7;1884-92

  • The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7.

    Li H, Sánchez-Torres J, del Carpio AF, Nogales-González A, Molina-Ortiz P, Moreno MJ, Török K and Villalobo A

    Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Arturo Duperier 4, Madrid E-28029, Spain.

    We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.

    Oncogene 2005;24;26;4206-19

  • Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation.

    Pan F, Means AR and Liu JO

    Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    Calcium signaling is critical for activation of T lymphocytes and has been proposed to be transduced through multiple calmodulin target proteins. Whereas the calcineurin-NFAT signaling module is critical for all mammalian T cells, the role of calmodulin-dependent kinase IV (CaMKIV) in mouse naïve CD4+ T-cell activation remains enigmatic. We have applied lentivius-mediated RNA interference of CaMKIV to human T cells and found that knockdown of CaMKIV abrogates T-cell receptor-mediated transcription of the IL-2 gene. We demonstrate that CaMKIV directly phosphorylates Cabin1, a transcriptional corepressor for myocyte enhancer factor 2, creating a docking site for 14-3-3, which causes its nuclear export. CaMKIV-mediated nuclear export of Cabin1 is likely to account for a significant part of the requirement of CaMKIV during human T-cell activation.

    Funded by: NICHD NIH HHS: HD-07503, R01 HD007503; NIGMS NIH HHS: GM-33976, R01 GM033976

    The EMBO journal 2005;24;12;2104-13

  • Regulation of phospholipase C-delta1 through direct interactions with the small GTPase Ral and calmodulin.

    Sidhu RS, Clough RR and Bhullar RP

    Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Manitoba R3E 0W2, Canada.

    Second messengers generated from membrane lipids play a critical role in signaling and control diverse cellular processes. Despite being one of the most evolutionarily conserved of all the phosphoinositide-specific phospholipase C (PLC) isoforms, a family of enzymes responsible for hydrolysis of the membrane lipid phosphatidylinositol bisphosphate, the mechanism of PLC-delta1 activation is still poorly understood. Here we report a novel regulatory mechanism for PLC-delta1 activation that involves direct interaction of the small GTPase Ral and the universal calcium-signaling molecule calmodulin (CaM) with PLC-delta1. In addition, we have identified a novel IQ type CaM binding motif within the catalytic region of PLC-delta1 that is not found in other PLC isoforms. Binding of CaM at the IQ motif inhibits PLC-delta1 activity, while addition of Ral reverses the inhibition. The overexpression of various Ral mutants in cells potentiates PLC-delta1 activity. Thus, the Ral-CaM complex defines a multifaceted regulatory mechanism for PLC-delta1 activation.

    The Journal of biological chemistry 2005;280;23;21933-41

  • Modulation of skeletal and cardiac voltage-gated sodium channels by calmodulin.

    Young KA and Caldwell JH

    Campus Box 8315, Dept. of Cell/Devel Biology and the Neuroscience Program, UCHSC, PO Box 6511, Aurora, CO 80045, USA.

    Calmodulin (CaM) has been shown to modulate different ion channels, including voltage-gated sodium channels (NaChs). Using the yeast two-hybrid assay, we found an interaction between CaM and the C-terminal domains of adult skeletal (NaV1.4) and cardiac (NaV1.5) muscle NaChs. Effects of CaM were studied using sodium channels transiently expressed in CHO cells. Wild type CaM (CaM(WT)) caused a hyperpolarizing shift in the voltage dependence of activation and inactivation for NaV1.4 and activation for NaV1.5. Intracellular application of CaM caused hyperpolarizing shifts equivalent to those seen with CaM(WT) coexpression with NaV1.4. Elevated Ca2+ and CaM-binding peptides caused depolarizing shifts in the inactivation curves seen with CaM(WT) coexpression with NaV1.4. KN93, a CaM-kinase II inhibitor, had no effect on NaV1.4, suggesting that CaM acts directly on NaV1.4 and not through activation of CaM-kinase II. Coexpression of hemi-mutant CaMs showed that an intact N-terminal lobe of CaM is required for effects of CaM upon NaV1.4. Mutations in the sodium channel IQ domain disrupted the effects of CaM on NaV1.4: the I1727E mutation completely blocked all calmodulin effects, while the L1736R mutation disrupted the effects of Ca2+-calmodulin on inactivation. Chimeric channels of NaV1.4 and NaV1.5 also indicated that the C-terminal domain is largely responsible for CaM effects on inactivation. CaM had little effect on NaV1.4 expressed in HEK cells, possibly due to large differences in the endogenous expression of beta-subunits between CHO and HEK cells. These results in heterologous cells suggest that Ca2+ released during muscle contraction rapidly modulates NaCh availability via CaM.

    Funded by: NICHD NIH HHS: HD41697-03; NINDS NIH HHS: NIH R01 NS26505

    The Journal of physiology 2005;565;Pt 2;349-70

  • A functional single nucleotide polymorphism in the core promoter region of CALM1 is associated with hip osteoarthritis in Japanese.

    Mototani H, Mabuchi A, Saito S, Fujioka M, Iida A, Takatori Y, Kotani A, Kubo T, Nakamura K, Sekine A, Murakami Y, Tsunoda T, Notoya K, Nakamura Y and Ikegawa S

    Laboratory for Bone and Joint Diseases, The Institute of Physical and Chemical Research (RIKEN), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    Osteoarthritis (OA), a common skeletal disease, is a leading cause of disability among the elderly populations. OA is characterized by gradual loss of articular cartilage, but the etiology and pathogenesis of OA are largely unknown. Epidemiological and genetic studies have demonstrated that genetic factors play an important role in OA. To identify susceptibility genes for OA, we performed a large-scale, case-control association study using gene-based single nucleotide polymorphisms (SNPs). In two independent case-control populations, we found significant association (P=9.8x10(-7)) between hip OA and a SNP (IVS3-293C>T) located in intron 3 of the calmodulin (CaM) 1 gene (CALM1). CALM1 was expressed in cultured chondrocytes and articular cartilage, and its expression was increased in OA. Subsequent linkage-disequilibrium mapping identified five SNPs showing significant association equivalent to IVS3-293C>T. One of these (-16C>T) is located in the core promoter region of CALM1. Functional analyses indicate that the susceptibility -16T allele decreases CALM1 transcription in vitro and in vivo. Inhibition of CaM in chondrogenic cells reduced the expression of the major cartilage matrix genes Col2a1 and Agc1. These results suggest that the transcriptional level of CALM1 is associated with susceptibility for hip OA through modulation of chondrogenic activity. Our findings reveal the CALM1-mediated signaling pathway in chondrocytes as a novel potential target for treatment of OA.

    Human molecular genetics 2005;14;8;1009-17

  • Sites on calmodulin that interact with the C-terminal tail of Cav1.2 channel.

    Xiong L, Kleerekoper QK, He R, Putkey JA and Hamilton SL

    Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.

    Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.

    Funded by: NIAMS NIH HHS: AR44864

    The Journal of biological chemistry 2005;280;8;7070-9

  • Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo.

    Matsubara M, Jing T, Kawamura K, Shimojo N, Titani K, Hashimoto K and Hayashi N

    Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.

    Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins. In the present report, we show that Nef can interact with calmodulin, the major intracellular receptor for calcium. Coimmunoprecipitation analyses with lysates from the NIH3T3 cell line constitutively expressing the native HIV-1 Nef protein revealed the presence of a stable Nef-calmodulin complex. When lysates from NIH3T3 cells were incubated with calmodulin-agarose beads in the presence of CaCl(2) or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca(2+) signaling. Glutathione S-transferase-Nef fusion protein bound directly to calmodulin with high affinity. Using synthetic peptides based on the N-terminal sequence of Nef, we determined that within a 20-amino-acid N-terminal basic domain was sufficient for calmodulin binding. Furthermore, the myristoylated peptide bound to calmodulin with higher affinity than nonmyris-toylated form. Thus, the N-terminal myristoylation domain of Nef plays an important role in interacting with calmodulin. This domain is highly conserved in several HIV-1 Nef variants and resembles the N-terminal domain of NAP-22/CAP23, a myristoylated calmodulin-binder. These results for the interaction between HIV Nef and calmodulin in the cells suggested that the Nef might interfere with intracellular Ca(2+) signaling through calmodulin-mediated interactions in infected cells.

    Protein science : a publication of the Protein Society 2005;14;2;494-503

  • Calmodulin interacts with the cytoplasmic tails of the parathyroid hormone 1 receptor and a sub-set of class b G-protein coupled receptors.

    Mahon MJ and Shimada M

    Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, Wellman 501, Boston, MA 02114-2696, USA. mahon@helix.mgh.harvard.edu

    Parathyroid hormone (PTH) binds to its receptor (PTH 1 receptor, PTH1R) and activates multiple pathways. The PTH1R, a class b GPCR, contains consensus calmodulin-binding motifs. The PTH1R cytoplasmic tail interacts with calmodulin in a calcium-dependent manner via the basic 1-5-8-14 motif. Calcium-dependent calmodulin interactions with the cytoplasmic tails of receptors for PTH 2, vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide, corticotropin releasing hormone, calcitonin, and the glucagon-like peptides 1 and 2 are demonstrated. The cytoplasmic tails of the secretin receptor and the growth hormone releasing hormone receptor either interact poorly or not at all with calmodulin, respectively. Fluphenazine, a calmodulin antagonist, enhances PTH-mediated accumulation of total inositol phosphates, suggesting that calmodulin regulates signaling via phospholipase C.

    Funded by: NIDDK NIH HHS: K01 DK59900-01

    FEBS letters 2005;579;3;803-7

  • Enhanced ligand affinity for receptors in which components of the binding site are independently mobile.

    Trevitt CR, Craven CJ, Milanesi L, Syson K, Mattinen ML, Perkins J, Annila A, Hunter CA and Waltho JP

    Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.

    Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized. The bifunctional ligands bound to calmodulin with a stoichiometry of 1:1 and with an affinity enhancement over their monofunctional counterparts; the latter bound with a stoichiometry of 2:1 ligand:protein. A lower limit to the effective concentrations of the domains of calmodulin relative to each other (0.2-2 mM) was determined. A comparable effective concentration was achieved for bifunctional ligands based on higher-affinity naphthalene sulphonamide derivatives.

    Funded by: Biotechnology and Biological Sciences Research Council: BBS/B/10218

    Chemistry & biology 2005;12;1;89-97

  • Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation.

    Thorogate R and Török K

    Department of Basic Medical Sciences, St Georges Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK.

    Translocation from the cytosol to the nucleus is a major response by calmodulin (CaM) to stimulation of cells by Ca2+. However, the mechanisms involved in this process are still controversial and both passive and facilitated diffusion have been put forward. We tested nuclear translocation mechanisms in electroporated HeLa cells, rat cortical neurons and glial cells using novel calmodulin and inhibitor peptide probes and confocal microscopy. Passive diffusion of calmodulin across the nuclear membrane was measured in conditions in which facilitated transport was blocked and was compared to that of a similarly sized fluorescein-labeled dextran. Wheat germ agglutinin, which blocks facilitated transport but not passive diffusion, inhibited the nuclear entry of both wild-type and Ca2+-binding-deficient mutant calmodulin both in low and elevated [Ca2+]. Ca2+-dependent nuclear translocation was prevented by a membrane-permeant CaM inhibitor, the mTrp peptide, which indicated that it was specific to Ca2+/CaM. Diffusion of free CaM and Ca2+/CaM was considerably slower than the observed nuclear translocation by facilitated transport. Our data show that the majority of CaM nuclear entry occurred by facilitated mechanisms in all cell types examined, in part by a Ca2+-independent and in part by a Ca2+-dependent translocation mechanism.

    Journal of cell science 2004;117;Pt 24;5923-36

  • Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods.

    Lidwell K, Dillon J, Sihota A, O'Connor V and Pilkington B

    Neuroscience Group, University of Southampton, Southampton, UK.

    mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission. The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6-8) mGluRs. In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3. Prominent among the candidate receptor interacting proteins was calmodulin, a Ca(2+) sensor known to bind identifiable sequences in group I and III mGluRs. The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs. Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs. This illustrates that certain Ca(2+)-dependent interactions are not recapitulated in yeast. Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.

    Biochemical Society transactions 2004;32;Pt 5;868-70

  • Calmodulin mediates Ca2+ sensitivity of sodium channels.

    Kim J, Ghosh S, Liu H, Tateyama M, Kass RS and Pitt GS

    Department of Pharmacology, Division of Cardiology, Columbia University, New York, New York 10032, USA.

    Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.

    Funded by: NHLBI NIH HHS: HL-56810, HL-67849, HL-71165, R01 HL071165, R01 HL071165-01, R01 HL071165-02, R01 HL071165-03

    The Journal of biological chemistry 2004;279;43;45004-12

  • Melatonin, an endogenous-specific inhibitor of estrogen receptor alpha via calmodulin.

    del Río B, García Pedrero JM, Martínez-Campa C, Zuazua P, Lazo PS and Ramos S

    Departamento de Bioquímica y Biología Molecular and Instituto Universitario de Oncología Del Principado de Asturias, Universidad de Oviedo, 33071 Oviedo, Spain.

    Melatonin is an indole hormone produced mainly by the pineal gland. We have previously demonstrated that melatonin interferes with estrogen (E(2)) signaling in MCF7 cells by impairing estrogen receptor (ER) pathways. Here we present the characterization of its mechanism of action showing that melatonin is a specific inhibitor of E(2)-induced ERalpha-mediated transcription in both estrogen response element- and AP1-containing promoters, whereas ERbeta-mediated transactivation is not inhibited or even activated at certain promoters. We show that the sensitivity of MCF-7 cells to melatonin depends on the ERalpha/ERbeta ratio, and ectopic expression of ERbeta results in MCF-7 cells becoming insensitive to this hormone. Melatonin acts as a calmodulin antagonist inducing conformational changes in the ERalpha-calmodulin (CaM) complex, thus impairing the binding of E(2).ERalpha.CaM complex to DNA and, therefore, preventing ERalpha-dependent transcription. Moreover the mutant ERalpha (K302G,K303G), unable to bind calmodulin, becomes insensitive to melatonin. The effect of melatonin is specific since other related indoles neither interact with CaM nor inhibit ERalpha-mediated transactivation. Interestingly, melatonin does not affect the binding of coactivators to ERalpha, indicating that melatonin action is different from that of current therapeutic anti-estrogens used in breast cancer therapy. Thus, they target ERalpha at different levels, representing two independent ways to control ERalpha activity. It is, therefore, conceivably a synergistic pharmacological effect of melatonin and current anti-estrogen drugs.

    The Journal of biological chemistry 2004;279;37;38294-302

  • The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells.

    Yu YY, Chen Y, Dai G, Chen J, Sun XM, Wen CJ, Zhao DH, Chang DC and Li CJ

    Jiangsu Key Laboratory for Molecular & Medical Biotechnology, School of Life Sciences, Nanjing Normal University, Nanjing 210097, Jiangsu Province, China.

    Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase-anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 microM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.

    The international journal of biochemistry & cell biology 2004;36;8;1562-72

  • ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation.

    Oury C, Sticker E, Cornelissen H, De Vos R, Vermylen J and Hoylaerts MF

    Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.

    Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.

    The Journal of biological chemistry 2004;279;25;26266-73

  • Calmodulin regulates transglutaminase 2 cross-linking of huntingtin.

    Zainelli GM, Ross CA, Troncoso JC, Fitzgerald JK and Muma NA

    Department of Pharmacology, Loyola University Medical Center, Maywood, Illinois 60153, USA.

    Striatal and cortical intranuclear inclusions and cytoplasmic aggregates of mutant huntingtin are prominent neuropathological hallmarks of Huntington's disease (HD). We demonstrated previously that transglutaminase 2 cross-links mutant huntingtin in cells in culture and demonstrated the presence of transglutaminase-catalyzed cross-links in the HD cortex that colocalize with transglutaminase 2 and huntingtin. Because calmodulin regulates transglutaminase activity in erythrocytes, platelets, and the gizzard, we hypothesized that calmodulin increases cross-linking of huntingtin in the HD brain. We found that calmodulin colocalizes at the confocal level with transglutaminase 2 and with huntingtin in HD intranuclear inclusions. Calmodulin coimmunoprecipitates with transglutaminase 2 and huntingtin in cells transfected with myc-tagged N-terminal huntingtin fragments containing 148 polyglutamine repeats (htt-N63-148Q-myc) and transglutaminase 2 but not in cells transfected with myc-tagged N-terminal huntingtin fragments containing 18 polyglutamine repeats. Our previous studies demonstrated that transfection with both htt-N63-148Q-myc and transglutaminase 2 resulted in cross-linking of mutant huntingtin protein fragments and the formation of insoluble high-molecular-weight aggregates of huntingtin protein fragments. Transfection with transglutaminase 2 and htt-N63-148Q-myc followed by treatment of cells with N-(6-aminohexyl)-1-naphthalenesulfonamide, a calmodulin inhibitor, resulted in a decrease in cross-linked huntingtin. Inhibiting the interaction of calmodulin with transglutaminase and huntingtin protein could decrease cross-linking and diminish huntingtin aggregate formation in the HD brain.

    Funded by: NIMH NIH HHS: MH/NS31862; NINDS NIH HHS: NS16375

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;8;1954-61

  • Calmodulin binding to the Fas death domain. Regulation by Fas activation.

    Ahn EY, Lim ST, Cook WJ and McDonald JM

    Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.

    Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca(2+)-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca(2+)-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased approximately 2-fold early upon Fas activation (at 30 min) and is decreased to approximately 50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.

    The Journal of biological chemistry 2004;279;7;5661-6

  • Endogenous calmodulin interacts with the epidermal growth factor receptor in living cells.

    Li H, Ruano MJ and Villalobo A

    Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.

    We have previously shown that exogenous calmodulin (CaM) binds to the epidermal growth factor receptor (EGFR) at its cytosolic juxtamembrane region inhibiting its tyrosine kinase activity. We demonstrate in this report that endogenous CaM binds to EGFR in intact cells as CaM co-immunoprecipitates with EGF-activated and non-activated receptors. We also show in living cells that cell-permeable CaM inhibitors prevent the full transphosphorylation of wild type EGFR but not the transphosphorylation of an insertional EGFR mutant in which the CaM-binding domain was divided into two parts. Overall these results suggest that CaM interacts with EGFR in vivo.

    FEBS letters 2004;559;1-3;175-80

  • Endofin recruits TOM1 to endosomes.

    Seet LF, Liu N, Hanson BJ and Hong W

    Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore. mcbslf@imcb.a-star.edu.sg

    Endofin is an endosomal protein implicated in regulating membrane trafficking. It is characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain positioned in the middle of the molecule. To determine its potential effectors or binding partners, we used the carboxyl-terminal half of endofin as bait to screen a human brain cDNA library in a yeast two-hybrid system. Three clones that encode TOM1 were recovered. TOM1 is a protein closely related to the VHS (VPS-27, Hrs, and STAM) domain-containing GGA family. Although the function of the GGAs in mediating Golgi to lysosomal trafficking is well established, the subcellular localization and function of TOM1 remain unknown. Glutathione S-transferase pull-down assays as well as co-immunoprecipitation experiments confirmed that the carboxyl-terminal half of endofin binds specifically to the carboxyl-terminal region of TOM1. Neither SARA nor Hrs, two other FYVE domain proteins, interact with this region of TOM1. Moreover, endofin does not interact with the analogous region of two other members of the TOM1 protein family, namely, TOM1-like 1 (TOM1-L1) or TOM1-like 2 (TOM1-L2). The carboxyl-terminal region of TOM1 was used as immunogen to generate TOM1-specific antibody. This antibody can distinguish TOM1 from the other family members as well as coimmunoprecipitate endogenous endofin. It also revealed the primarily cytosolic distribution of TOM1 in a variety of cell types by immunofluorescence analyses. In addition, sucrose density gradient analysis showed that both TOM1 and endofin can be detected in cellular compartments marked by the early endosomal marker EEA1. A marked recruitment of TOM1 to endosomes was observed in cells overexpressing endofin or its carboxyl-terminal fragment, indicating TOM1 to be an effector for endofin and suggesting a possible role for TOM1 in endosomal trafficking.

    The Journal of biological chemistry 2004;279;6;4670-9

  • Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells.

    Cifuentes E, Mataraza JM, Yoshida BA, Menon M, Sacks DB, Barrack ER and Reddy GP

    Vattikuti Urology Institute, Henry Ford Health Sciences Center, Detroit, MI 48202, USA.

    Ca(2+) and calmodulin (CaM) play a critical role in proliferation and viability of a wide variety of cells, including prostate cancer cells. We examined two prostate cancer cell lines, androgen-sensitive LNCaP and androgen-independent PC-3. Proliferation of LNCaP cells was six to eight times more sensitive to the inhibitory effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) than were PC-3 cells. Because LNCaP cell proliferation is sensitive to stimulation by androgen, we assessed the physical and functional interaction between androgen receptor (AR) and CaM. We observed tight binding of AR to CaM when LNCaP cell extracts were subjected to CaM-affinity column chromatography. AR binding to CaM was Ca(2+)-dependent and was inhibited by pretreatment of the cell extracts with W-7. Using immunofluorescence staining and confocal microscopy, we demonstrated colocalization of AR and CaM in the nucleus of LNCaP cells. Furthermore, the functional relevance of AR-CaM interactions in intact cells was revealed by the observation that W-7 was as effective as Casodex, an antiandrogen, in blocking AR-regulated expression of prostate-specific antigen in LNCaP cells. AR seems to interact with CaM directly because purified human AR could bind to CaM-agarose, and CaM could be detected in AR-immunoprecipitate prepared from purified soluble proteins. These studies provide direct evidence for physical and functional interaction between AR and CaM and suggest the potential usefulness of CaM antagonists in blocking AR activity in prostate cancer.

    Funded by: NCI NIH HHS: CA93645, R01 CA093645; NIDDK NIH HHS: DK57864, R01 DK057864

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;2;464-9

  • TRPC channel interactions with calmodulin and IP3 receptors.

    Zhu MX and Tang J

    Center for Molecular Neurobiology and Department of Neuroscience, The Ohio State University, 168 Rightmire Hall, 1060 Carmack Road, Columbus, Ohio 43210, USA.

    Consistent with the conformational coupling mechanism, which suggests that store-operated channels are activated via physical interactions with intracellular calcium release channels, previous studies have demonstrated a functional coupling and a physical interaction between the transient receptor potential canonical type 3 (TRPC3) and inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R-TRPC binding domains were determined using in vitro binding assays. This work aimed to study the effect of IP3R-TRPC interaction on TRPC function. Pull-down experiments were used to study the binding of TRPC to IP3R and to calmodulin. Patch clamp recordings in whole-cell and inside-out configurations were used to examine the effect of a TRPC-binding IP3R fragment, Ca2+ and calmodulin on TRPC activity. We found that IP3R and calmodulin compete for a common binding site at the TRPC C-terminus. TRPC channels are activated either by a peptide representing the TRPC-binding domain of IP3R or by inactivation of calmodulin from the excised membrane patches. TRPC3 activity is inhibited by Ca2+ and calmodulin. Therefore, we have identified a critical IP3R-TRPC interaction that is involved in the activation of TRPC-formed channels. We propose that IP3Rs activate TRPC channels by displacing inhibitory calmodulin from a common calmodulin-IP3R binding site located at the C-terminus of TRPC.

    Funded by: NINDS NIH HHS: NS42183

    Novartis Foundation symposium 2004;258;44-58; discussion 58-62, 98-102, 263-6

  • A new role for IQ motif proteins in regulating calmodulin function.

    Putkey JA, Kleerekoper Q, Gaertner TR and Waxham MN

    Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA. John.Putkey@uth.tmc.edu

    IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca2+ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2+. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca2+ from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca2+ from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca2+-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca2+ binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca2+-dependent cell signaling.

    Funded by: NHLBI NIH HHS: HL45724; NINDS NIH HHS: NS26086

    The Journal of biological chemistry 2003;278;50;49667-70

  • Calmodulin is a phospholipase C-beta interacting protein.

    McCullar JS, Larsen SA, Millimaki RA and Filtz TM

    Department of Pharmaceutical Sciences, College of Pharmacy, and the Molecular and Cellular Biology Program, Oregon State University, Corvallis, Oregon 97331, USA.

    Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

    Funded by: NIGMS NIH HHS: GM61244, R01 GM061244

    The Journal of biological chemistry 2003;278;36;33708-13

  • Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade.

    Vergne I, Chua J and Deretic V

    Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, 915 Camino de Salud, NE, Albuquerque, NM 87131, USA.

    The capacity of Mycobacterium tuberculosis to infect latently over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome in infected macrophages. Here we describe how M. tuberculosis toxin lipoarabinomannan (LAM) causes phagosome maturation arrest, interfering with a new pathway connecting intracellular signaling and membrane trafficking. LAM from virulent M. tuberculosis, but not from avirulent mycobacteria, blocked cytosolic Ca2+ increase. Ca2+ and calmodulin were required for a newly uncovered Ca2+/calmodulin phosphatidylinositol (PI)3 kinase hVPS34 cascade, essential for production of PI 3 phosphate (PI3P) on liposomes in vitro and on phagosomes in vivo. The interference of the trafficking toxin LAM with the calmodulin-dependent production of PI3P described here ensures long-term M. tuberculosis residence in vacuoles sequestered away from the bactericidal and antigen-processing organelles in infected macrophages.

    Funded by: NIAID NIH HHS: AI-75320, AI45148, R01 AI045148

    The Journal of experimental medicine 2003;198;4;653-9

  • Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site.

    Strotmann R, Schultz G and Plant TD

    Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany.

    Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.

    The Journal of biological chemistry 2003;278;29;26541-9

  • The Na+/H+ exchanger cytoplasmic tail: structure, function, and interactions with tescalcin.

    Li X, Liu Y, Kay CM, Müller-Esterl W and Fliegel L

    Department of Biochemistry, Faculty of Medicine, CIHR Membrane Protein Research Group, University of Alberta, 347 Medical Science Building, Edmonton, Alberta, Canada T6G 2H7.

    We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.

    Biochemistry 2003;42;24;7448-56

  • Structural determinant of TRPV1 desensitization interacts with calmodulin.

    Numazaki M, Tominaga T, Takeuchi K, Murayama N, Toyooka H and Tominaga M

    Department of Cellular and Molecular Physiology, Mie University School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan.

    The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca2+-dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca2+-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;13;8002-6

  • GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity.

    Caprini M, Gomis A, Cabedo H, Planells-Cases R, Belmonte C, Viana F and Ferrer-Montiel A

    Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Alicante, Spain.

    The identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion cDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca(2+) concentration ([Ca(2+)](i)). We report the unexpected identification of GAP43 (also known as neuromodulin or B50), a membrane-anchored neuronal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca(2+)](i) in response to hypotonicity. Palmitoylation of GAP43 plays an important role in the protein osmosensitivity. Depletion of intracellular stores or inhibition of phospholipase C (PLC) activity abrogates hypotonicity-evoked, GAP43-mediated [Ca(2+)](i) elevations. Notably, hypotonicity promoted the selective association of GAP43 with the PLC-delta(1) isoform, and a concomitant increase in inositol-1,4,5-trisphosphate (IP(3)) formation. Collectively, these findings indicate that hypo-osmotic activation of GAP43 induces Ca(2+) release from IP(3)-sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phospho inositide metabolism, spontaneous triggering of cytosolic Ca(2+) transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces.

    The EMBO journal 2003;22;12;3004-14

  • Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels.

    Trudeau MC and Zagotta WN

    Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington Medical School, Seattle, Washington 98195, USA.

    Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels.

    Funded by: NEI NIH HHS: EY010329-10, R01 EY010329, R01 EY010329-10, T32 EY07031

    The Journal of biological chemistry 2003;278;21;18705-8

  • Ca2+/calmodulin binds and dissociates K-RasB from membrane.

    Sidhu RS, Clough RR and Bhullar RP

    Department of Oral Biology, University of Manitoba, 780 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W2.

    We have investigated the interaction of calmodulin (CaM) with Ras-p21 and the significance of this association. All Ras-p21 isoforms tested (H-, K-, and N-Ras) were detected in the particulate fraction of human platelets and MCF-7 cells, a human breast cancer cell line. In MCF-7 cells, H- and N-Ras were also detected in the cytosolic fraction. K-RasB from platelet and MCF-7 cell lysates was found to bind CaM in a Ca2+ -dependent but GTPgammaS-independent manner. The yeast two-hybrid analysis demonstrated that K-RasB binds to CaM in vivo. Incubation of isolated membranes from platelet and MCF-7 cells with CaM caused dissociation of only K-RasB from membranes in a Ca2+ -dependent manner. CaM antagonist, W7, inhibited dissociation of K-RasB. Addition of platelet or MCF-7 cytosol alone to isolated platelet membranes did not cause dissociation of K-RasB and only addition of exogenous CaM caused dissociation. The results suggest a potential role for Ca2+/CaM in the regulation of K-RasB function.

    Biochemical and biophysical research communications 2003;304;4;655-60

  • A noncontiguous, intersubunit binding site for calmodulin on the skeletal muscle Ca2+ release channel.

    Zhang H, Zhang JZ, Danila CI and Hamilton SL

    Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.

    Both apocalmodulin (Ca(2+)-free calmodulin) and Ca(2+)-calmodulin bind to and regulate the activity of skeletal muscle Ca(2+) release channel (ryanodine receptor, RYR1). Both forms of calmodulin protect sites after amino acids 3630 and 3637 on RYR1 from trypsin cleavage. Only apocalmodulin protects sites after amino acids 1982 and 1999 from trypsin cleavage. Ca(2+)-calmodulin and apocalmodulin both bind to two different synthetic peptides representing amino acids 3614-3643 and 1975-1999 of RYR1, but Ca(2+)-calmodulin has a higher affinity than apocalmodulin for both peptides. Cysteine 3635, within the 3614-3643 sequence of RYR1, can form a disulfide bond with a cysteine on an adjacent subunit within the RYR1 tetramer. The second cysteine is now shown to be between amino acids 2000 and 2401. The close proximity of the cysteines forming the intersubunit disulfide to the two sites that bind calmodulin suggests that calmodulin is binding at a site of intersubunit contact, perhaps with one lobe bound between amino acids 3614 and 3643 on one subunit and the second lobe bound between amino acids 1975 and 1999 on an adjacent subunit. This model is consistent with the finding that Ca(2+)-calmodulin and apocalmodulin each bind to a single site per RYR1 subunit (Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K., and Hamilton, S. L. (2000) Biochemistry 39, 7807-7812).

    Funded by: NIAMS NIH HHS: AR41729, AR41802

    The Journal of biological chemistry 2003;278;10;8348-55

  • Characterization of calmodulin binding to the orphan nuclear receptor Errgamma.

    Hentschke M, Schulze C, Süsens U and Borgmeyer U

    Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany.

    The estrogen receptor-related receptor gamma (ERRgamma/ ERR3/NR3B3), a member of the nuclear receptor superfamily, activates transcription in the absence of ligands. In order to identify ligand-independent mechanisms of activation, we tested whether calmodulin (CaM), a key regulator of numerous cellular processes and a predominant intracellular receptor for Ca2+-signals, interacts with ERRgamma. In vitro pull-down experiments with calmodulin-Sepharose demonstrated a Ca2+-dependent interaction with cellularly expressed ERRgamma. As shown by truncation analysis, the CaM binding site is highly unusual in that it is composed of two discontinuous elements. Moreover, by surface plasmon resonance (SPR) biosensor technology, we detected a direct interaction of immobilized bacterially expressed ERR-gamma fusion protein with Ca2+-calmodulin. This is best described by a model which assumes a conformational change of the initially formed complex to a more stable form. Whereas in vitro DNA binding was calmodulin-independent, transient transfection analysis revealed a Ca2+-influx-dependent ERRgamma-mediated transcriptional activation of a luciferase reporter gene. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of ERRgamma.

    Biological chemistry 2003;384;3;473-82

  • Elucidation of the interaction of calmodulin with the IQ motifs of IQGAP1.

    Li Z and Sacks DB

    Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Calmodulin regulates the function of numerous proteins by binding to short regions on the target molecule. IQ motifs, which are found in over 100 human proteins, appear in tandem repeats and bind calmodulin in the absence of Ca(2+). One of these IQ-containing proteins, IQGAP1, interacts with several targets, including Cdc42, beta-catenin, E-cadherin, and actin, in a calmodulin-regulated manner. To elucidate the molecular mechanism by which apocalmodulin and Ca(2+)/calmodulin differentially regulate IQGAP1, a series of constructs of IQGAP1 with selected point mutations of the four tandem IQ motifs were generated. Mutating the basic charged arginine residues in all four IQ motifs abrogated binding of IQGAP1 to apocalmodulin, but had no effect on its interaction with Ca(2+)/calmodulin. Analysis of IQGAP1 constructs with point mutations in single, double, or triple IQ motifs revealed that apocalmodulin bound only to IQ3 and IQ4. By contrast to the arginine mutant constructs, mutation of selected hydrophobic residues in the IQ motifs produced an IQGAP1 protein incapable of binding either apocalmodulin or Ca(2+)/calmodulin. These results, which differ from the conventional model of Ca(2+)-independent binding of calmodulin to IQ motifs, provide insight into the complexity of the molecular interactions between calmodulin and IQ motifs.

    The Journal of biological chemistry 2003;278;6;4347-52

  • Regulation of c-Rel nuclear localization by binding of Ca2+/calmodulin.

    Antonsson A, Hughes K, Edin S and Grundström T

    Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.

    The NF-kappa B/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory I kappa B proteins sequester NF-kappa B/Rel in the cytoplasm. Cellular stimulation results in the degradation of I kappa B and modification of NF-kappa B/Rel proteins, allowing NF-kappa B/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca(2+) binding protein that serves as a key mediator of intracellular Ca(2+) signals. Here we report that two members of the NF-kappa B/Rel family, c-Rel and RelA, interact directly with Ca(2+)-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of I kappa B. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca(2+) signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from I kappa B and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-kappa B/Rel proteins following stimulation.

    Molecular and cellular biology 2003;23;4;1418-27

  • Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism.

    Clapperton JA, Martin SR, Smerdon SJ, Gamblin SJ and Bayley PM

    Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

    Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieve autoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI (residues 294-318) has been determined by X-ray crystallography at 1.7 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303 (interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residues associated with the inhibitory pseudosubstrate sequence.

    Biochemistry 2002;41;50;14669-79

  • Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor.

    Lee FJ, Xue S, Pei L, Vukusic B, Chéry N, Wang Y, Wang YT, Niznik HB, Yu XM and Liu F

    Department of Neuroscience, Centre for Addiction and Mental Health, Clarke Division, University of Toronto, Toronto, M5T 1R8, Ontario, Canada

    Dopamine D1-like receptors, composed of D1 and D5 receptors, have been documented to modulate glutamate-mediated fast excitatory synaptic neurotransmission. Here, we report that dopamine D1 receptors modulate NMDA glutamate receptor-mediated functions through direct protein-protein interactions. Two regions in the D1 receptor carboxyl tail can directly and selectively couple to NMDA glutamate receptor subunits NR1-1a and NR2A. While one interaction is involved in the inhibition of NMDA receptor-gated currents, the other is implicated in the attenuation of NMDA receptor-mediated excitotoxicity through a PI-3 kinase-dependent pathway.


  • Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.

    Wen H and Levitan IB

    Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    Calmodulin (CaM) was identified as a KCNQ2 and KCNQ3 potassium channel-binding protein, using a yeast two-hybrid screen. CaM is tethered constitutively to the channel, in the absence or presence of Ca2+, in transfected cells and also coimmunoprecipitates with KCNQ2/3 from mouse brain. The structural elements critical for CaM binding to KCNQ2 lie in two conserved motifs in the proximal half of the channel C-terminal domain. Truncations and point mutations in these two motifs disrupt the interaction. The first CaM-binding motif has a sequence that conforms partially to the consensus IQ motif, but both wild-type CaM and a Ca2+-insensitive CaM mutant bind to KCNQ2. The voltage-dependent activation of the KCNQ2/3 channel also shows no Ca2+ sensitivity, nor is it affected by overexpression of the Ca2+-insensitive CaM mutant. On the other hand, KCNQ2 mutants deficient in CaM binding are unable to generate detectable currents when coexpressed with KCNQ3 in CHO cells, although they are expressed and targeted to the cell membrane and retain the ability to assemble with KCNQ3. A fusion protein containing both of the KCNQ2 CaM-binding motifs competes with the full-length KCNQ2 channel for CaM binding and decreases KCNQ2/3 current density in CHO cells. The correlation of CaM binding with channel function suggests that CaM is an auxiliary subunit of the KCNQ2/3 channel.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;18;7991-8001

  • alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes.

    Lee D, Lee SY, Lee EN, Chang CS and Paik SR

    Department of Biochemistry, College of Medicine, Inha University, Nam-Ku, Inchon, Korea.

    alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed tha 14d9 t alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.

    Journal of neurochemistry 2002;82;5;1007-17

  • Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex.

    Takahashi M, Yamagiwa A, Nishimura T, Mukai H and Ono Y

    Biosignal Research Center, Kobe University, Japan.

    Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.

    Molecular biology of the cell 2002;13;9;3235-45

  • The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels.

    Yus-Najera E, Santana-Castro I and Villarroel A

    Instituto Cajal, Consejo Superior de Investigaciones, Avenida, Dr. Arce 37, 28002 Madrid, Spain.

    We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B). These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.

    The Journal of biological chemistry 2002;277;32;28545-53

  • Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets.

    Clough RR, Sidhu RS and Bhullar RP

    Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W2, Canada.

    Ral GTPases may be involved in calcium/calmodulin-mediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calcium-independent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.

    The Journal of biological chemistry 2002;277;32;28972-80

  • Phosphorylation of calmodulin. Functional implications.

    Benaim G and Villalobo A

    Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.

    Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein-serine/threonine and protein-tyrosine kinases. Casein kinase II and myosin light-chain kinase are two of the well established protein-serine/threonine kinases implicated in this process. On the other hand, within the protein-tyrosine kinases involved in the phosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein-tyrosine kinases, such as several members of the Src family kinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM-dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We will emphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation-mediated changes of CaM activity, its action on CaM-dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.

    European journal of biochemistry 2002;269;15;3619-31

  • Presence of cyclic nucleotide phosphodiesterases PDE1A, existing as a stable complex with calmodulin, and PDE3A in human spermatozoa.

    Lefièvre L, de Lamirande E and Gagnon C

    Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.

    Mammalian sperm motility, capacitation, and the acrosome reaction are regulated by signal transduction systems involving cAMP as a second messenger. Levels of cAMP are controlled by two key enzymes, adenylyl cyclase and phosphodiesterases (PDEs), the latter being involved in cAMP degradation. Calmodulin-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities were previously identified in spermatozoa via the use of specific inhibitors. Here we report that human sperm PDEs are associated with the plasma membrane (50%-60%) as well as with the particulate fraction (30%-50%) and have more affinity for cAMP than cGMP. Immunocytochemical data indicated that PDE1A, a variant of PDE1, is localized on the equatorial segment of the sperm head as well as on the mid and principal pieces of the flagellum, and that PDE3A is found on the postacrosomal segment of the sperm head. Immunoblotting confirmed the presence of PDE1A and PDE3A isoforms in spermatozoa. Milrinone, a PDE3 inhibitor, increased intracellular levels of cAMP by about 15% but did not affect sperm functions, possibly because PDE3 represents only a small proportion of the sperm total PDE activity (10% and 25% in Triton X-100 soluble and particulate fractions, respectively). PDE1A activity in whole sperm extract or after partial purification by anion-exchange chromatography was not stimulated by calcium + calmodulin. Results obtained with electrophoresis in native conditions indicated that calmodulin is tightly bound to PDE1A. Incubation with EGTA + EDTA, trifluoperazine, or urea did not dissociate the PDE1A-calmodulin complex. These results suggest that PDE1A is permanently activated in human spermatozoa.

    Biology of reproduction 2002;67;2;423-30

  • Calmodulin-containing substructures of the centrosomal matrix released by microtubule perturbation.

    Moisoi N, Erent M, Whyte S, Martin S and Bayley PM

    Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

    Calmodulin redistribution in MDCK and HeLa cells subjected to microtubule perturbations by antimitotic drugs was followed using a calmodulin-EGFP fusion protein that preserves the Ca(2+) affinity, target binding and activation properties of native calmodulin. CaM-EGFP targeting to spindle structures in normal cell division and upon spindle microtubule disruption allows evaluation of the dynamic redistribution of calmodulin in cell division. Under progressive treatment of stably transfected mammalian cells with nocodazole or vinblastine, the centrosomal matrix at the mitotic poles subdivides into numerous small 'star-like' structures, with the calmodulin concentrated centrally, and partially distinct from the reduced microtubule mass to which kinetochores and chromosomes are attached. Prolonged vinblastine treatment causes the release of localised calmodulin into a uniform cytoplasmic distribution, and tubulin paracrystal formation. By contrast, paclitaxel treatment of metaphase cells apparently causes limited disassembly of the pericentriolar material into a number of multipolar 'ring-like' structures containing calmodulin, each one having multiple attached microtubules terminating in the partially disordered kinetochore/chromosome complex. Thus drugs with opposite effects in either destabilising or stabilising mitotic microtubules cause subdivision of the centrosomal matrix into two distinctive calmodulin-containing structures, namely small punctate 'stars' or larger polar 'rings' respectively. The 'star-like' structures may represent an integral subcomponent for the attachment of kinetochore microtubules to the metaphase centrosome complex. The results imply that microtubules have a role in stabilising the structure of the pericentriolar matrix, involving interaction, either direct or indirect, with one or more proteins that are targets for binding of calmodulin. Possible candidates include the pericentriolar matrix-associated coiled-coil proteins containing calmodulin-binding motifs, such as myosin V, kendrin (PCNT2) and AKAP450.

    Journal of cell science 2002;115;Pt 11;2367-79

  • Protein Ser/Thr phosphatases PPEF interact with calmodulin.

    Kutuzov MA, Solov'eva OV, Andreeva AV and Bennett N

    Laboratoire de Biophysique Moléculaire et Cellulaire URA CNRS No. 520, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble cedex 9, France. m.kutuzov@usa.net

    Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.

    Biochemical and biophysical research communications 2002;293;3;1047-52

  • Calmodulin is a selective modulator of estrogen receptors.

    García Pedrero JM, Del Rio B, Martínez-Campa C, Muramatsu M, Lazo PS and Ramos S

    Departamento de Bioquímica y Biología Molecular and Instituto Universitario de Oncología Principado de Asturias, Universidad de Oviedo, 33007 Oviedo, Spain.

    In the search for differences between ERalpha and ERbeta, we analyzed the interaction of both receptors with calmodulin (CaM) and demonstrated that ERalpha but not ERbeta directly interacts with CaM. Using transiently transfected HeLa cells, we examined the effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W7) on the transactivation properties of ERalpha and ERbeta in promoters containing either estrogen response elements or activator protein 1 elements. Transactivation by ERalpha was dose-dependently inhibited by W7, whereas that of ERbeta was not inhibited or even activated at low W7 concentrations. In agreement with these results, transactivation of an estrogen response element containing promoter in MCF-7 cells (which express a high ERalpha/ERbeta ratio) was also inhibited by W7. In contrast, transactivation in T47D cells (which express a low ERalpha/ERbeta ratio) was not affected by this CaM antagonist. The sensitivity of MCF-7 cells to W7 was abolished when cells were transfected with increasing amounts of ERbeta, indicating that the sensitivity to CaM antagonists of estrogen-responsive tissues correlates with a high ERalpha/ERbeta ratio. Finally, substitution of lysine residues 302 and 303 of ERalpha for glycine rendered a mutant ERalpha unable to interact with CaM whose transactivation activity became insensitive to W7. Our results indicate that CaM antagonists are selective modulators of ER able to inhibit ERalpha-mediated activity, whereas ERbeta actions were not affected or even potentiated by W7.

    Molecular endocrinology (Baltimore, Md.) 2002;16;5;947-60

  • IQGAP1-mediated stimulation of transcriptional co-activation by beta-catenin is modulated by calmodulin.

    Briggs MW, Li Z and Sacks DB

    Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.

    Beta-catenin, an oncoprotein integral to cell-cell adhesion and proliferative signaling, is increased in several malignancies. The recently discovered calmodulin-binding protein IQGAP1 binds stoichiometrically to beta-catenin and regulates the association of beta-catenin with the cell-cell adhesion complex. In the present work, we investigated the role of IQGAP1 on the transcriptional co-activator function of beta-catenin, and whether calmodulin modulated the functional interaction between IQGAP1 and beta-catenin. In vitro competition assays revealed that both Ca(2+)/calmodulin and apo-calmodulin, when pre-bound to IQGAP1, prevented binding of beta-catenin to IQGAP1, and partially displaced beta-catenin pre-bound to IQGAP1 when added subsequently. Conversely, beta-catenin partially displaced apo-calmodulin, but not Ca(2+)/calmodulin, from IQGAP1. Overexpression of IQGAP1 in SW480 colon carcinoma cells enhanced beta-catenin-mediated transcriptional co-activation by 1.72-fold, and this stimulation was significantly attenuated upon antagonism of calmodulin using the cell-permeable antagonist CGS9343B. Moreover, an IQGAP1 mutant that does not bind calmodulin was unable to stimulate beta-catenin transcriptional function. Results of pulse-chase analyses suggested that IQGAP1 slowed the turnover of soluble, but not total, beta-catenin. Immunocytochemistry revealed that IQGAP1 overexpression increased the amount of beta-catenin located in the nucleus, whereas incubation of cells with CGS9343B blocked this accumulation. Together, our results imply that IQGAP1 enhances the function of beta-catenin in the nucleus and that calmodulin regulates this stimulation.

    Funded by: NCI NIH HHS: CA 75205

    The Journal of biological chemistry 2002;277;9;7453-65

  • Interaction between metabotropic glutamate receptor 7 and alpha tubulin.

    Saugstad JA, Yang S, Pohl J, Hall RA and Conn PJ

    Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon, USA. jsaugstad@downeurobiology.org

    Metabotropic glutamate receptors (mGluRs) mediate a variety of responses to glutamate in the central nervous system. A primary role for group-III mGluRs is to inhibit neurotransmitter release from presynaptic terminals, but the molecular mechanisms that regulate presynaptic trafficking and activity of group-III mGluRs are not well understood. Here, we describe the interaction of mGluR7, a group-III mGluR and presynaptic autoreceptor, with the cytoskeletal protein, alpha tubulin. The mGluR7 carboxy terminal (CT) region was expressed as a GST fusion protein and incubated with rat brain extract to purify potential mGluR7-interacting proteins. These studies yielded a single prominent mGluR7 CT-associated protein of 55 kDa, which subsequent microsequencing analysis revealed to be alpha tubulin. Coimmunoprecipitation assays confirmed that full-length mGluR7 and alpha tubulin interact in rat brain as well as in BHK cells stably expressing mGluR7a, a splice variant of mGluR7. In addition, protein overlay experiments showed that the CT domain of mGluR7a binds specifically to purified tubulin and calmodulin, but not to bovine serum albumin. Further pull-down studies revealed that another splice variant mGluR7b also interacts with alpha tubulin, indicating that the binding region is not localized to the splice-variant regions of either mGluR7a (900-915) or mGluR7b (900-923). Indeed, deletion mutagenesis experiments revealed that the alpha tubulin-binding site is located within amino acids 873-892 of the mGluR7 CT domain, a region known to be important for regulation of mGluR7 trafficking. Interestingly, activation of mGluR7a in cells results in an immediate and significant decrease in alpha tubulin binding. These data suggest that the mGluR7/alpha tubulin interaction may provide a mechanism to control access of the CT domain to regulatory molecules, or alternatively, that this interaction may lead to morphological changes in the presynaptic membrane in response to receptor activation.

    Funded by: NIMH NIH HHS: R01 MH059951, R01 MH059951-01; NINDS NIH HHS: NS313373, NS348876, NS36755; PHS HHS: R01-521635

    Journal of neurochemistry 2002;80;6;980-8

  • Nef of HIV-1 interacts directly with calcium-bound calmodulin.

    Hayashi N, Matsubara M, Jinbo Y, Titani K, Izumi Y and Matsushima N

    Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan. nhayashi@fujita-hu.ac.jp

    It was recently found that the myristoyl group of CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is essential for the interaction between the protein and Ca(2+)-bound calmodulin (Ca(2+)/CaM). Based on the N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein-calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca(2+)/CaM in the presence of a myristoylated peptide corresponding to the N-terminal region of Nef. The dissociation constant between Ca(2+)/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca(2+)/CaM changed markedly in the Ca(2+)/CaM-Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X-ray scattering measurements is 2-3 A smaller that of Ca(2+)/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca(2+)/CaM.

    Protein science : a publication of the Protein Society 2002;11;3;529-37

  • The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins.

    Tchernev VT, Mansfield TA, Giot L, Kumar AM, Nandabalan K, Li Y, Mishra VS, Detter JC, Rothberg JM, Wallace MR, Southwick FS and Kingsmore SF

    CuraGen Corporation, New Haven, CT 06511, USA. velizart@molecularstaging.com

    Background: Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis.

    Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay.

    Results: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II).

    Conclusions: On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.

    Funded by: NIAID NIH HHS: P01 AI039824; NICHD NIH HHS: U19 HD077693

    Molecular medicine (Cambridge, Mass.) 2002;8;1;56-64

  • Rab3B in human platelet is membrane bound and interacts with Ca(2+)/calmodulin.

    Sidhu RS and Bhullar RP

    Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0W2, Canada.

    The subcellular distribution of Rab3B in fresh and aged platelets was determined and majority of the protein was localized with the particulate fraction with only a minor amount detected in the cytosol. Rab3B was pulled out from platelet particulate fraction with GST-RabGDI-alpha fusion protein. Using GST-Rab3B in in vitro pull-down experiments, the binding of calmodulin from platelet cytosol to Rab3B was demonstrated. In the reverse experiment, binding of Rab3B from platelet particulate and cytosolic fractions to Sepharose-CaM beads was also observed. The interaction between Rab3B and calmodulin was Ca(2+)-dependent but independent of the guanine nucleotide status of Rab3B. These findings provide evidence that Rab3B is primarily localized with the particulate fraction and that Ca(2+)/calmodulin could regulate function of this GTPase in the platelet.

    Biochemical and biophysical research communications 2001;289;5;1039-43

  • A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin.

    Benítez MJ, Cochet C and Jiménez JS

    Departamento de Química Física Aplicada, y Centro de Biología Molecular Severo Ochoa (UAM-CSIC), Universidad Autónoma de Madrid, Cantoblanco, Spain.

    Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, alpha-subunits, and two regulatory, beta-subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is downregulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30 degrees C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2beta subunit and protein substrates share a common, or at least an overlapping, site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to alpha subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine.

    Molecular and cellular biochemistry 2001;227;1-2;31-6

  • Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

    Villalonga P, López-Alcalá C, Bosch M, Chiloeches A, Rocamora N, Gil J, Marais R, Marshall CJ, Bachs O and Agell N

    Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.

    Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

    Molecular and cellular biology 2001;21;21;7345-54

  • Some properties of caldesmon and calponin and the participation of these proteins in regulation of smooth muscle contraction and cytoskeleton formation.

    Gusev NB

    Department of Biochemistry, School of Biology and School of Fundamental Medicine, Lomonosov Moscow State University, Moscow, 119899, Russia. NBGusev@mail.ru

    The interaction of caldesmon with different Ca2+-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of caldesmon. The arrangement of caldesmon and Ca2+-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B' of caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of caldesmon. The complex of calmodulin and caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with caldesmon. Phosphorylation of caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actin-binding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and alpha-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton.

    Biochemistry. Biokhimiia 2001;66;10;1112-21

  • Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin binding.

    Wang D, Quillan JM, Winans K, Lucas JL and Sadée W

    Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.

    The mu opioid receptor (MOR) plays a central role in mediating acute and chronic effects of narcotic drugs. Three rare single nucleotide polymorphisms in the hMOR gene have been identified that cause amino acid substitutions in the third intracellular (i3) loop of MOR (R260H, R265H, and S268P). Genotyping 252 individuals of the Coriell collection identified one allele encoding the R265H-MOR variant and a new variant encoding D274N-MOR. Variants R260H-, R265H-, and S268P-MOR were constructed and transfected into HEK293 cells. Morphine stimulated G protein coupling of the three receptor variants to a maximal level approaching that of wild type MOR. In contrast, spontaneous, agonist-independent (basal) MOR signaling, proposed to play a role in opioid tolerance and dependence, was significantly reduced for R260H- and R265H-MOR. Moreover, domains within the i3 loop of MOR have been shown to interact with both G proteins and calmodulin (CaM). CaM binding was deficient for variants R265H- and S268P-MOR, suggesting that domains for G protein coupling and CaM binding overlap partially. Morphine pretreatment significantly enhanced basal G protein coupling of wild type MOR, which is thought to result from release of CaM. In contrast basal G protein coupling activity of the three variants was unaffected by morphine pretreatment consistent with diminished CaM regulation, low basal activity, or both. In conclusion, each of the three single nucleotide polymorphisms mapping to the i3 loop of MOR caused substantial changes in basal G protein coupling, CaM binding, or both. Carriers of the mutant alleles might display altered responses to narcotic analgesics.

    The Journal of biological chemistry 2001;276;37;34624-30

  • Human Ca2+/calmodulin-dependent protein kinase kinase beta gene encodes multiple isoforms that display distinct kinase activity.

    Hsu LS, Chen GD, Lee LS, Chi CW, Cheng JF and Chen JY

    Graduate Institute of Life Sciences, National Defense Medical Center, Institute of Biomedical Sciences, Academia Sinica, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China.

    Ca(+2)/calmodulin-dependent protein kinases (CaMKs) are activated upon binding of Ca(+2)/calmodulin. To gain maximal activity, CaMK I and CaMK IV can be further phosphorylated by an upstream kinase, CaMK kinase (CaMKK). We previously isolated cDNA clones encoding human CaMKK beta isoforms that are heterogeneous in their 3'-sequences (Hsu, L.-S., Tsou, A.-P., Chi, C.-W., Lee, C.-H., and Chen, J.-Y. (1998) J. Biomed. Sci. 5, 141-149). In the present study, we examined the genomic organization and transcription of the human CaMKK beta gene. The human CaMKK beta locus spans more than 40 kilobase pairs and maps to chromosome 12q24.2. It is organized into 18 exons and 17 introns that are flanked by typical splice donor and acceptor sequences. Two major species of transcripts, namely the beta1 (5.6 kilobase pairs) and beta2 (2.9 kilobase pairs), are generated through differential usage of polyadenylation sites located in the last and penultimate exons. Additional forms of CaMKK beta transcripts were also identified that resulted from alternative splicing of the internal exons 14 and/or 16. These isoforms display differential expression patterns in human tissues and tumor-derived cell lines. They also exhibit a distinct ability to undergo autophosphorylation and to phosphorylate the downstream kinases CaMK I and CaMK IV. The differential expression of CaMKK beta isoforms with distinct activity further suggests the complexity of the regulation of the CaMKK/CaMK cascade and an important role for CaMKK in the action of Ca(+2)-mediated cellular responses.

    The Journal of biological chemistry 2001;276;33;31113-23

  • Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

    Young P, Ehler E and Gautel M

    European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

    Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

    The Journal of cell biology 2001;154;1;123-36

  • Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin.

    Schumacher MA, Rivard AF, Bächinger HP and Adelman JP

    Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA. schumacm@ohsu.edu

    Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.

    Nature 2001;410;6832;1120-4

  • The tumor-sensitive calmodulin-like protein is a specific light chain of human unconventional myosin X.

    Rogers MS and Strehler EE

    Tumor Biology Program, Department of Biochemistry and Molecular Biology, Mayo Graduate School and Mayo Clinic Cancer Center, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.

    Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human "unconventional" myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca(2+)-dependent. CLP interacted strongly with IQ motif 3 (K(d) approximately 0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin X in vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.

    Funded by: NCI NIH HHS: T32CA75926

    The Journal of biological chemistry 2001;276;15;12182-9

  • Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin.

    Niemeyer BA, Bergs C, Wissenbach U, Flockerzi V and Trost C

    Institut für Pharmakologie und Toxikologie der Universität des Saarlandes, 66421 Homburg, Germany.

    A finely tuned Ca(2+) signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca(2+) channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca(2+) permeation properties suggest an important function in Ca(2+) uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca(2+)-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;6;3600-5

  • Myosin VIIA is specifically associated with calmodulin and microtubule-associated protein-2B (MAP-2B).

    Todorov PT, Hardisty RE and Brown SD

    MRC Mammalian Genetics Unit and UK Mouse Genome Centre, Harwell OX11 0RD, U.K.

    Myosin VIIA is a motor molecule with a conserved head domain and tail region unique to myosin VIIA, which probably defines its unique function in vivo. In an attempt to further characterize myosin VIIA function we set out to identify molecule(s) that specifically associate with it. We demonstrate that 17 and 55 kDa proteins from mouse kidney and cochlea co-purify with myosin VIIA on affinity columns carrying immobilized anti-myosin VIIA antibody. N-terminal sequencing and immunoblotting analysis identified the 17 kDa protein as calmodulin, whereas MS and immunoblotting analysis identified the 55 kDa protein as microtubule-associated protein-2B (MAP-2B). Myosin VIIA can also be co-immunoprecipitated from kidney homogenate using anti-calmodulin or anti-MAP2 (recognizing isoforms 2A and 2B) antibodies, confirming the strong association between calmodulin and myosin VIIA and between MAP-2B and myosin VIIA. Myosin VIIA binds to calmodulin with an apparent K(d) of 10(-9) M. Scatchard analysis of the binding of myosin VIIA to MAP-2B provided evidence for two binding sites, with K(d) values of 10(-10) and 10(-9) M, which have been mapped to medial and C-terminal tail domains of myosin VIIA. The characterization of the interaction of calmodulin and MAP-2B with myosin VIIA provides new insights into the function of myosin VIIA.

    The Biochemical journal 2001;354;Pt 2;267-74

  • Kinase recognition by calmodulin: modeling the interaction with the autoinhibitory region of human cardiac titin kinase.

    Amodeo P, Castiglione Morelli MA, Strazzullo G, Fucile P, Gautel M and Motta A

    Istituto per la Chimica di Molecole di Interesse Biologico del CNR, Arco Felice (Napoli), I-80072, Italy.

    Calmodulin (CaM)-protein interactions are usually described by studying complexes between synthetic targets of ca 25 amino acids and CaM. To understand the relevance of contacts outside the protein-binding region, we investigated the complex between recombinant human CaM (hCaM) and P7, a 38-residue peptide corresponding to the autoinhibitory domain of human cardiac titin kinase (hTK). To expedite the structure determination of hCaM-P7 we relied upon the high degree of similarity with other CaM-kinase peptide complexes. By using a combined homonuclear NMR spectroscopy and molecular modeling approach, we verified for the bound hCaM similar trends in chemical shifts as well as conservation of NOE patterns, which taken together imply the conservation of CaM secondary structure. P7 was anchored to the protein with 52 experimental intermolecular contacts. The hCaM-P7 structure is very similar to known CaM complexes, but the presence of NOE contacts outside the binding cavity appears to be novel. Comparison with the hTK crystal structure indicates that the P7 charged residues all correspond to accessible side-chains, while the putative anchoring hydrophobic side-chains are partially buried. To test this finding, we also modeled the early steps of the complex formation between Ca(2+)-loaded hCaM and hTK. The calculated trajectories strongly suggest the existence of an "electrostatic funnel", driving the long-range recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residues remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca(2+)/hCaM, favoring the intrasteric regulation of the kinase. Furthermore, our structure, the first of CaM bound to a peptide derived from a kinase whose three-dimensional structure is known, suggests that special care is needed in the choice of template peptides to model protein-protein interactions.

    Journal of molecular biology 2001;306;1;81-95

  • Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41.

    Yuan T, Tencza S, Mietzner TA, Montelaro RC and Vogel HJ

    Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.

    The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.

    Funded by: NIAID NIH HHS: R01-AI36198

    Biopolymers 2001;58;1;50-62

  • Calmodulin and HIV type 1: interactions with Gag and Gag products.

    Radding W, Williams JP, McKenna MA, Tummala R, Hunter E, Tytler EM and McDonald JM

    Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA. radding@bioc.rice.edu

    The level of calmodulin increases in cells expressing HIV-1 envelope glycoprotein. Although a calmodulin increase is bound to alter many cellular metabolic and signaling pathways, the benefits to the virus of these alterations must be indirect. However, the possibility exists that increased cellular calmodulin benefits the virus by directly associating with nonenvelope viral proteins. We have, therefore, investigated whether calmodulin can interact with HIV structural proteins Gag, p17, and p24. Calmodulin binds Gag and p17 but not p24 in (125)I-labeled calmodulin overlays of SDS-polyacrylamide gels. Removal of calcium by addition of EGTA eliminates this binding. A computer algorithm for predicting helical regions that should bind calmodulin predicts that there are two calmodulin-binding regions near the N terminus of p17. Intrinsic tryptophan fluorimetry shows that two peptides, each of which includes one of the predicted regions, bind calmodulin: p17(11-25) binds calmodulin with a 2-to-1 stoichiometry and dissociation constant of approximately 10(-9) M(2), and p17(31-46) also binds calmodulin with a dissociation constant of about 10(-9) M. These binding sites are nearly contiguous, forming an extended calmodulin-binding domain p17(11-46). In H-9 cells, Gag and calmodulin colocalize within the resolution of confocal light microscopy.

    Funded by: NCI NIH HHS: R01CA72823; NIAID NIH HHS: R01AI28928, R01AI33826; ...

    AIDS research and human retroviruses 2000;16;15;1519-25

  • Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase alpha.

    Liang L, Lim KL, Seow KT, Ng CH and Pallen CJ

    Cell Regulation Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore.

    cDNA expression library screening revealed binding between the membrane distal catalytic domain (D2) of protein-tyrosine phosphatase alpha (PTPalpha) and calmodulin. Characterization using surface plasmon resonance showed that calmodulin bound to PTPalpha-D2 in a Ca(2+)-dependent manner but did not bind to the membrane proximal catalytic domain (D1) of PTPalpha, to the two tandem catalytic domains (D1D2) of PTPalpha, nor to the closely related D2 domain of PTPepsilon. Calmodulin bound to PTPalpha-D2 with high affinity, exhibiting a K(D) approximately 3 nm. The calmodulin-binding site was localized to amino acids 520-538 in the N-terminal region of D2. Site-directed mutagenesis showed that Lys-521 and Asn-534 were required for optimum calmodulin binding and that restoration of these amino acids to the counterpart PTPepsilon sequence could confer calmodulin binding. The overlap of the binding site with the predicted lip of the catalytic cleft of PTPalpha-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (K(i) approximately 340 nm), suggests that binding of calmodulin physically blocks or distorts the catalytic cleft of PTPalpha-D2 to prevent interaction with substrate. When expressed in cells, full-length PTPalpha and PTPalpha lacking only D1, but not full-length PTPepsilon, bound to calmodulin beads in the presence of Ca(2+). Also, PTPalpha was found in association with calmodulin immunoprecipitated from cell lysates. Thus calmodulin does associate with PTPalpha in vivo but not with PTPalpha-D1D2 in vitro, highlighting a potential conformational difference between these forms of the tandem catalytic domains. The above findings suggest that calmodulin is a possible specific modulator of PTPalpha-D2 and, via D2, of PTPalpha.

    The Journal of biological chemistry 2000;275;39;30075-81

  • Calmodulin directly gates gap junction channels.

    Peracchia C, Sotkis A, Wang XG, Peracchia LL and Persechini A

    Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642-8711, USA. camilo_peracchia@urmc.rochester.edu

    Cytosolic changes control gap junction channel gating via poorly understood mechanisms. In the past two decades calmodulin participation in gating has been suggested, but compelling evidence for it has been lacking. Here we show that calmodulin indeed is associated with gap junctions and plays a direct role in chemical gating. Expression of a calmodulin mutant with the N-terminal EF hand pair replaced by a copy of the C-terminal pair dramatically increases the chemical gating sensitivity of gap junction channels composed of connexin 32 and decreases their sensitivity to transjunctional voltage. The increased chemical gating sensitivity, most likely because of the higher overall Ca(2+) binding affinity of this mutant as compared with native calmodulin, and the decreased voltage sensitivity are only observed when the mutant is expressed before connexin 32. This indicates that the mutant, and by extension native calmodulin, must interact with connexin 32 before gap junctions are formed. Immunofluorescence data suggest further that this interaction leads to incorporation of native or mutant calmodulin into the connexon as an integral regulatory subunit.

    Funded by: NIGMS NIH HHS: GM20113

    The Journal of biological chemistry 2000;275;34;26220-4

  • Interaction between endothelial differentiation-related factor-1 and calmodulin in vitro and in vivo.

    Mariotti M, De Benedictis L, Avon E and Maier JA

    Department of Biomedical Sciences and Technologies, University of Milan, Italy.

    Calmodulin (CaM) is the principal Ca(2+) receptor protein inside the cell. When activated by Ca(2+), CaM binds and activates target proteins, thus altering the metabolism and physiology of the cell. Under basal conditions, calcium-free CaM binds to other proteins termed CaM-binding proteins. Recently, we described endothelial differentiation-related factor (EDF)-1 as a protein involved in the repression of endothelial cell differentiation (Dragoni, I., Mariotti, M., Consalez, G. G., Soria, M., and Maier, J. A. M. (1998) J. Biol. Chem. 273, 31119-31124). Here we report that (i) EDF-1 binds CaM in vitro and in vivo; (ii) EDF-1 is phosphorylated in vitro and in vivo by protein kinase C; and (iii) EDF-1-CaM interaction is modulated by the concentrations of Ca(2+) and by the phosphorylation of EDF-1 by protein kinase C both in vitro and in vivo. In addition, 12-O-tetradecanoylphorbol-13-acetate treatment of human umbilical vein endothelial cell stimulates the nuclear translocation of EDF-1. On the basis of the high homology of EDF-1 with multiprotein bridging factor-1, a transcriptional coactivator that binds TATA-binding protein (TBP), we also demonstrate that EDF-1 interacts with TBP in vitro and in human endothelial cells. We hypothesize that EDF-1 serves two main functions in endothelial cells as follows: (i) to bind CaM in the cytosol at physiologic concentrations of Ca(2+) and (ii) to act in the nucleus as a transcriptional coactivator through its binding to TBP.

    The Journal of biological chemistry 2000;275;31;24047-51

  • Protein kinase C-regulated dynamitin-macrophage-enriched myristoylated alanine-rice C kinase substrate interaction is involved in macrophage cell spreading.

    Yue L, Lu S, Garces J, Jin T and Li J

    Department of Oral Biology, College of Dentistry, the Cancer Center and Department of Microbiology & Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.

    Macrophage spreading requires the microtubule cytoskeleton and protein kinase C (PKC). The mechanism of involvement of the microtubules and PKC in this event is not fully understood. Dynamitin is a subunit of dynactin, which is important for linking the microtubule-dependent motor protein dynein to vesicle membranes. We report that dynamitin is a Ca(2+)/calmodulin-binding protein and that dynamitin binds directly to macrophage-enriched myristoylated alanine-rice C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in macrophage spreading and integrin activation. Dynamitin was found to copurify with MacMARCKS both during MacMARCKS purification with conventional chromatography and during the immunoabsorption of MacMARCKS using anti-MacMARCKS antibody. Vice versa, MacMARCKS was also found to cosediment with the 20 S dynactin complex. We determined that the effector domain of MacMARCKS is required to interact with the N-terminal domain of dynamitin. MacMARCKS and dynamitin also partially colocalized at peripheral regions of macrophages and in the cell-cell border of 293 epithelial cells. Treatment with phorbol esters abolished this colocalization. Disrupting the interaction with a short peptide derived from the MacMARCKS-binding domain of dynamitin caused macrophages to spread and flatten. These data suggest that the dynamitin-MacMARCKS interaction is involved in cell spreading. Furthermore, the regulation of this interaction by PKC and Ca(2+)/calmodulin provides a possible regulatory mechanism for cell adhesion and spreading.

    Funded by: NIGMS NIH HHS: GM54715

    The Journal of biological chemistry 2000;275;31;23948-56

  • Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP(3)) in morphine-pretreated cell.

    Wang D, Surratt CK and Sadée W

    Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California at San Francisco, San Francisco, California, USA.

    Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist-stimulated G protein coupling of the mu-opioid receptor (OP(3)) through direct interaction with the third intracellular (i3) loop of the receptor. Here we have investigated the role of CaM in regulating changes in OP(3)-G protein coupling during morphine treatment, shown to result in CaM release from plasma membranes. Basal and agonist-stimulated G protein coupling by OP(3) was measured before and after morphine pretreatment by incorporation of guanosine 5'-O-(3-[(35)S]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP(3) cDNA. The opioid antagonist beta-chlornaltrexamine fully suppressed basal G protein coupling of OP(3), providing a direct measure of basal signaling. Pretreatment of the cells with morphine enhanced basal G protein coupling (sensitization). In contrast, agonist-stimulated coupling was diminished (desensitization), resulting in a substantially flattened morphine dose-response curve. To test whether CaM is involved in these changes, we constructed OP(3)-i3 loop mutants with reduced affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of these mutant OP(3) receptors was higher than that of the wild-type receptor and, moreover, unaffected by morphine pretreatment, whereas desensitization to agonist stimulation was only slightly attenuated. Therefore, CaM-OP(3) interactions appear to play only a minor role in the desensitization of OP(3). In contrast, release of CaM from the plasma membrane appears to enhance the inherent basal G protein coupling of OP(3), thereby resolving the paradox that OP(3) displays both desensitization and sensitization during morphine treatment.

    Journal of neurochemistry 2000;75;2;763-71

  • Zinedin, SG2NA, and striatin are calmodulin-binding, WD repeat proteins principally expressed in the brain.

    Castets F, Rakitina T, Gaillard S, Moqrich A, Mattei MG and Monneron A

    FRE 9041, CNRS, 31 chemin Joseph Aiguier, Université de la Méditerranée, Marseille, 13402 Marseille Cedex 20, France.

    Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.

    The Journal of biological chemistry 2000;275;26;19970-7

  • Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity.

    Popov SG, Krishna UM, Falck JR and Wilkie TM

    Pharmacology and Biochemistry Departments, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G(i) and/or G(q) class G protein alpha subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP(3) but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP(3). Calmodulin binds all RGS proteins we tested in a Ca(2+)-dependent manner but does not directly affect GAP activity. Indeed, Ca(2+)/calmodulin binds a complex of RGS4 and a transition state analog of Galpha(i1)-GDP-AlF(4)(-). Ca(2+)/calmodulin reverses PIP(3)-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca(2+)/calmodulin competition with PIP(3) may provide an intracellular mechanism for feedback regulation of Ca(2+) signaling evoked by G protein-coupled agonists.

    Funded by: NIDDK NIH HHS: DK47890; NIGMS NIH HHS: GM31278

    The Journal of biological chemistry 2000;275;25;18962-8

  • Alternative splice variant of gamma-calmodulin-dependent protein kinase II alters activation by calmodulin.

    Kwiatkowski AP and McGill JM

    Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. akwiatk2@iupui.edu

    Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca(2+)-signaling pathways. These family members are expressed from four highly homologous genes (alpha, beta, gamma, and delta) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I-XI, are differentially expressed in various cell types. gammaB, gammaC, gammaD, gammaE, gammaF, gammaGs, and gammaH CaMKII isoforms are expressed in the biliary epithelium; however, little is known about their roles in these cells. We began our studies into the function of these variable regions by examining the effects of variable region I on kinase activation and calmodulin binding. Activities and calmodulin binding properties of gammaB and gammaGs, which differ only by the exclusion or inclusion of this region, were compared. The K(0.5) for calmodulin was 2.5-fold lower for gammaGs than gammaB. In contrast, gammaB bound calmodulin more tightly in a calmodulin overlay assay. Mutation of variable regions I's charged residue, gammaGs-R318E, resulted in an enzyme with intermediate activation properties but a calmodulin affinity similar to gammaB. Thus, variable region I appears to modulate calmodulin sensitivity, in part, through charge-charge interactions. This altered threshold of activation may modulate cellular responses to gradients of Ca(2+)/calmodulin in the biliary tract.

    Funded by: NIDDK NIH HHS: DK51080

    Archives of biochemistry and biophysics 2000;378;2;377-83

  • Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin.

    Flory MR, Moser MJ, Monnat RJ and Davis TN

    Program in Molecular and Cellular Biology, Department of Pathology, University of Washington, Seattle, WA 98195, USA.

    Eukaryotic chromosome segregation depends on the mitotic spindle apparatus, a bipolar array of microtubules nucleated from centrosomes. Centrosomal microtubule nucleation requires attachment of gamma-tubulin ring complexes to a salt-insoluble centrosomal core, but the factor(s) underlying this attachment remains unknown. In budding yeast, this attachment is provided by the coiled-coil protein Spc110p, which links the yeast gamma-tubulin complex to the core of the yeast centrosome. Here, we show that the large coiled-coil protein kendrin is a human orthologue of Spc110p. We identified kendrin by its C-terminal calmodulin-binding site, which shares homology with the Spc110p calmodulin-binding site. Kendrin localizes specifically to centrosomes throughout the cell cycle. N-terminal regions of kendrin share significant sequence homology with pericentrin, a previously identified murine centrosome component known to interact with gamma-tubulin. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with spindle microtubules.

    Funded by: NCI NIH HHS: R01 CA48022; NIA NIH HHS: P01 AG001751, R01 AG048022; NIGMS NIH HHS: R01 GM040506, R01 GM40506, T32 GM-07270-22, T32 GM007270

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;11;5919-23

  • A novel type of calmodulin interaction in the inhibition of basic helix-loop-helix transcription factors.

    Onions J, Hermann S and Grundström T

    Division of Tumour Biology, Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.

    Calmodulin is the predominant intracellular receptor for Ca(2+) signals, mediating the regulation of numerous cellular processes. Previous studies have shown that calcium-loaded calmodulin can bind to and inhibit the activity of certain basic helix-loop-helix (bHLH) transcription factors. The basic sequence within the bHLH domain is the primary target for calmodulin binding, and sequences modulating the calmodulin interaction reside directly N-terminal to the basic sequence. Here we show that the interaction of calmodulin with bHLH proteins is of a novel type, displaying characteristics very different from those of previously characterized calmodulin-target complexes. We show that calmodulin interacts much stronger with a dimeric basic sequence than with the monomeric form. The calmodulin-bHLH protein complex contains equimolar amounts of calmodulin and bHLH chains. The interaction is unusual in being to a large extent polar in nature, and it is highly resistant to tested calmodulin inhibitors. Both the N-terminal and C-terminal domains of calmodulin can independently bind to and inhibit the DNA binding of bHLH proteins. The C-terminal domain preferentially binds to the basic sequence, whereas the N-terminal domain is essential for the effect of the modulatory sequence. We propose a model for the calmodulin-bHLH complex where two calmodulin molecules interact with one bHLH dimer, with one domain of calmodulin preferentially binding to the basic sequence of bHLH proteins and the other domain interacting with the modulatory sequence.

    Biochemistry 2000;39;15;4366-74

  • Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins.

    Nunomura W, Takakuwa Y, Parra M, Conboy JG and Mohandas N

    Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Shinjuku, Tokyo 162-8666, Japan.

    In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.

    Funded by: NIDDK NIH HHS: DK 26263, DK 32094

    The Journal of biological chemistry 2000;275;9;6360-7

  • WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A.

    Moreno CS, Park S, Nelson K, Ashby D, Hubalek F, Lane WS and Pallas DC

    Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca(2+)-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G(2) nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the post-synaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G(2) phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca(2+)-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca(2+)-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.

    Funded by: NCI NIH HHS: CA57327, R01 CA057327

    The Journal of biological chemistry 2000;275;8;5257-63

  • Requirement of calmodulin binding by HIV-1 gp160 for enhanced FAS-mediated apoptosis.

    Micoli KJ, Pan G, Wu Y, Williams JP, Cook WJ and McDonald JM

    Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

    Accelerated apoptosis is one mechanism proposed for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type 1 (HIV-1) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of gp160 in Jurkat T-cells results in increased sensitivity to FAS- and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine. This enhanced apoptosis in response to FAS antibody or C(2)-ceramide is associated with activation of caspase 3, a critical mediator of apoptosis. A point mutation in the C-terminal calmodulin-binding domain of gp160 (alanine 835 to tryptophan, A835W) eliminates gp160-dependent enhanced FAS-mediated apoptosis in transiently transfected cells, as well as in vitro calmodulin binding to a peptide corresponding to the C-terminal calmodulin-binding domain of gp160. Stable Tet-off Jurkat cell lines were developed that inducibly express wild type gp160 or gp160A835W. Increasing expression of wild type gp160, but not gp160A835W, correlates with increased calmodulin levels, increased apoptosis, and caspase 3 activation in response to anti-FAS treatment. The data indicate that gp160-enhanced apoptosis is dependent upon calmodulin up-regulation, involves the activation of caspase 3, and requires calmodulin binding to the C-terminal binding domain of gp160.

    Funded by: NCI NIH HHS: CA/72823

    The Journal of biological chemistry 2000;275;2;1233-40

  • The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments.

    Assmann V, Jenkinson D, Marshall JF and Hart IR

    Richard Dimbleby Department of Cancer Research/ICRF Laboratory, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK. V.Assmann@icrf.icnet.uk

    We reported recently on the intracellular localisation of the hyaluronan receptor RHAMM/IHABP in human cancer cells. Here we describe the colocalisation of RHAMM/IHABP proteins with microtubules, both in interphase and dividing cells, suggesting that RHAMM/IHABP represents a novel member of the family of microtubule-associated proteins (MAPs). We have identified four different splice variants of RHAMM/IHABP, all of which colocalise, at least transiently, with microtubules when expressed as GFP fusion proteins in HeLa cells. Using microtubule-binding assays and transient transfection experiments of deletion-bearing RHAMM/IHABP mutants, we localised the microtubule-binding region to the extreme N terminus of RHAMM/IHABP. This interaction domain is composed of two distinct subdomains, one of which is sufficient to mediate binding to the mitotic spindle while both domains are required for binding of RHAMM/IHABP proteins to interphase microtubules. Sequence analysis revealed that the projection domain of RHAMM/IHABP is predicted to form coiled-coils, implying that RHAMM/IHABP represents a filamentous protein capable of interacting with other proteins and we found that RHAMM/IHABP interacts with actin filaments in vivo and in vitro. Moreover, in vitro translated RHAMM/IHABP isoforms efficiently bind to immobilised calmodulin in a Ca(2+)-dependent manner via a calmodulin-binding site within the projection domain of RHAMM/IHABP (residues 574-602). Taken together, our results strongly suggest that RHAMM/IHABP is a ubiquitously expressed, filamentous protein capable of interacting with microtubules and microfilaments and not, as numerous previous reports suggest, a cell surface receptor for the extracellular matrix component hyaluronan.

    Journal of cell science 1999;112 ( Pt 22);3943-54

  • A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.

    Nakajima Y, Yamamoto T, Nakayama T and Nakanishi S

    Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan.

    Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades.

    The Journal of biological chemistry 1999;274;39;27573-7

  • Study of the gerbil utricular macula following treatment with gentamicin, by use of bromodeoxyuridine and calmodulin immunohistochemical labelling.

    Ogata Y, Slepecky NB and Takahashi M

    Department of Otolaryngology, Yamaguchi University School of Medicine, Ube, Japan. yoichi-o@po.cc.yamaguchi-u.ac.jp

    Effects of ototoxic drugs on the gerbil vestibular sensory epithelium were probed by use of immunocytochemical labelling with antibodies to both a mitogenic marker (bromodeoxyuridine) and a hair cell specific protein (calmodulin). Nine animals had gentamicin administered once daily for 5 days, as a transtympanic injection into the right middle ear. They additionally were given a daily intraperitoneal injection of bromodeoxyuridine, starting on the same day as the gentamicin injection and continuing until the day of sacrifice. Nine other animals, serving as controls for bromodeoxyuridine incorporation, received only the intraperitoneal injections of bromodeoxyuridine. The inner ears from three gerbils were obtained at 1, 2 or 4 weeks following the last gentamicin injection and utricles from the injected ears were processed for immunohistochemical analysis. In specimens where gentamicin was administered, we found evidence of bromodeoxyuridine incorporation in 17 cells (10 single cells and 7 pairs of cells) in a total of 216 sections taken from the central regions of the 9 utricles. However, in control specimens, no bromodeoxyuridine labelling was found in any cells of the 216 sections examined. Of 10 single cells labelled with bromodeoxyuridine, two cells in the hair cell layer were labelled with antibodies against calmodulin. One had a faint labelling in the nucleus and the other in the stereocilia, but not in the cell bodies. Of 7 pairs of cells, two pairs with nuclei localized in the hair cell layer had faint labelling for calmodulin in the nuclei, but no labelling in any other part of the cell. The other 13 cells labelled with antibodies to bromodeoxyuridine were not labelled with antibodies to calmodulin. Our results suggest that the bromodeoxyuridine-labelled cells could not be positively identified as hair cells based on immunohistochemical labelling for calmodulin.

    Hearing research 1999;133;1-2;53-60

  • Structural elements within the methylation loop (residues 112-117) and EF hands III and IV of calmodulin are required for Lys(115) trimethylation.

    Cobb JA, Han CH, Wills DM and Roberts DM

    Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA.

    Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114-->Ala; L116T, Leu116-->Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

    The Biochemical journal 1999;340 ( Pt 2);417-24

  • Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

    Corti C, Leclerc L'Hostis E, Quadroni M, Schmid H, Durussel I, Cox J, Dainese Hatt P, James P and Carafoli E

    Protein Chemistry Laboratory, Department of Biology, Swiss Federal Institute of Technology, (ETH) Zürich.

    The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.

    European journal of biochemistry 1999;262;3;790-802

  • In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation.

    Luk SC, Ngai SM, Tsui SK, Fung KP, Lee CY and Waye MM

    Department of Biochemistry, Chinese University of Hong Kong, Shatin, N.T.

    Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin. In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent. Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation.

    Journal of cellular biochemistry 1999;73;1;31-5

  • Calmodulin-binding sites on adenylyl cyclase type VIII.

    Gu C and Cooper DM

    Neuroscience Program and Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

    Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.

    Funded by: NIGMS NIH HHS: GM 32483

    The Journal of biological chemistry 1999;274;12;8012-21

  • Interactions between neurogranin and calmodulin in vivo.

    Prichard L, Deloulme JC and Storm DR

    Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.

    Neurogranin is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) within its IQ domain at serine 36. Since CaM binds to neurogranin through the IQ domain, PKC phosphorylation and CaM binding are mutually exclusive. Consequently, we hypothesize that neurogranin may function to concentrate CaM at specific sites in neurons and release free CaM in response to increased Ca2+ and PKC activation. However, it has not been established that neurogranin interacts with CaM in vivo. In this study, we examined this question using yeast two-hybrid methodology. We also searched for additional proteins that might interact with neurogranin by screening brain cDNA libraries. Our data illustrate that CaM binds to neurogranin in vivo and that CaM is the only neurogranin-interacting protein isolated from brain cDNA libraries. Single amino acid mutagenesis indicated that residues within the IQ domain are important for CaM binding to neurogranin in vivo. The Ile-33 --> Gln point mutant completely inhibited and Arg-38 --> Gln and Ser-36 --> Asp point mutants reduced neurogranin/CaM interactions. These data demonstrate that CaM is the major protein that interacts with neurogranin in vivo and support the hypothesis that phosphorylation of neurogranin at Ser-36 regulates its binding to CaM.

    The Journal of biological chemistry 1999;274;12;7689-94

  • Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1.

    Fanger CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K, Chandy KG, Cahalan MD and Aiyar J

    Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.

    Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail of hSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1. 3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with hIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

    Funded by: NIGMS NIH HHS: GM41514, GMOD54872; NINDS NIH HHS: NS14609; ...

    The Journal of biological chemistry 1999;274;9;5746-54

  • Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites.

    Masuko N, Makino K, Kuwahara H, Fukunaga K, Sudo T, Araki N, Yamamoto H, Yamada Y, Miyamoto E and Saya H

    Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811, Japan.

    NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

    The Journal of biological chemistry 1999;274;9;5782-90

  • Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.

    Krupp JJ, Vissel B, Thomas CG, Heinemann SF and Westbrook GL

    Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, USA.

    Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and alpha-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of alpha-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of alpha-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or alpha-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from alpha-actinin by two distinct but converging calcium-dependent processes: competitive displacement of alpha-actinin by calmodulin and reduction in the affinity of alpha-actinin for C0 after binding of calcium to alpha-actinin.

    Funded by: NIMH NIH HHS: MH46613; NINDS NIH HHS: NS28709

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;4;1165-78

  • Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4) activity and nuclear accumulation of cyclin D1-Cdk4 during G1.

    Taulés M, Rius E, Talaya D, López-Girona A, Bachs O and Agell N

    Department of Cell Biology, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Faculty of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain.

    Although it is known that calmodulin is involved in G1 progression, the calmodulin-dependent G1 events are not well understood. We have analyzed here the role of calmodulin in the activity, the expression, and the intracellular location of proteins involved in G1 progression. The addition of anti-calmodulin drugs to normal rat kidney cells in early G1 inhibited cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities, as well as retinoblastoma protein phosphorylation. Protein levels of cdk4, cyclin D1, cyclin D2, cyclin E, p21, and p27 were not affected after CaM inhibition, whereas decreases in the amount of cyclin A and Cdc2 were observed. The decrease of Cdk4 activity was due neither to changes in its association to cyclin D1 nor to changes in the amount of p21 or p27 bound to cyclin D1-Cdk4 complexes. Calmodulin inhibition also produced a translocation of nuclear cyclin D1 and Cdk4 to the cytoplasm. This translocation could be responsible for the decreased Cdk4 activity upon calmodulin inhibition. Immunoprecipitation, calmodulin affinity chromatography, and direct binding experiments indicated that calmodulin associates with Cdk4 and cyclin D1 through a calmodulin-binding protein. The facts that Hsp90 interacts with Cdk4 and that its inhibition induced Cdk4 and cyclin D1 translocation to the cytoplasm point to Hsp90 as a good candidate for being the calmodulin-binding protein involved in the nuclear accumulation of Cdk4 and cyclin D1.

    The Journal of biological chemistry 1998;273;50;33279-86

  • Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta.

    Anderson KA, Means RL, Huang QH, Kemp BE, Goldstein EG, Selbert MA, Edelman AM, Fremeau RT and Means AR

    Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina 27710, USA.

    Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.

    Funded by: NIGMS NIH HHS: GM-33976; NINDS NIH HHS: NS-24738

    The Journal of biological chemistry 1998;273;48;31880-9

  • Localization of the sites for Ca2+-binding proteins on G protein-coupled receptor kinases.

    Levay K, Satpaev DK, Pronin AN, Benovic JL and Slepak VZ

    Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33136, USA.

    Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs.

    Funded by: NIGMS NIH HHS: GM44944

    Biochemistry 1998;37;39;13650-9

  • Apoptosis induction by the binding of the carboxyl terminus of human immunodeficiency virus type 1 gp160 to calmodulin.

    Ishikawa H, Sasaki M, Noda S and Koga Y

    Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.

    The role of calmodulin (CaM) in apoptosis induced by gp160 of human immunodeficiency virus type 1 was investigated with cells undergoing single-cell killing. These cells were found to express, under the control of an inducible promoter, wild-type gp160 or mutant gp160 devoid of various lengths of the carboxyl terminus. Immunoprecipitation accompanied by immunoblotting revealed binding of CaM to wild-type gp160 but not to mutant gp160 bearing a carboxyl terminus with a deletion spanning more than five amino acid residues. A significant coenzyme activity was detected in the CaM bound to gp160 even in the presence of a Ca2+ chelater, EGTA. The cells forming this gp160-CaM complex exhibited an elevated intracellular Ca2+ level followed by DNA fragmentation, which is a hallmark of apoptosis, and finally cell killing, while the cells not forming this complex did not show any significant elevation in Ca2+ level or DNA fragmentation. These results thus indicated that CaM plays a key role in gp160-induced apoptosis.

    Journal of virology 1998;72;8;6574-80

  • Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors.

    Zhang S, Ehlers MD, Bernhardt JP, Su CT and Huganir RL

    Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. We report here that the mutation of a specific calmodulin binding site in the CO region of the NR1 subunit of the NMDA receptor blocks CDI. Moreover, intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. Furthermore, this inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2 known to interact with the CO region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton.

    Neuron 1998;21;2;443-53

  • Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3.

    Toutenhoofd SL, Foletti D, Wicki R, Rhyner JA, Garcia F, Tolon R and Strehler EE

    Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Foundation, Rochester, MN 55905, USA.

    Human calmodulin is encoded by three genes CALM1, CALM2 and CALM3 located on different chromosomes. To complete the characterization of this family, the exon-intron structure of CALM2 was solved by a combination of genomic DNA library screening and genomic PCR amplification. Intron interruptions were found at identical positions in human CALM2 as in CALM1 and CALM3; however, the overall size of CALM2 (16 kb) was almost twice that of the other two human CALM genes. Over 1 kb of the 5' flanking sequence of human CALM2 were determined, revealing the presence of a TATA-like sequence 27 nucleotides upstream of the transcriptional start site and several conserved sequence elements possibly involved in the regulation of this gene. To determine if differential transcriptional activity plays a major role in regulating cellular calmodulin levels, we directly measured and compared the mRNA abundance and transcriptional activity of the three CALM genes in proliferating human teratoma cells. CALM3 was at least 5-fold more actively transcribed than CALM1 or CALM2. CALM transcriptional activity agreed well with the mRNA abundance profile in the teratoma cells. In transient transfections using luciferase reporter genes driven by 1 kb of the 5' flanking DNA of the three CALM genes, the promoter activity correlated with the endogenous CALM transcriptional activity, but only when the 5' untranslated regions were included in the constructs. We conclude that the CALM gene family is differentially active at the transcriptional level in teratoma cells and that the 5' untranslated regions are necessary to recover full promoter activation.

    Cell calcium 1998;23;5;323-38

  • Human myosin-IXb is a mechanochemically active motor and a GAP for rho.

    Post PL, Bokoch GM and Mooseker MS

    Department of Molecular Biology, Yale University, New Haven, CT 06520, USA. penny.post@yale.edu

    The heavy chains of the class IX myosins, rat myr5 and human myosin-IXb, contain within their tail domains a region with sequence homology to GTPase activating proteins for the rho family of G proteins. Because low levels of myosin-IXb expression preclude purification by conventional means, we have employed an immunoadsorption strategy to purify myosin-IXb, enabling us to characterize the mechanochemical and rho-GTPase activation properties of the native protein. In this report we have examined the light chain content, actin binding properties, in vitro motility and rho-GTPase activity of human myosin-IXb purified from leukocytes. The results presented here indicate that myosin-IXb contains calmodulin as a light chain and that it binds to actin with high affinity in both the absence and presence of ATP. Myosin-IXb is an active motor which, like other calmodulin-containing myosins, exhibits maximal velocity of actin filaments (15 nm/second) in the absence of Ca2+. Native myosin-IXb exhibits GAP activity on rho. Class IX myosins may be an important link between rho and rho-dependent remodeling of the actin cytoskeleton.

    Funded by: NIDDK NIH HHS: DK 09407, DK 25387, DK 38979; ...

    Journal of cell science 1998;111 ( Pt 7);941-50

  • Alpha1-syntrophin has distinct binding sites for actin and calmodulin.

    Iwata Y, Pan Y, Yoshida T, Hanada H and Shigekawa M

    Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.

    Overlay and co-sedimentation assays using recombinant alpha1-syntrophin proteins revealed that two regions of alpha1-syntrophin, i.e. aa 274-315 and 449-505, contain high-affinity binding sites for F-actin (Kd 0.16-0.45 microM), although only a single high-affinity site (Kd 0.35 microM) was detected in the recombinant full-length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti-syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin-glycoprotein complex or for other cell functions. We also found that alpha1-syntrophin binds calmodulin at two distinct sites with high (Kd 15 nM) and low (Kd 0.3 microM) affinity.

    FEBS letters 1998;423;2;173-7

  • Molecular mechanisms of calmodulin's functional versatility.

    Zhang M and Yuan T

    Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, People's Republic of China. mzhang@uxmail.ust.hk

    Calmodulin (CaM) is a primary Ca2+-binding protein found in all eukaryotic cells. It couples the intracellular Ca2+ signal to many essential cellular events by binding and regulating the activities of more than 40 different proteins and enzymes in a Ca2+-dependent manner. CaM contains two structurally similar domains connected by a flexible central linker. Each domain of the protein binds two Ca2+ ions with positive cooperativity. The binding of Ca2+ transforms the protein into its active form through a reorientation of the existing helices of the protein. The two helices in each helix-loop-helix Ca2+-binding motif are almost antiparallel in Ca2+-free CaM. The binding of Ca2+ induces concerted helical pair movements and changes the two helices in each Ca2+ binding motif to a nearly perpendicular orientation. These concerted helix pair movements are accompanied by dramatic changes on the molecular surface of the protein. Rather than exhibiting a flat, hydrophilic molecular surface as seen in Ca2+-free CaM, the Ca2+-saturated form of the protein contains a Met-rich, cavity-containing hydrophobic surface in each domain. These hydrophobic surfaces are largely responsible for the binding of CaM to its targets. The unique flexibility and high polarizability of the Met residues located at the entrance of each hydrophobic pocket together with other hydrophobic amino acid residues create adjustable, sticky interaction surface areas that can accommodate CaM's targets, which have various sizes and shapes. Therefore, CaM is able to bind to a large array of targets without obvious sequence homology. Upon binding to its target peptides, the unwinding of the central linker allows the two domains of the protein to engulf the hydrophobic face of target peptides of differing lengths. The binding of Ca2+ reduces the backbone flexibility of CaM. Formation of complexes with its target peptides further decreases the backbone motion of CaM.

    Biochemistry and cell biology = Biochimie et biologie cellulaire 1998;76;2-3;313-23

  • Expression, purification, and regulation of two isoforms of the inositol 1,4,5-trisphosphate 3-kinase.

    Woodring PJ and Garrison JC

    Department of Pharmacology and Cancer Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. jcg8w@virginia.edu

    The level of inositol 1,4,5-trisphosphate in the cytoplasm is tightly regulated by two enzymes, the inositol 1,4,5,5-phosphatase and the inositol 1,4,5-trisphosphate 3-kinase. Two isoforms of the inositol 1,4,5-trisphosphate 3-kinase have been identified, the A form and the B form. The regulatory properties of the two isoforms were compared following overexpression and purification of the proteins from a v-src transformed mammalian cell line. The highly purified, recombinant inositol 1,4,5-trisphosphate 3-kinases were differentially regulated by calcium/calmodulin and via phosphorylation by protein kinase C or the cyclic AMP-dependent protein kinase. Both enzymes had similar affinities for inositol 1,4, 5-trisphosphate (Km 2-5 mu M). Calcium/calmodulin stimulated the activity of isoform A about 2.5-fold, whereas the activity of isoform B was increased 20-fold. The cyclic AMP-dependent protein kinase phosphorylated the inositol 1,4,5-trisphosphate 3-kinase A to the extent of 0.9 mol/mol and isoform B to 1 mol/mol. Protein kinase C phosphorylated isoform A to the extent of 2 mol/mol and isoform B to 2.7 mol/mol. Phosphorylation of isoform A by the cyclic AMP-dependent protein kinase caused a 2.5-fold increase in its activity when assayed in the absence of calcium/calmodulin, whereas phosphorylation by protein kinase C decreased activity by 72%. The activity of isoform B in the absence of calcium/calmodulin was not affected by phosphorylation using either kinase. When assayed in the presence of calcium/calmodulin, phosphorylation of isoform A by the cyclic AMP-dependent protein kinase increased activity 1.5-fold, whereas phosphorylation of isoform B decreased activity by 45%. Phosphorylation of either isoform A or B by protein kinase C resulted in a 70% reduction of calcium/calmodulin-stimulated activity. Differential expression and regulation of the two inositol 1,4,5-trisphosphate 3-kinase isoforms provides multiple mechanisms for regulating the cytosolic level of inositol 1,4,5-trisphosphate in cells.

    Funded by: NCI NIH HHS: P01-CA 40042; NIDDK NIH HHS: R01-DK-19952

    The Journal of biological chemistry 1997;272;48;30447-54

  • Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin.

    Martoglio B, Graf R and Dobberstein B

    Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany.

    Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now.

    The EMBO journal 1997;16;22;6636-45

  • The prooncoprotein EWS binds calmodulin and is phosphorylated by protein kinase C through an IQ domain.

    Deloulme JC, Prichard L, Delattre O and Storm DR

    University of Washington, Department of Pharmacology, Seattle, Washington 98195, USA.

    A growing family of proteins is regulated by protein kinase C and calmodulin through IQ domains, a regulatory motif originally identified in neuromodulin (Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544-7549). Here we report that EWS, a nuclear RNA-binding prooncoprotein, contains an IQ domain, is phosphorylated by protein kinase C, and interacts with calmodulin. Interestingly, PKC phosphorylation of EWS inhibits its binding to RNA homopolymers, and conversely, RNA binding to EWS interferes with PKC phosphorylation. Several other RNA-binding proteins, including TLS/FUS and PSF, co-purify with EWS. PKC phosphorylation of these proteins also inhibits their binding to RNA in vitro. These data suggest that PKC may regulate interactions of EWS and other RNA-binding proteins with their RNA targets and that IQ domains may provide a regulatory link between Ca2+ signal transduction pathways and RNA processing.

    Funded by: NINDS NIH HHS: NS 20498

    The Journal of biological chemistry 1997;272;43;27369-77

  • Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M and Tsien RY

    Department of Pharmacology, University of California, San Diego, La Jolla 92093-0647, USA.

    Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

    Nature 1997;388;6645;882-7

  • Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulin.

    Malvoisin E and Wild F

    Inserm Unit 404, Immunity and Vaccination, Institut Pasteur de Lyon, France. fwipaly@imaginet.fr

    Calmodulin, an EF-hand protein, inhibited the fusion between CD4+ human cells and cells stably expressing HIV-1 envelope proteins. Fusion was also inhibited when HIV-1, HIV-2 or SIV envelope glycoproteins were expressed by vaccinia virus (VV) recombinants, but calmodulin did not inhibit syncytia formation induced by measles virus glycoproteins. Calmodulin also inhibited fusion induced by vPE17, a VV-recombinant expressing a truncated form of HIV-1gp160 which lacks the two known calmodulin-binding sites located in the cytoplasmic domain of gp41. The inhibitory activity was specific to calmodulin among the EF-hand proteins. These observations may be important in understanding the mechanism of retroviral envelope glycoprotein-mediated cell fusion. Several possible mechanisms of action are discussed.

    Virus research 1997;50;2;119-27

  • Regulation of the AKAP79-protein kinase C interaction by Ca2+/Calmodulin.

    Faux MC and Scott JD

    Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, USA.

    The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (PKC) at the postsynaptic densities in neurons. Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme. This report focuses on the regulation of AKAP79-PKC interaction by calmodulin. AKAP79 binds calmodulin with high affinity (KD of 28 +/- 4 nM (n = 3)) in a Ca2+-dependent manner. Immunofluorescence staining shows that both proteins exhibit overlapping staining patterns in cultured hippocampal neurons. Calmodulin reversed the inhibition of PKCbetaII by the AKAP79(31-52) peptide and reduced inhibition by the full-length AKAP79 protein. The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31-52) peptide was shown to be partially dependent on Ca2+. Ca2+/calmodulin reduced PKC coimmunoprecipitated with AKAP79 and resulted in a 2.6 +/- 0.5-fold (n = 6) increase in PKC activity in a preparation of postsynaptic densities. Collectively, these findings suggest that Ca2+/calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein.

    Funded by: NIGMS NIH HHS: GM48231

    The Journal of biological chemistry 1997;272;27;17038-44

  • Identification and characterization of a calmodulin-binding domain in Ral-A, a Ras-related GTP-binding protein purified from human erythrocyte membrane.

    Wang KL, Khan MT and Roufogalis BD

    Department of Pharmacy, University of Sydney, New South Wales 2006, Australia.

    A 28-kDa protein (p28) has been purified from Triton X-100 extracts of human erythrocyte plasma membrane by calmodulin affinity chromatography. Based on internal peptide sequencing and its protein amino acid composition, this protein has been shown to be highly related, if not identical to, Ral-A, a Ras-related GTP-binding protein. This protein assignment is consistent with the findings that p28 binds [32P]GTP specifically and has low GTPase activity. In this study we describe the identification and characterization of a calmodulin-binding domain in Ral-A. The Ca2+-dependent interaction of p28 with calmodulin was first detected by a calmodulin affinity column. Gel overlay experiments of both p28 and recombinant Ral-A with biotinylated calmodulin provided strong evidence that Ral-A is a calmodulin-binding protein. A peptide of 18 residues (P18) with the sequence SKEKNGKKKRKSLAKRIR has been identified as a putative calmodulin-binding domain in Ral-A, because it comprises a basic/hydrophobic composition with the propensity to form an amphiphilic helix. P18 was synthesized, and its interaction with calmodulin by gel overlay was shown to be Ca2+-dependent. Circular dichroism analysis demonstrated that this interaction results in less alpha-helical content upon calmodulin complex formation. These results indicate that Ral-A is a calmodulin-binding protein, raising the possibility that it may be associated with Ca2+-dependent intracellular signaling pathways.

    The Journal of biological chemistry 1997;272;25;16002-9

  • Rad and Rad-related GTPases interact with calmodulin and calmodulin-dependent protein kinase II.

    Moyers JS, Bilan PJ, Zhu J and Kahn CR

    Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.

    Members of the Rad family of GTPases (including Rad, Gem, and Kir) possess several unique features of unknown function in comparison to other Ras-like proteins, with major N-terminal and C-terminal extensions, a lack of typical prenylation motifs, and several non-conservative changes in the sequence of the GTP binding domain. Here we show that Rad and Gem bind to calmodulin (CaM)-Sepharose in vitro in a calcium-dependent manner and that Rad can be co-immunoprecipitated with CaM in C2C12 cells. The interaction is influenced by the guanine nucleotide binding state of Rad with the GDP-bound form exhibiting 5-fold better binding to CaM than the GTP-bound protein. In addition, the dominant negative mutant of Rad (S105N) which binds GDP, but not GTP, exhibits enhanced binding to CaM in vivo when expressed in C2C12 cells. Peptide competition studies and expression of deletion mutants of Rad localize the binding site for CaM to residues 278-297 at the C terminus of Rad. This domain contains a motif characteristic of a calmodulin-binding region, consisting of numerous basic and hydrophobic residues. In addition, we have identified a second potential regulatory domain in the extended N terminus of Rad which, when removed, decreases Rad protein expression but increases the binding of Rad to CaM. The ability of Rad mutants to bind CaM correlates with their localization in cytoskeletal fractions of C2C12 cells. Immunoprecipitates of calmodulin-dependent protein kinase II, the cellular effector of Ca2+-calmodulin, also contain Rad, and in vitro both Rad and Gem can serve as substrates for this kinase. Thus, the Rad family of GTP-binding proteins possess unique characteristics of binding CaM and calmodulin-dependent protein kinase II, suggesting a role for Rad-like GTPases in calcium activation of serine/threonine kinase cascades.

    Funded by: NIDDK NIH HHS: DK 07260, DK 45935, P30DK36836

    The Journal of biological chemistry 1997;272;18;11832-9

  • G protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells.

    Sallese M, Mariggiò S, Collodel G, Moretti E, Piomboni P, Baccetti B and De Blasi A

    Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche "Mario Negri", Santa Maria Imbaro, 66030, Italy.

    G protein-coupled receptor kinase 4 (GRK4) presents some peculiar characteristics that make it a unique member within the GRK multigene family. For example, this is the only GRK for which four splice variants (GRK4alpha, -beta, -gamma, -delta) have been identified. We developed a simple assay to study kinase activity, and we found that GRK4alpha, but not GRK4beta, -gamma, and -delta, was able to phosphorylate rhodopsin in an agonist-dependent manner. GRK4alpha kinase activity was inhibited by Ca2+/calmodulin (CaM) (IC50 = 80 nM), and a direct interaction between GRK4alpha and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The other three GRK4 isoforms did not interact with CaM in parallel experiments. The present investigation also aimed to define cellular and ultrastructural localization of GRK4. A substantial expression of GRK4 mRNA was only found in testis and in the spermatogonia cell line GC-1 spg. Specific GRK4 immunoreactivity was only found on sperm membranes, and immunochemical and ultrastructural analyses showed that it is associated to the acrosomal membranes and to the outer mitochondrial membranes. GRK4gamma was the only detectable isoform in human sperm. We concluded that: i) only GRK4alpha can phosphorylate rhodopsin and that this activity is inhibited by CaM; ii) the other three isoforms do not phosphorylate rhodopsin and do not interact with CaM; and iii) the association of GRK4 with highly specialized sperm organelles, which are essential for fertilization, strongly indicates that this kinase is involved in this process.

    The Journal of biological chemistry 1997;272;15;10188-95

  • Interleukin 10 is induced by recombinant HIV-1 Nef protein involving the calcium/calmodulin-dependent phosphodiesterase signal transduction pathway.

    Brigino E, Haraguchi S, Koutsonikolis A, Cianciolo GJ, Owens U, Good RA and Day NK

    Department of Pediatrics, University of South Florida, St. Petersburg 33701, USA.

    HIV-1 Nef protein shares a significant homology with the immunosuppressive and highly conserved retroviral transmembrane protein p15E. In the present study, extracellular Nef protein is shown to induce interleukin (IL)-10 mRNA expression in human peripheral blood mononuclear cells as well as in cells of H9 T and U937 promonocytic human cell lines. Release of IL-10 protein into supernatants of peripheral blood mononuclear cells stimulated with Nef is dose-dependent. Expression of cytokines IL-2, IL-4, IL-5, IL-12 p40, IL-13, and interferon gamma is not affected by Nef stimulation. IL-10 protein production induced by Nef is inhibited by the calcium/calmodulin phosphodiesterase inhibitor W-7 but not by the protein kinase A inhibitor H-89 nor the protein kinase C inhibitors staurosporine and calphostin C. The calcium chelating agent EGTA also inhibits the IL-10 production induced by Nef, and this inhibition is reversed by the addition of calcium along with Nef. These findings indicate that extracellular Nef may contribute to the immunopathogenesis of HIV infection by inducing IL-10.

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;7;3178-82

  • Competitive binding of alpha-actinin and calmodulin to the NMDA receptor.

    Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM and Sheng M

    Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.

    The mechanisms by which neurotransmitter receptors are immobilized at postsynaptic sites in neurons are largely unknown. The activity of NMDA (N-methyl-D-aspartate) receptors is mechanosensitive and dependent on the integrity of actin, suggesting a functionally important interaction between NMDA receptors and the postsynaptic cytoskeleton. alpha-Actinin-2, a member of the spectrin/dystrophin family of actin-binding proteins, is identified here as a brain postsynaptic density protein that colocalizes in dendritic spines with NMDA receptors and the putative NMDA receptor-clustering molecule PSD-95. alpha-Actinin-2 binds by its central rod domain to the cytoplasmic tail of both NR1 and NR2B subunits of the NMDA receptor, and can be immunoprecipitated with NMDA receptors and PSD-95 from rat brain. Intriguingly, NR1-alpha-actinin binding is directly antagonized by Ca2+/calmodulin. Thus alpha-actinin may play a role in both the localization of NMDA receptors and their modulation by Ca2+.

    Nature 1997;385;6615;439-42

  • Tryptophan residues in caldesmon are major determinants for calmodulin binding.

    Graether SP, Heinonen TY, Raharjo WH, Jin JP and Mak AS

    Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.

    Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B' (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 296, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a Kd of 0.13 x 10(-6) M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22- and 31-fold (Kd = 2.9 x 10(-6) and 4.0 x 10(-6) M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol-1, respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a Kd of 6.1 x 10(-6) M and destabilizes the complex by 2.17 kcal mol-1. On the other hand, mutation of Trp 722 at site B' to Ala causes a much smaller decrease in affinity (Kd = 0.6 x 10(-6) M) and results in a destabilization energy of 0.87 kcal mol-1. To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A + B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (Kd = 1.7 x 10(-6) M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (Kd = 4.1 x 10(-6) M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmon-calmodulin interaction and that Trp 722 in site B' plays a minor role.

    Biochemistry 1997;36;2;364-9

  • Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins.

    Lee CH, Della NG, Chew CE and Zack DJ

    Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9289, USA.

    cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases are Mucor racemosus Ras2 and Ras3, to which they show approximately 48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

    Funded by: NEI NIH HHS: EY09679

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;21;6784-94

  • Mirror image motifs mediate the interaction of the COOH terminus of multiple synaptotagmins with the neurexins and calmodulin.

    Perin MS

    Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.

    I have previously reported that the COOH-terminal 34 amino acids of synaptotagmin 1 are capable of interacting with the presynaptic proteins, the neurexins. Multiple synaptotagmins and a synaptotagmin-like protein, rabphilin 3A, are conserved in this domain, raising the possibility that many different synaptotagmins may interact with neurexins. Here 1 report that the COOH termini of synaptotagmins 1, 2, 4, 5, 6, 7, and 9 and rabphilin 3A are capable of interacting with neurexins. The COOH terminus of rabphilin 3A is still capable or substantial enrichment of neurexins from solubilized brain membranes even though only 11 of 33 residues are identical with the COOH terminus of synaptotagmin 1. Like the purification of neurexins on the COOH terminus of synaptotagmin 1, purification by the COOH terminus of rabphilin 3A is calcium-independent. The conservation between carboxyl termini of these proteins suggests symmetrical motifs are necessary for neurexin binding. These include the sequence Leu-X-His-Trp, followed by 13 amino acids, and the sequence Trp-His-X-Lcu. Deletion of the first motif or substitution of residues in the second of these motifs greatly reduces neurexin enrichment. Interestingly, these same COOH termini yield substantial calcium-dependent enrichment of calmodulin mediated by the first of these sequence motifs. This correlates with the binding of 125I-labeled calmodulin by recombinant pieces of synaptotagmn 1 containing the carboxyl terminus. These data suggest that multiple synaptotagmins may interact with neurexins to mediate docking or regulation of neurotransmitter release and that synaptotagmins may be calcium-regulated via interaction with calmodulin.

    Funded by: NINDS NIH HHS: R01 NS30541

    Biochemistry 1996;35;43;13808-16

  • Analysis of the role of calmodulin binding and sequestration in neuromodulin (GAP-43) function.

    Gamby C, Waage MC, Allen RG and Baizer L

    R. S. Dow Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, Portland, Oregon 97209, USA.

    We demonstrated previously that forced expression of the neuronal phosphoprotein neuromodulin (also known as GAP-43, F1, B-50, and p57) in mouse anterior pituitary AtT-20 cells enhances depolarization-mediated secretion and alters cellular morphology. Here we analyze the role of calmodulin binding by neuromodulin in these responses. In cells expressing wild-type neuromodulin, a complex with calmodulin that is sensitive to intracellular calcium and phosphorylation is localized to the plasma membrane. Transfection of several mutant forms of neuromodulin shows that the effects of this protein on secretion are dependent on both calmodulin binding and association with the plasma membrane. In contrast, the morphological changes depend only on membrane association. Thus, the multitude of effects of neuromodulin noted in previous studies may result from divergent properties of this protein.

    Funded by: NINDS NIH HHS: NS26806

    The Journal of biological chemistry 1996;271;43;26698-705

  • Induction of apoptosis by calmodulin-dependent intracellular Ca2+ elevation in CD4+ cells expressing gp 160 of HIV.

    Sasaki M, Uchiyama J, Ishikawa H, Matsushita S, Kimura G, Nomoto K and Koga Y

    Department of Virology, Kyushu University, Fukuoke, Japan.

    Human CD4+ cell clones expressing either gp160 or gp120 of HIV-1 under the transcriptional control of an inducible promoter were used to examine the role of Ca2+ signalling in the induction of apoptosis by envelope glycoproteins. Singlecell killing with apoptosis was induced in the cells expressing gp 160 while no such apoptosis was found in the cells expressing gp 120. An increase of intracellular Ca2+ was found in the gp 160-expressing cells but not in the gp 120-expressing cells as determined by Intracellular Ca2+ imaging analysis before the appearance of DNA fragmentation. W7, a calmodulin antagonist, blocked the elevation of Ca2+ as well as the resultant DNA fragmentation, which thus implies that the calmodulin-dependent intracellular Ca2+ release system is first activated by gp 160 and thereafter apoptosis takes place. The above results thus indicate that Ca2+ signalling plays a crucial role in the apoptosis accompanying the single-cell death induced by gp 160 in CD4+ cells.

    Virology 1996;224;1;18-24

  • Use of a two-hybrid system to investigate molecular interactions of GAP-43.

    Chao S, Benowitz LI, Krainc D and Irwin N

    Department of Neurosurgery, Children's Hospital, Boston, MA 02115, USA.

    We used the 'interaction trap' (two-hybrid system) to identify polypeptides that interact with the neuronal phosphoprotein, GAP-43, in an intracellular environment. GAP-43 (neuromodulin, B-50, F1), a protein kinase C (PKC) substrate important for the growth and plasticity of neuronal connections, has been implicated in vitro in several signal transduction pathways. In the yeast-based cloning system, the only strong interaction that was detected between GAP-43 and the calcium effector protein, calmodulin (CaM). PKC phosphorylates GAP-43 on serine 41. When we changed this serine to an aspartate residue to mimic constitutive phosphorylation, the interaction with CaM was blocked. Surprisingly, the N-terminal third of GAP-43 alone bound CaM more strongly than did intact GAP-43, suggesting that the protein's C-terminus may play a role in modulating the interaction with CaM. These results, along with other recent findings, suggest a novel role for the interaction between GAP-43 and CaM.

    Funded by: NEI NIH HHS: EY 05690

    Brain research. Molecular brain research 1996;40;2;195-202

  • The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.

    Brill S, Li S, Lyman CW, Church DM, Wasmuth JJ, Weissbach L, Bernards A and Snijders AJ

    Massachusetts General Hospital Cancer Centre, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

    We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.

    Funded by: NIAMS NIH HHS: AR16265E21; NINDS NIH HHS: NS31747

    Molecular and cellular biology 1996;16;9;4869-78

  • Role of calmodulin in HIV-potentiated Fas-mediated apoptosis.

    Pan Z, Radding W, Zhou T, Hunter E, Mountz J and McDonald JM

    Department of Pathology, University of Alabama at Birmingham 35294-0007, USA.

    The recently demonstrated extraordinary rate of turnover of T cells in human immunodeficiency virus (HIV)-1-infected patients and the apparently concomitant high rate of viral production and death are consistent with a large amount of cell death directly due to infection. Apoptosis may be one of the major forms of T cell death in HIV-1 infection. Many apoptotic pathways depend on calcium and therefore would be expected to involve calmodulin. As the HIV-1 envelope glycoprotein, gp160, contains two known calmodulin-binding domains, we investigated the possibility that the cytoplasmic domain of the HIV-1 envelope protein gp160 could enhance Fas-mediated apoptosis, the major form of apoptosis in lymphocytes. Our studies have shown that 1) transfection of H9 and MOLT-4 cells with a non-infectious HIV proviral clone, pFN, which expresses wild-type gp160, leads to enhanced Fas-mediated apoptosis, 2) transfection of MOLT-4 cells with a pFN construct pFN delta 147, which expresses a carboxyl-terminally truncated gp160 lacking the calmodulin-binding domains, produces less Fas-mediated apoptosis than transfection with pFN, and 3) the calmodulin antagonists trifluoperazine and tamoxifen completely inhibit the pFN enhancement of Fas-mediated apoptosis in MOLT-4 cells. We have replicated all of these results using the vectors pSRHS and pSRHS delta 147, which express wild-type gp160 and truncated gp160, respectively, in the absence of other viral proteins. These investigations provide a mechanism by which HIV-1 may induce apoptosis and a possible intracellular target for future therapeutics.

    Funded by: NIAID NIH HHS: 5P30-AI27767-07; NIAMS NIH HHS: P01-AR0355, R01-AR442567

    The American journal of pathology 1996;149;3;903-10

  • A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons.

    Castets F, Bartoli M, Barnier JV, Baillat G, Salin P, Moqrich A, Bourgeois JP, Denizot F, Rougon G, Calothy G and Monneron A

    UPR Centre National de la Recherche Scientifique (CNRS) 9013, Laboratoire de Neurobiologie Cellulaire et Fonctionnelle, Marseille, France.

    A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD-repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)-signaling pathway specific to excitatory synapses.

    The Journal of cell biology 1996;134;4;1051-62

  • Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes.

    Li X and Bennett V

    Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Adducin is an actin-binding protein that has been proposed to function as a regulated assembly factor for the spectrin/actin network. This study has addressed the question of the subunit and domains of spectrin required for formation of spectrin/adducin/actin complexes in in vitro assays. Quantitative evidence is presented that the beta-spectrin N-terminal domain plus the first two alpha-helical domains are required for optimal participation of spectrin in spectrin/adducin/actin complexes. The alpha subunit exhibited no detectable activity either alone or following association with beta-spectrin. The critical domains of beta-spectrin involved in complex formation were determined using recombinant proteins expressed in bacteria. The N-terminal domain (residues 1-313) of beta-spectrin associated with F-actin with a Kd of 26 microM, and promoted adducin binding to F-actin with half-maximal activation at 110 nM. Addition of the first alpha-helical domain (residues 1-422) lowered the Kdfor F-actin by 4-fold to 6 microM, but also reduced the capacity by 3-fold and had no effect on interaction with adducin. Further addition of the second alpha-helical domain (residues 1-528) did not alter binding to F-actin but resulted in a 2-fold increased activity in promoting adducin binding with half-maximal activation at 50 nM. Addition of up to eight additional alpha-helical domains (residues 1-1388) resulted in no further change in F-actin binding or association with adducin. These results demonstrate an unanticipated role of the first repeat of beta-spectrin in actin binding activity and of the second repeat in association with adducin/actin, and imply the possibility of an extended contact between adducin, spectrin, and actin involving several actin subunits.

    The Journal of biological chemistry 1996;271;26;15695-702

  • IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs.

    Hart MJ, Callow MG, Souza B and Polakis P

    Onyx Pharmaceuticals, Richmond, CA 94806, USA.

    Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.

    The EMBO journal 1996;15;12;2997-3005

  • Identification of insulin-stimulated phosphorylation sites on calmodulin.

    Joyal JL, Crimmins DL, Thoma RS and Sacks DB

    Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

    Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin-sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin.

    Funded by: NIDDK NIH HHS: DK09062, DK43682

    Biochemistry 1996;35;20;6267-75

  • A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments.

    Kuhlman PA, Hughes CA, Bennett V and Fowler VM

    Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

    Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.

    Funded by: NIDDK NIH HHS: DK29808; NIGMS NIH HHS: GM34225

    The Journal of biological chemistry 1996;271;14;7986-91

  • Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit.

    Ehlers MD, Zhang S, Bernhadt JP and Huganir RL

    Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    NMDA (N-methyl-D-aspartate) receptors are excitatory neurotransmitter receptors in the brain critical for synaptic plasticity and neuronal development. These receptors are Ca2+-permeable glutamate-gated ion channels whose physiological properties are regulated by intracellular Ca2+. We report here the purification of a 20 kDa protein identified as calmodulin that interacts with the NR1 subunit of the NMDA receptor. Calmodulin binding to the NR1 subunit is Ca2+ dependent and occurs with homomeric NR1 complexes, heteromeric NR1/NR2 subunit complexes, and NMDA receptors from brain. Furthermore, calmodulin binding to NR1 causes a 4-fold reduction in NMDA channel open probability. These results demonstrate that NMDA receptor function can be regulated by direct binding of calmodulin to the NR1 subunit, and suggest a possible mechanism for activity-dependent feedback inhibition and Ca2+-dependent inactivation of NMDA receptors.

    Cell 1996;84;5;745-55

  • Expression of HIV-1 envelope glycoprotein alters cellular calmodulin.

    Radding W, Pan ZQ, Hunter E, Johnston P, Williams JP and McDonald JM

    Department of Pathology, University of Alabama at Birmingham 35294-0007, USA.

    Removal of parts of a known calmodulin binding site at the C-terminus of HIV-1 envelope glycoprotein, gp160, can result in diminished infectivity. We investigated whether expression of full length gp160 would result in changes in intracellular calmodulin compared to expression of gp160 truncated to remove both known calmodulin binding sites. Both Western and Northern blots demonstrated that expression of gp160 led to increased calmodulin when compared to expression of truncated gp160. The induced calmodulin was associated preferentially with a particulate subcellular fraction. Confocal immunomicroscopy confirmed the increase in calmodulin and also showed that there was enhanced colocalization of calmodulin with gp160. Understanding of the role of calmodulin in the viral life-cycle may lead to new therapeutics.

    Funded by: NIAID NIH HHS: P30-AI-27767

    Biochemical and biophysical research communications 1996;218;1;192-7

  • The regulatory region of calcium/calmodulin-dependent protein kinase I contains closely associated autoinhibitory and calmodulin-binding domains.

    Yokokura H, Picciotto MR, Nairn AC and Hidaka H

    Department of Pharmacology, Nagoya University School of Medicine, Japan.

    The mechanism for the regulation of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was investigated using a series of COOH-terminal truncated mutants. These mutants were expressed in bacteria as fusion proteins with glutathione S-transferase and purified by affinity chromatography using glutathione Sepharose 4B. A mutant (residues 1-332) showed complete Ca2+/CaM-dependent activity. Truncation mutants (residues 1-321, 1-314, and 1-309) exhibited decreasing affinities for Ca2+/CaM and also exhibited decreasing Ca2+/CaM-dependent activities. Truncation mutants (residues 1-305 or 1-299) were unable to bind Ca2+/CaM and were inactive. In contrast, truncation mutants (residues 1-293 or 1-277) were constitutively active at a slightly higher level (2-fold) than fully active CaM kinase I. These results indicate the location of the Ca2+/CaM-binding domain on CaM kinase I (residues 294-321) and predict the existence of an autoinhibitory domain near, or overlapping, the Ca2+/CaM-binding domain. These conclusions were supported by studies which showed that a synthetic peptide (CaM kinase I (294-321)) corresponding to residues 294-321 of CaM kinase I inhibited the fully active kinase in a manner that was competitive with Ca2+/CaM and also inhibited the constitutively active mutant (residues 1-293) in a manner that was competitive with Syntide-2, a peptide substrate, (Ki = 1.2 microM) but was non-competitive with ATP. Thus, these results suggest that CaM kinase I is regulated through an intrasteric mechanism common to other members of the family of Ca2+/CaM-dependent protein kinases.

    The Journal of biological chemistry 1995;270;40;23851-9

  • Characterization of a Ca2+/calmodulin-dependent protein kinase cascade. Molecular cloning and expression of calcium/calmodulin-dependent protein kinase kinase.

    Tokumitsu H, Enslen H and Soderling TR

    Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.

    Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) can mediate Ca(2+)-dependent regulation of gene expression through the phosphorylation of transcriptional activating proteins. We have previously identified and purified a 68-kDa rat brain CaM-kinase kinase that phosphorylates and increases total and Ca(2+)-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269, 28640-28647). Using a partial amino acid sequence of the purified brain kinase, a CaM-kinase kinase cDNA was cloned from a rat brain cDNA library. Northern blot analysis showed that CaM-kinase kinase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spleen. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-40% homologous with members of the CaM-kinase family. Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, which catalyzed in vitro activation in the presence of Mg2+/ATP and Ca2+/ CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compared with CaM-kinase IV alone. These results are consistent with the hypothesis that CaM-kinases I and IV are regulated through a unique signal transduction cascade involving CaM-kinase kinase.

    Funded by: NIGMS NIH HHS: GM 41292

    The Journal of biological chemistry 1995;270;33;19320-4

  • Assignment of the human beta-adducin gene (ADD2) to 2p13-p14 by in situ hybridization.

    Gilligan DM, Lieman J and Bennett V

    Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06512, USA.

    Funded by: PHS HHS: K08

    Genomics 1995;28;3;610-2

  • Multiple mRNA species are generated by alternate polyadenylation from the human calmodulin-I gene.

    Senterre-Lesenfants S, Alag AS and Sobel ME

    Molecular Pathology Section, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892, USA.

    Three distinct genes encode an identical calmodulin protein in mammalian cells. In addition, multiple mRNA transcripts, with approximate sizes of 1.6 kb and 4.4 kb, are visualized on Northern blots hybridized to calmodulin-I cDNA probes. To elucidate the mechanism generating multiple calmodulin mRNAs, the complete sequence of the 4194 base human calmodulin-I mRNA was determined from cDNA clones and 3' rapid amplification of complementary ends (3' RACE). The 5' untranslated region of calmodulin-I mRNA contains a GC-rich domain containing multiple repeats of GGC interrupted by a GCA sequence, as well as a tandem repeat sequence of eight GCA triplets. The 3' untranslated region of calmodulin-I mRNA contains two canonical and one aberrant (ATTAAA) polyadenylation signal, consistent with the sizes of 1.6 kb and 4.4 kb mRNAs visualized on Northern blots, and a potential minor 4.2 kb mRNA detected by 3' RACE. Hybridization experiments using specific probes upstream and downstream of the polyadenylation signals demonstrated that alternate use of polyadenylation signals is the molecular mechanism for multiple calmodulin-I mRNA transcripts in human cells. Thirteen adenine rich elements with the motif AUUUA were detected in the 3' untranslated region. Three such motifs are embedded in regions that are conserved with the rat 3' untranslated region of calmodulin-I mRNA. One of these is surrounded by an adenine-uridine rich region that can form an 11-base pair stem structure. We propose that sequences in the 3' untranslated region of calmodulin-I mRNA may play a role in the regulation of calmodulin expression.

    Journal of cellular biochemistry 1995;58;4;445-54

  • Evidence for a functional interaction between calmodulin and the glucocorticoid receptor.

    Ning YM and Sánchez ER

    Department of Pharmacology, Medical College of Ohio, Toledo 43699.

    In the signaling cascade of membrane-bound receptors, calmodulin (CaM) plays an important role. However, little is known about the role of CaM in the activation of intracellular steroid receptors, which are known to act as ligand-regulated transcription factors. We report here that CaM can interact in a calcium-dependent manner with the untransformed glucocorticoid receptor (GR) complex containing hsp90. In addition, we demonstrate that four unrelated CaM antagonists (trifluoperazine, compound 48/80, W7, and phenoxybenzamine) can inhibit GR-mediated gene expression in mouse L929 cells stably-transfected with the MMTV-CAT reporter gene. These results provide evidence that CaM may play an important role in the signal transduction pathways of steroid hormone receptors.

    Funded by: NIDDK NIH HHS: DK43867, R01 DK043867

    Biochemical and biophysical research communications 1995;208;1;48-54

  • The interaction of calmodulin with clathrin-coated vesicles, triskelions, and light chains. Localization of a binding site.

    Pley UM, Hill BL, Alibert C, Brodsky FM and Parham P

    Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305.

    The binding of clathrin-coated vesicles, clathrin triskelions, and free clathrin light chains to calmodulin-Sepharose was compared. When isolated from bovine brain, all three components bound to calmodulin-Sepharose in the presence of calcium and could be eluted by its removal. In contrast, coated vesicles and triskelions isolated from bovine adrenal gland did not bind to calmodulin-Sepharose, although the free light chains from adrenal gland bound as effectively as those from brain. As distinct isoforms of the clathrin light chains are expressed by brain and adrenal gland, these results implicate the clathrin light chains as the calmodulin-binding component of coated vesicles and triskelions. Furthermore, the insertion sequences found in the neuron-specific isoforms, although not necessary for the binding of free clathrin light chains to calmodulin, must facilitate the interaction of heavy chain-associated light chains with calmodulin. Recombinant mutants of LCa, with deletions spanning the entire sequence, were tested for binding to calmodulin-Sepharose. Those mutants retaining structural integrity, as assessed by the binding of a panel of monoclonal antibodies, exhibited varying amounts of calmodulin binding activity. However, deletion of the carboxyl-terminal 20 residues abolished calmodulin interaction. Thus, the carboxyl terminus of LCa appears to constitute a calmodulin-binding site. Peptides corresponding to the carboxyl terminus of LCa or LCb inhibited the interaction of the light chains with calmodulin, suggesting that this region forms the calmodulin-binding site of both LCa and LCb. The carboxyl-terminal peptides of LCa and LCb inhibited the interaction of light chains with calmodulin approximately 10-fold less effectively than a calmodulin-binding peptide derived from smooth muscle myosin light chain kinase, but much more effectively than a calmodulin-binding peptide derived from adenylate cyclase. This comparison places the clathrin light chain-calmodulin interaction within the physiological range seen for other calmodulin-binding proteins.

    Funded by: NIGMS NIH HHS: GM38093

    The Journal of biological chemistry 1995;270;5;2395-402

  • Regulation of p68 RNA helicase by calmodulin and protein kinase C.

    Buelt MK, Glidden BJ and Storm DR

    Department of Pharmacology, University of Washington, Seattle 98195.

    Human p68 RNA helicase is a nuclear RNA-dependent ATPase that belongs to a family of putative helicases known as the DEAD box proteins. These proteins have been implicated in aspects of RNA function including translation initiation, splicing, and ribosome assembly in a variety of organisms ranging from Escherichia coli to humans. While members of this family are believed to function in the manipulation of RNA secondary structure, little is known about the regulation of these enzymes. By immunological methods and sequence comparison, we have found that p68 possesses a region of sequence similarity to the conserved protein kinase C phosphorylation site and calmodulin binding domain (also known as the IQ domain) of the neural-specific proteins neuromodulin (GAP-43) and neurogranin (RC3). We report that p68 is phosphorylated by protein kinase C in vitro and binds calmodulin in a Ca(2+)-dependent manner. Both phosphorylation and calmodulin binding inhibited p68 ATPase activity, suggesting that the RNA unwinding activity of p68 may be regulated by dual Ca2+ signal transduction pathways through its IQ domain.

    Funded by: NINDS NIH HHS: NS 31496, NS09174

    The Journal of biological chemistry 1994;269;47;29367-70

  • Mechanism of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N- diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin interactions with smooth muscle myosin light chain kinase and derived peptides.

    Török K and Trentham DR

    National Institute for Medical Research, Mill Hill, London, United Kingdom.

    The mechanism of the interactions of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]- 1,3,5-triazin-4-yl]calmodulin (TA-calmodulin) with smooth muscle myosin light-chain kinase (MLCK) and two 17-residue peptides, Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide) and Tyr peptide, in which W is replaced by Y, were studied by measurements of equilibrium and transient fluorescence changes in the nanomolar range. Most reactions were carried out in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0, and 21 degrees C. In each case association of MLCK or peptide to TA-calmodulin could be described by a two-step process, a bimolecular step and an isomerization. In the case of the interaction between TA-calmodulin and Tyr peptide it was shown that the isomerization involved the binary complex of TA-calmodulin and Tyr peptide as opposed to an isomerization of either TA-calmodulin or Tyr peptide in isolation. These distinctions depended in part on development for transient kinetic experiments of a general theory to quantify relative phase amplitudes in two-step mechanisms. The kinetics for all three association reactions were then interpreted in terms of a bimolecular association (rate constants k+1 and k-1) followed by an isomerization of the binary complex (rate constants k+2 and k-2). For the interaction of TA-calmodulin and Tyr peptide, values of the rate constants are k+1, 8.8 x 10(8) M-1 s-1; k-1, 5.7 s-1; k+2, 0.38 s-1; and k-2, 0.65 s-1. The fluorescence intensities (lambda ex 365 nm, lambda ex 365 nm, lambda em > 400 nm) of TA-calmodulin, the initial binary complex of TA-calmodulin and Tyr peptide, and the isomerized binary complex are in the ratio 1:2.8:1.3. Analogous mechanisms were found for TA-calmodulin binding to Trp peptide and to MLCK, but values for the rate constants and relative fluorescence intensities of the binary complexes were generally not so completely defined. Values for the Trp peptide and MLCK, respectively, are k+1, 8.8 x 10(8) M-1 s-1 and 1.1 x 10(8) M-1 s-1; (k+2 + k-2), 0.97 s-1 and 1.3 s-1; and k-1k-2/(k+2 + k-2), 0.0079 s-1 and 0.025-0.056 s-1. Equilibrium dissociation constants (Kd) for interactions of TA-calmodulin and targets determined from these data are Tyr peptide, 4.1 nM; Trp peptide, 0.011 nM; and MLCK, 0.23-0.51 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

    Funded by: NHLBI NIH HHS: HLB 15835

    Biochemistry 1994;33;43;12807-20

  • Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2.

    Rhyner JA, Ottiger M, Wicki R, Greenwood TM and Strehler EE

    Laboratory for Biochemistry, Swiss Federal Institute of Technology, ETH Zentrum, Zurich.

    The human CALM1 calmodulin gene has been isolated and characterized. The gene contains six exons spread over about 10 kb of genomic DNA. The exon-intron structure is identical to that of the human CALM3 and of the rat CALM1 and CALM3 genes. A cluster of transcription-start sites was identified 200 bp upstream of the ATG translation-start codon, and several putative regulatory elements were found in the 5' flanking region as well as in intron 1. Sequence comparison with the rat CALM1 gene revealed significant similarities in the promoter regions of the two genes and an even more striking degree of identity (70%) in the available intron 1 sequences. A short CAG trinucleotide repeat region was identified in the 5' untranslated region of the human CALM1 gene; this sequence is not conserved in the rat counterpart. Expression of the CALM1 gene was detected in all human tissues tested, although at varying levels. A 1.7-kb mRNA was uniformly present at comparable levels, whereas a 4.2-kb mRNA species was particularly abundant in brain and skeletal muscle. Clones for two different CALM1-related pseudogenes CALM1P1 and CALM1P2 were also isolated and characterized. Both pseudogenes are intronless and non-functional as judged from the presence of mutations abolishing the open reading frame. Genomic Southern analysis indicates that the human CALM1 gene/pseudogene subfamily comprises at least three but probably no more than four members. The entire family consists of three bona fide CALM genes, at least one expressed calmodulin-like CALML gene as well as at least five pseudogenes.

    European journal of biochemistry 1994;225;1;71-82

  • Isolation of phosphorylated calmodulin from rat liver and identification of the in vivo phosphorylation sites.

    Quadroni M, James P and Carafoli E

    Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.

    A procedure is described for the isolation of calmodulin (CaM) from rat liver which produces a fraction containing non-phosphorylated, mono-, di-, and triphosphocalmodulin as determined by mass spectrometric analysis. The distribution of CaM between the various phospho-species varies from preparation to preparation even though the isolation procedure is rigidly defined, suggesting that CaM phosphorylation may be a very labile phenomenon dependent on the state of the liver as it is removed from the animal. Approximately 15% of CaM in the cell is phosphorylated. The in vivo phosphorylation sites were determined by mass spectrometric analysis of a combined CNBr and trypsin digestion of the phosphocalmodulin (phospho-CaM)-containing fractions. Phosphorylated peptides were sequenced using two mass scanning devices linked together for collisionally activated fragmentation studies to determine peptide sequences, and the phosphorylation sites were determined as Thr-79, Ser-81, and Ser-101. These correspond to three of the four in vitro target sites of calmodulin phosphorylation by casein kinase II, which indicates that this may be the enzyme responsible for the phosphorylation in vivo. A preliminary study on the modulatory activity of phosphorylated calmodulin using a sample extensively phosphorylated in vitro with casein kinase II confirmed that phospho-CaM has an altered biological activity, i.e. reduced activation of the erythrocyte plasma membrane Ca2+ pump.

    The Journal of biological chemistry 1994;269;23;16116-22

  • Identification of a calmodulin-binding and inhibitory peptide domain in the HIV-1 transmembrane glycoprotein.

    Miller MA, Mietzner TA, Cloyd MW, Robey WG and Montelaro RC

    Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.

    A number of studies suggest a critical role of the HIV-1 envelope glycoprotein in cytopathogenesis, but the detailed mechanisms of cell injury remain to be defined. HIV-1 envelope proteins associate with the host cell membrane, and studies have demonstrated that HIV perturbs membrane structure and function. We describe here a structurally conserved region of the HIV-1 transmembrane protein (TM) that displays functional properties of target regions of proteins that interact directly with calcium-saturated calmodulin as part of cellular response cascades. The synthetic peptide homolog encompassing the carboxyl terminus (amino acid residues 828-855) of HIV-1 TM protein (LLP-1) is shown in standard in vitro assays to bind efficiently to purified calmodulin (CaM) and to inhibit in vitro CaM-mediated stimulation of phosphodiesterase activity. This suggests that this peptide homolog binds to CaM at affinities similar to those reported for a reference CaM-binding peptide. In addition, the CaM-dependent process of phospholipid synthesis can be inhibited in cell cultures by exogenous addition of the LLP-1. Finally, we have shown that the full-length TM protein binds CaM, whereas a truncated TM protein lacking the LLP-1 segment does not bind CaM. These results suggest a novel mechanism of viral cytopathogenesis mediated by the interaction of HIV-1 TM protein with cellular CaM, that could lead to an uncoupling of critical cellular signal transduction pathways.

    Funded by: NIAID NIH HHS: AI25722, AI28243

    AIDS research and human retroviruses 1993;9;11;1057-66

  • Cytosolic domain of the human immunodeficiency virus envelope glycoproteins binds to calmodulin and inhibits calmodulin-regulated proteins.

    Srinivas SK, Srinivas RV, Anantharamaiah GM, Compans RW and Segrest JP

    Department of Medicine, University of Alabama at Birmingham 35294.

    Calmodulin (CaM), the major intracellular receptor for calcium, is involved in regulation of diverse cellular functions. Positively charged amphipathic helical segments have been identified as an important structural motif in the recognition of CaM by different CaM-activated enzymes and peptides. The carboxyl-terminal domain of the envelope glycoproteins of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) contain regions that can fold into amphipathic helical segments, which closely resemble the amphipathic segments found in CaM-activated enzymes. We show here that synthetic peptide analogs corresponding to the two putative amphipathic helical regions of HIV-1/WMJ gp160 bind to CaM with high affinity (Kd 31-41 nM) in the presence of calcium. They also bind CaM in the absence of calcium, although with much lower affinity. The peptides inhibit CaM-regulated activation of bovine brain phosphodiesterase in vitro. The peptides also inhibit mitogen-induced lymphocyte activation, a property shared by CaM antagonists. Purified HIV-1 gp160 binds to CaM, while gp120, which lacks the putative amphipathic helical segments, does not bind CaM. In HIV-infected cells, the putative CaM-binding regions of gp160 are located intracellularly and may therefore interact with the cytosolic CaM. We postulate that CaM binding by HIV envelope proteins is likely to exert diverse modulatory effects, and the mechanism for HIV-induced cytotoxicity may involve, in part, inhibition of CaM-regulated cellular functions.

    Funded by: NCI NIH HHS: CA 21765; NIAID NIH HHS: AI 25784, AI 28928

    The Journal of biological chemistry 1993;268;30;22895-9

  • The calmodulin-binding domain of the mouse 90-kDa heat shock protein.

    Minami Y, Kawasaki H, Suzuki K and Yahara I

    Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan.

    The mouse 90-kDa heat shock protein (HSP90) and Ca(2+)-calmodulin were cross-linked at an equimolar ratio using a carbodiimide zero-length cross-linker. To identify the calmodulin-binding domain(s) of HSP90, CNBr-cleaved peptide fragments of HSP90 were mixed with Ca(2+)-calmodulin and cross-linked. Amino acid sequence determination revealed that an HSP90 alpha-derived peptide starting at the 486th amino acid residue was contained in the cross-linked products, which contains a calmodulin-binding motif (from Lys500 to Ile520). A similar motif is present also in HSP90 beta (from Lys491 to Val511). The synthetic peptides corresponding to these putative calmodulin-binding sequences were found to be cross-linked with Ca(2+)-calmodulin and to prevent the cross-linking of HSP90 and Ca(2+)-calmodulin. Both HSP90 alpha and HSP90 beta bind Ca2+. The HSP90 peptides bind HSP90 and thereby inhibit the binding of Ca2+. In addition, the HSP90 peptides augment the self-oligomerization of HSP90 induced at elevated temperatures. These results suggest that the calmodulin-binding domain of HSP90 might interact with another part of the same molecule and that Ca(2+)-calmodulin might modulate the structure and function of HSP90 through abolishing the intramolecular interaction.

    The Journal of biological chemistry 1993;268;13;9604-10

  • Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3.

    Berchtold MW, Egli R, Rhyner JA, Hameister H and Strehler EE

    Institute of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.

    The three bona fide genes coding for calmodulin CALM1, CALM2, and CALM3, were assigned to human chromosomes 14, 2, and 19, respectively, by polymerase chain reaction-based amplification of calmodulin gene-specific sequences using DNA from human-hamster cell hybrids as template. Employing calmodulin gene-specific DNA probes of mainly intronic or flanking parts of the individual genes, regional sublocalization was performed by in situ hybridization on metaphase spreads of human lymphocytes. The three calmodulin genes map to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3, respectively. These results represent the first complete chromosomal localization study on a mammalian calmodulin gene family and indicate that these structurally closely related genes were most likely dispersed throughout the genome concomitantly with their generation from an ancestral precursor gene.

    Genomics 1993;16;2;461-5

  • Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

    Kessler F, Falchetto R, Heim R, Meili R, Vorherr T, Strehler EE and Carafoli E

    Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.

    The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.

    Biochemistry 1992;31;47;11785-92

  • Insulin-stimulated phosphorylation of calmodulin.

    Sacks DB, Davis HW, Crimmins DL and McDonald JM

    Department of Pathology, Brigham and Women's Hospital, Boston, MA.

    Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling.

    Funded by: NIDDK NIH HHS: DK01680, DK25897

    The Biochemical journal 1992;286 ( Pt 1);211-6

  • Interaction of calmodulin with lactoferrin.

    de Lillo A, Tejerina JM and Fierro JF

    Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Spain.

    Calmodulin, as a major intracellular calcium-binding protein, regulates many Ca(2+)-dependent enzymes and plays an important role in a wide spectrum of cellular functions of the eukaryotes. Interaction between calmodulin and human lactoferrin, a 78 kDa protein with antibacterial properties, was found in the presence of Ca2+ using (i) a method for the detection of calmodulin binding proteins with biotinylated calmodulin, (ii) affinity chromatography on an agarose-calmodulin column with subsequent detection by an enzyme-linked immunosorbent assay (ELISA). The binding of calmodulin to lactoferrin blocked the ability of lactoferrin to agglutinate Micrococcus lysodeikticus.

    FEBS letters 1992;298;2-3;195-8

  • Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase.

    Takazawa K and Erneux C

    Institut de Recherche Interdisciplinaire (IRIBHN), Université Libre de Bruxelles, Belgium.

    In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein [clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem. J. 272, 107-112]. Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes. The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way. The catalytic domain is thus located at the C-terminal end of the protein. A series of 5' deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids. Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain. Affinity chromatography on CaM-Sepharose of 5' and 3' deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation.

    The Biochemical journal 1991;280 ( Pt 1);125-9

  • Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26.

    Girsch SJ and Peracchia C

    Department of Physiology, University of Rochester, School of Medicine and Dentistry, NY 14642.

    Lens fiber cells are coupled by communicating junctions that comprise over 50% of their appositional surfaces. The main intrinsic protein (MIP26) of lens fibers is a 28.2 kDa protein that forms large gap junction-like channels in reconstituted systems. Previously, we have shown that Ca(++)-activated calmodulin (CaM) regulates the permeability of reconstituted MIP26 channels and changes the conformation of MIP26, as measured by intrinsic fluorescence and circular dichroism spectroscopy. Examination of the MIP26 amino acid sequence has revealed a basic amphiphilic alpha-helical segment (Pep C) on the C-terminus with residue distribution similar to that found in other CaM binding proteins. To test the interaction between the amphiphilic segment and CaM, both a 20-mer peptide and trp-substituted fluorescent analog have been synthesized and purified by HPLC. Evidence from spectrofluorometric titration shows that the Pep C binds with CaM in 1:1 stoichiometry and with a kd of approximately 10 nM. Neither Ca++ nor H+ alone affects the conformation of the Pep C. However, when mixed with CaM the Pep C undergoes both a dramatic blue-shift in tryptophan fluorescence emission, indicative of strong hydrophobic interaction, and an increase in circular dichroism absorption in the alpha-helical region. Additional fluorescence blue-shift and alpha-helical content occur when Ca++ is added to the CaM:Pep C complex.

    Funded by: NEI NIH HHS: EY 06467; NIGMS NIH HHS: GM 20113

    Current eye research 1991;10;9;839-49

  • Cleavage of phosphorylase kinase and calcium-free calmodulin by HIV-1 protease.

    Daube H, Billich A, Mann K and Schramm HJ

    Max-Planck-Institut für Biochemie, Martinsried bei München, Germany.

    Phosphorylase kinase and calcium-free calmodulin are digested by human immunodeficiency virus-1 protease. In phosphorylase kinase, the alpha subunit is preferentially hydrolyzed at arg748-val749. The beta subunit is cleaved only slowly at leu678-pro679, and calmodulin, the integral delta subunit of phosphorylase kinase, is not cleaved at all. However, free calmodulin in the calcium-depleted form showed to be a good substrate for the protease. Here the cleavage occurs at phe65-pro66 and met71-met72. This fast hydrolysis of free calmodulin can be blocked by micromolar concentrations of Ca2+ or millimolar concentrations of Mg2+.

    Biochemical and biophysical research communications 1991;178;3;892-8

  • Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2.

    Tomasselli AG, Howe WJ, Hui JO, Sawyer TK, Reardon IM, DeCamp DL, Craik CS and Heinrikson RL

    Biochemistry Unit, Upjohn Company, Kalamazoo, Michigan 49001.

    Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NIGMS NIH HHS: GM-39552

    Proteins 1991;10;1;1-9

  • Structural organization of the human CaMIII calmodulin gene.

    Koller M, Schnyder B and Strehler EE

    Laboratory for Biochemistry, Swiss Federal Institute of Technology, Zurich.

    The complete structural organization of the human calmodulin III gene has been determined. This gene specifies the mRNA represented by the previously reported cDNA ht6. The gene contains six exons spread over a total of approx. 10 kb of DNA. Its exon-intron organization is identical to that of the only known chicken calmodulin gene and to that of two of the three characterized rat calmodulin genes. As in many other genes encoding Ca2+ binding proteins, intron 1 separates the ATG initiation codon from the remainder of the coding region. The major and two minor sites of transcription initiation have been determined by primer extension and ribonuclease protection assays. The DNA sequence in the promoter and 5' untranslated region is extremely GC-rich. No typical TATA and CAAT boxes are present upstream of the major transcriptional start site; however, a consensus CAAT box sequence is found further upstream and may play a role in transcriptional initiation from the minor start sites. Six sequence elements with high similarity to monkey SV40-like Sp1-binding regions are present in the putative promoter region, two of which contain perfect GGGCGG core sequences. The structure of the human calmodulin III gene promoter indicates that this gene belongs to a class of 'house-keeping' genes but that its level of expression may also be specifically regulated.

    Biochimica et biophysica acta 1990;1087;2;180-9

  • Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau.

    Padilla R, Maccioni RB and Avila J

    Centro de Bíologia Molecular, Universidad Autónoma, Madrid, Spain.

    Previous studies have demonstrated that the microtubule-associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments alpha (430-441) and beta (422-434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.

    Molecular and cellular biochemistry 1990;97;1;35-41

  • Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation.

    Winder SJ and Walsh MP

    Department of Medical Biochemistry, University of Calgary, Alberta, Canada.

    Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.

    The Journal of biological chemistry 1990;265;17;10148-55

  • The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin.

    Dasgupta M, Honeycutt T and Blumenthal DK

    University of Texas Health Center, Department of Biochemistry, Tyler 75710.

    Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.

    Funded by: NIGMS NIH HHS: GM39290

    The Journal of biological chemistry 1989;264;29;17156-63

  • Calmodulin site at the C-terminus of the putative lens gap junction protein MIP26.

    Peracchia C and Girsch SJ

    Department of Physiology, University of Rochester, School of Medicine and Dentistry, New York 14642.

    Lens fiber junctions contain cell-to-cell channels believed to be composed of a 28.2 kD protein (MIP26). Previous evidence indicates that calmodulin (CaM) is involved in the regulation of channel permeability by changing the conformation of the C terminal chain of MIP26. A study of the amino acid sequence of MIP26 has revealed an amphiphilic segment of the C-terminal chain with potential CaM-binding characteristics. To test the capacity of this chain to interact with CaM, a 20-amino acid peptide (peptide C) of appropriate sequence has been synthesized and purified by HPLC. Evidence from spectrofluorometry and circular dichroism experiments indicates that CaM interacts with and affects the conformation of peptide C, suggesting the involvement of MIP26 C-terminal chain and CaM in gating lens junction channels.

    Funded by: NEI NIH HHS: EY 06467; NIGMS NIH HHS: GM 20113

    Lens and eye toxicity research 1989;6;4;613-21

  • Vascular smooth muscle calponin. A novel troponin T-like protein.

    Takahashi K, Hiwada K and Kokubu T

    Second Department of Internal Medicine, Ehime University School of Medicine, Japan.

    In a search for additional Ca2+ regulatory components in vascular smooth muscle, a novel troponin T-like protein was purified from bovine aorta smooth muscle. The isolated protein was separated into several isoforms on isoelectric focusing. The major isoelectric variants were focused in the pH region of 8.4 to 9.1. The protein had slightly different molecular masses in the Mr range of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molar ratio relative to tropomyosin in the muscle extract was estimated to be 0.9:1.0. The novel protein bound to the immobilized calmodulin and exhibited a number of common physicochemical properties with gizzard (Mr = 34,000) calmodulin-binding and F-actin-binding protein. The aorta and gizzard proteins were immunologically cross-reactive. Both proteins shared a common antigenic determinant with COOH-terminal segments of rabbit skeletal and bovine cardiac troponin T and bound to the immobilized smooth muscle tropomyosin. Both proteins interacted with rabbit skeletal troponin C in the presence and absence of Ca2+, but they did not interact with troponin I. These results suggest that the novel protein, which is designated calponin, may be a specialized component of smooth muscle thin filament involved in the regulation of contractile apparatus.

    Hypertension (Dallas, Tex. : 1979) 1988;11;6 Pt 2;620-6

  • Characterization of four tyrosine protein kinases from the particulate fraction of rat spleen.

    Brunati AM and Pinna LA

    Istituto di Chimica Biologica, Universitàa di Padova e Centro delle Biomembrane del CNR, Italy.

    Four distinct tyrosine protein kinases active on poly(Glu4,Tyr1) and angiotensin II, and operationally termed TPK-I, TPK-IIA, TPK-IIB and TPK-III have been resolved and partially purified from rat spleen particulate fraction by combining DEAE-Sepharose, heparin-Sepharose, phosphocellulose and polylysine-agarose chromatographies. Once partially purified all of them are free of Ser/Thr-specific protein kinase activity as judged using casein, histones, protamine and the peptide Arg-Arg-Ala-Ser-Val-Ala as substrates. TPK-I (apparent molecular mass 64 kDa, by gel filtration) and TPK-IIA (54 kDa) share several properties, including substrate specificity and stimulation by heparin; the latter however is much more responsive to polylysine then the former (10- and 3-fold maximum stimulation, respectively). Conversely TPK-IIB (51 kDa) is markedly inhibited by heparin and it is also characterized by its unique substrate specificity: unlike the other three tyrosine protein kinases it by far prefers the tetrapeptide Glu-Tyr-Ala-Ala over the decapeptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly and readily phosphorylates band-3 protein of red cell membrane. The unusual preference for Mg2+ over Mn2+ as activator and the capability to phosphorylate calmodulin distinguish TPK-III (61 kDa) from the other isoenzymes. Moreover TPK-III is insensitive to heparin and polylysine and is inhibited by quercetin much more efficiently than the other enzymes (I50 = 10 microM). Upon incubation with [gamma-32P]ATP, TPK-I, TPK-IIA and TPK-III give rise to alkali-stable radiolabeled components of 61, 55 and 52 kDa respectively, as evaluated by PAGE/SDS. In every case such a radiolabeling takes place also in the presence of a large excess of phosphorylatable substrate (angiotensin II) while it is readily reversed by isotopic dilution with 10-fold excess unlabeled ATP, supporting the view that it represents an autophosphorylation process. No (auto)phosphorylation product(s) could be detected in TPK-IIB even if its amount, in terms of catalytic activity, was 10-fold higher than that of the others.

    European journal of biochemistry 1988;172;2;451-7

  • Molecular analysis of human and rat calmodulin complementary DNA clones. Evidence for additional active genes in these species.

    SenGupta B, Friedberg F and Detera-Wadleigh SD

    Division of Intramural Research Programs, National Institute of Mental Health, Bethesda, Maryland 20892.

    A cDNA clone, lambda rCB1, encoding calmodulin was isolated from a rat brain expression library. The sequence was determined and compared to the structures of the previously described rat genes, lambda SC4 and lambda SC8 (Nojima, H., and Sokabe, H. (1986) J. Mol. Biol. 190, 391-400). Faithful sequence conservation is observed in the coding regions of lambda rCB1 and lambda SC4, the bona fide gene. Both cDNAs encode identical amino acid sequence. Very limited sequence homology, however, is noted in the 3'-untranslated segments of these clones. Surprisingly, when the lambda rCB1 nucleotide structure is compared to the processed intronless gene, lambda SC8, extensive sequence homology is found both in the coding and noncoding regions. The inferred protein sequences of lambda SC8 and lambda rCB1, however, are divergent. Using a fragment of lambda rCB1 to screen an expression library derived from a human embryonic cell line, a calmodulin cDNA clone, lambda hCE1, was isolated and characterized. Comparison of the sequence of lambda hCE1 to the cDNA from human liver, hCWP (Wawrzynczak, E. J., and Perham, R. N. (1984) Biochem. Int. 9, 177-185), reveals substantial structural divergence. Strikingly poor homology is seen in the 5'- and 3'-noncoding segments, but the coding regions were 85% homologous. Both lambda hCE1 and hCWP encode proteins of identical primary structure which is equivalent to the protein sequence deduced from lambda rCB1 and lambda SC4. Taken together these results suggest the existence of an additional actively transcribed calmodulin gene, not previously identified, in each of the human and rat genomes. Transcripts of lambda rCB1 and lambda hCE1 were observed in all tissues examined indicating the absence of tissue-specific expression. Calmodulin gene polymorphisms were detected using TaqI, HindIII, and MspI.

    The Journal of biological chemistry 1987;262;34;16663-70

  • Comparison of S100b protein with calmodulin: interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein kinase C.

    Baudier J, Mochly-Rosen D, Newton A, Lee SH, Koshland DE and Cole RD

    To gauge similarities between S100b protein and calmodulin, interactions were observed between S100b and melittin and between S100b and tau, the microtubule-associated proteins. The interaction of melittin with S100b protein in the presence and absence of calcium was studied by fluorescence polarization, UV difference spectroscopy, and sulfhydryl derivatization. Whether calcium was present or not in the solution, melittin and S100b form a complex of molar ratios up to 2:1. Further binding of melittin occurred, but it resulted in precipitation of S100b, as is true of the corresponding case of melittin binding to calmodulin. In the absence of calcium, the interaction of melittin and S100b shielded the tryptophan (Trp) of the former protein and exposed cysteine-84 beta (Cys-84 beta) of the latter protein, leaving the tyrosine-16 beta (Tyr-16 beta) of S100b unaffected. Calcium addition to the complex partially restored the exposure of Trp of melittin and caused changes in the environment of Tyr-16 beta (unlike the environmental changes induced for Tyr-16 beta by calcium in the absence of melittin). The conformational changes induced in S100b by interaction with melittin increased its affinity for calcium and offset the inhibition of calcium binding otherwise observed in the presence of potassium ions. This corroborated the previous finding that S100b affinity for calcium greatly depends on the protein conformation. The phenomena described above are similar to the interactions of melittin with calmodulin and thus suggest that S100b and calmodulin have a common structural domain not only that binds melittin but also that may interact with common target proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NIEHS NIH HHS: EHS-ES-01896; NIGMS NIH HHS: GMS-20338

    Biochemistry 1987;26;10;2886-93

  • Polycation-dependent, Ca2+-antagonized phosphorylation of calmodulin by casein kinase-2 and a spleen tyrosine protein kinase.

    Meggio F, Brunati AM and Pinna LA

    Ten distinct protein kinases have been tested for their ability to phosphorylate calmodulin. Only casein kinase-2 and a spleen tyrosine protein kinase (TPK-III) proved effective, their phosphorylation efficiency being dramatically enhanced by histones and other polybasic peptides while being depressed by 50 microM Ca2+. Phosphorylation by CK-2 takes place with a Km of 12 microM calmodulin, leading to the incorporation of more than 1.5 mol P/mol substrate. Ser81 and Thr79 are among the residues affected. On the other hand, the two tyrosyl residues of calmodulin are both phosphorylated by TPK-III, Tyr99 being preferred over Tyr138.

    FEBS letters 1987;215;2;241-6

  • Isolation and nucleotide sequence of a cDNA encoding human calmodulin.

    Wawrzynczak EJ and Perham RN

    We have isolated and determined the nucleotide sequence of a cDNA clone that contains the entire coding sequence of human calmodulin and substantial parts of the untranslated flanking regions of the mRNA. This clone was detected serendipitously in the course of screening a cDNA library with a mixture of oligodeoxyribonucleotide probes based on a target amino acid sequence from an unrelated protein. The predicted primary structure of the translation product of the calmodulin mRNA is identical with that determined for calmodulin from human brain by direct amino acid sequence analysis except for the presence of an additional methionine residue at the N-terminus which appears to be the site of initiation.

    Biochemistry international 1984;9;2;177-85

  • Studies of the interaction of troponin I with proteins of the I-filament and calmodulin.

    Moir AJ, Ordidge M, Grand RJ, Trayer IP and Perry SV

    1. All lysine residues in native troponin I from rabbit fast-twitch skeletal muscle reacted with methyl acetimidate and ethyl acetimidate. 2. The reactivity of lysine-18 of troponin I to acetimidate was much diminished when the troponin I was complexed in the presence of Ca2+ with troponin C alone or in the whole troponin complex. 3. In the presence of EGTA, lysine-18 of troponin I in the troponin I-troponin C complex was more reactive to acetimidate than it was in the presence of Ca2+. 4. No masking of lysine residues could be detected when troponin I interacted with calmodulin or actin. 5. Sedimentation-equilibrium studies indicated that the complex of troponin I with calmodulin was more readily dissociated in the absence of Ca2+ than was its complex with troponin C under otherwise identical conditions. 6. These studies suggest that the nature of the involvement of the N-terminal region of troponin I is a major difference between its modes of interaction with calmodulin and with troponin C.

    The Biochemical journal 1983;209;2;417-26

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000032 G2C Homo sapiens Pocklington H1 Human orthologues of cluster 1 (mouse) from Pocklington et al (2006) 21
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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