G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
spectrin, beta, non-erythrocytic 1
G00000546 (Mus musculus)

Databases (7)

ENSG00000115306 (Ensembl human gene)
6711 (Entrez Gene)
7 (G2Cdb plasticity & disease)
SPTBN1 (GeneCards)
182790 (OMIM)
Marker Symbol
HGNC:11275 (HGNC)
Protein Sequence
Q01082 (UniProt)

Literature (49)

Pubmed - other

  • Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies.

    Rivadeneira F, Styrkársdottir U, Estrada K, Halldórsson BV, Hsu YH, Richards JB, Zillikens MC, Kavvoura FK, Amin N, Aulchenko YS, Cupples LA, Deloukas P, Demissie S, Grundberg E, Hofman A, Kong A, Karasik D, van Meurs JB, Oostra B, Pastinen T, Pols HA, Sigurdsson G, Soranzo N, Thorleifsson G, Thorsteinsdottir U, Williams FM, Wilson SG, Zhou Y, Ralston SH, van Duijn CM, Spector T, Kiel DP, Stefansson K, Ioannidis JP, Uitterlinden AG and Genetic Factors for Osteoporosis (GEFOS) Consortium

    Department of Internal Medicine, Rotterdam, The Netherlands.

    Bone mineral density (BMD) is a heritable complex trait used in the clinical diagnosis of osteoporosis and the assessment of fracture risk. We performed meta-analysis of five genome-wide association studies of femoral neck and lumbar spine BMD in 19,195 subjects of Northern European descent. We identified 20 BMD loci that reached genome-wide significance (GWS; P < 5 x 10(-8)), of which 13 map to regions not previously associated with this trait: 1p31.3 (GPR177), 2p21 (SPTBN1), 3p22 (CTNNB1), 4q21.1 (MEPE), 5q14 (MEF2C), 7p14 (STARD3NL), 7q21.3 (FLJ42280), 11p11.2 (LRP4, ARHGAP1, F2), 11p14.1 (DCDC5), 11p15 (SOX6), 16q24 (FOXL1), 17q21 (HDAC5) and 17q12 (CRHR1). The meta-analysis also confirmed at GWS level seven known BMD loci on 1p36 (ZBTB40), 6q25 (ESR1), 8q24 (TNFRSF11B), 11q13.4 (LRP5), 12q13 (SP7), 13q14 (TNFSF11) and 18q21 (TNFRSF11A). The many SNPs associated with BMD map to genes in signaling pathways with relevance to bone metabolism and highlight the complex genetic architecture that underlies osteoporosis and variation in BMD.

    Funded by: Arthritis Research UK; Canadian Institutes of Health Research; NHLBI NIH HHS: N01-HC-25195, N01HC25195, N02-HL-6-4278; NIA NIH HHS: R01 AR/AG41398; NIAMS NIH HHS: R01 AR 050066, R01 AR041398, R01 AR041398-18S1A1, R01 AR050066, R01 AR050066-04; Wellcome Trust

    Nature genetics 2009;41;11;1199-206

  • New sequence variants associated with bone mineral density.

    Styrkarsdottir U, Halldorsson BV, Gretarsdottir S, Gudbjartsson DF, Walters GB, Ingvarsson T, Jonsdottir T, Saemundsdottir J, Snorradóttir S, Center JR, Nguyen TV, Alexandersen P, Gulcher JR, Eisman JA, Christiansen C, Sigurdsson G, Kong A, Thorsteinsdottir U and Stefansson K

    deCODE Genetics, Reykjavik, Iceland. unnur.styrkarsdottir@decode.is

    In an extended genome-wide association study of bone mineral density among 6,865 Icelanders and a follow-up in 8,510 subjects of European descent, we identified four new genome-wide significant loci. These are near the SOST gene at 17q21, the MARK3 gene at 14q32, the SP7 gene at 12q13 and the TNFRSF11A (RANK) gene at 18q21. Furthermore, nonsynonymous SNPs in the C17orf53, LRP4, ADAM19 and IBSP genes were suggestively associated with bone density.

    Nature genetics 2009;41;1;15-7

  • Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis.

    Baek HJ, Lim SC, Kitisin K, Jogunoori W, Tang Y, Marshall MB, Mishra B, Kim TH, Cho KH, Kim SS and Mishra L

    Radiation Medicine Branch, National Cancer Center, Goyang, Korea.

    Unlabelled: We have previously demonstrated that 40%-70% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) within 15 months, revealing the importance of the transforming growth factor-beta (TGF-beta) signaling pathway in suppressing tumorigenesis in the liver. The current study was carried out to investigate mechanisms by which embryonic liver fodrin (ELF), a crucial Smad3/4 adaptor, suppresses liver tumor formation. Histological analysis of hyperplastic liver tissues from elf(+/-) mice revealed abundant newly formed vascular structures, suggesting aberrant angiogenesis with loss of ELF function. In addition, elf(+/-) mice displayed an expansion of endothelial progenitor cells. Ectopic ELF expression in fetal bovine heart endothelial (FBHE) cells resulted in cell cycle arrest and apoptosis. Further analysis of developing yolk sacs of elf(-/-) mice revealed a failure of normal vasculature and significantly decreased endothelial cell differentiation with embryonic lethality. Immunohistochemical analysis of hepatocellular cancer (HCC) from the elf(+/-) mice revealed an abnormal angiogenic profile, suggesting the role of ELF as an angiogenic regulator in suppressing HCC. Lastly, acute small interfering RNA (siRNA) inhibition of ELF raised retinoblastoma protein (pRb) levels nearly fourfold in HepG2 cells (a hepatocellular carcinoma cell line) as well as in cow pulmonary artery endothelial (CPAE) cells, respectively.

    Conclusion: Taken together these results, ELF, a TGF-beta adaptor and signaling molecule, functions as a critical adaptor protein in TGF-beta modulation of angiogenesis as well as cell cycle progression. Loss of ELF in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers. Our studies propose that ELF is potentially a powerful target for mimetics enhancing the TGF-beta pathway tumor suppression of HCC.

    Funded by: NCI NIH HHS: P01 CA130821, P01 CA130821-01A1, R01 CA106614, R01 CA106614-01A2, R01 CA106614-04, R01 CA106614A, R01 CA4285718A; NIDDK NIH HHS: R01 DK056111, R01 DK56111, R01 DK58637

    Hepatology (Baltimore, Md.) 2008;48;4;1128-37

  • Fine expression profiling of full-length transcripts using a size-unbiased cDNA library prepared with the vector-capping method.

    Oshikawa M, Sugai Y, Usami R, Ohtoko K, Toyama S and Kato S

    Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan.

    Recently, we have developed a vector-capping method for constructing a full-length cDNA library. In the present study, we performed in-depth analysis of the vector-capped cDNA library prepared from a single type of cell. As a result of single-pass sequencing analysis of 24,000 clones randomly isolated from the unamplified library, we identified 19,951 full-length cDNA clones whose intactness was confirmed by the presence of an additional G at their 5' end. The full-length cDNA content was >95%. Mapping these sequences to the human genome, we identified 4,513 transcriptional units that include 36 antisense transcripts against known genes. Comparison of the frequencies of abundant clones showed that the expression profiles of different libraries, including the distribution of transcriptional start sites (TSSs), were reproducible. The analysis of long-sized cDNAs showed that this library contained many cDNAs with a long-sized insert up to 11,199 bp of golgin B, including multiple slicing variants for filamin A and filamin B. These results suggest that the size-unbiased full-length cDNA library constructed using the vector-capping method will be an ideal resource for fine expression profiling of transcriptional variants with alternative TSSs and alternative splicing.

    DNA research : an international journal for rapid publication of reports on genes and genomes 2008;15;3;123-36

  • Multiple genetic loci for bone mineral density and fractures.

    Styrkarsdottir U, Halldorsson BV, Gretarsdottir S, Gudbjartsson DF, Walters GB, Ingvarsson T, Jonsdottir T, Saemundsdottir J, Center JR, Nguyen TV, Bagger Y, Gulcher JR, Eisman JA, Christiansen C, Sigurdsson G, Kong A, Thorsteinsdottir U and Stefansson K

    deCODE Genetics, Reykjavik, Iceland.

    Background: Bone mineral density influences the risk of osteoporosis later in life and is useful in the evaluation of the risk of fracture. We aimed to identify sequence variants associated with bone mineral density and fracture.

    Methods: We performed a quantitative trait analysis of data from 5861 Icelandic subjects (the discovery set), testing for an association between 301,019 single-nucleotide polymorphisms (SNPs) and bone mineral density of the hip and lumbar spine. We then tested for an association between 74 SNPs (most of which were implicated in the discovery set) at 32 loci in replication sets of Icelandic, Danish, and Australian subjects (4165, 2269, and 1491 subjects, respectively).

    Results: Sequence variants in five genomic regions were significantly associated with bone mineral density in the discovery set and were confirmed in the replication sets (combined P values, 1.2x10(-7) to 2.0x10(-21)). Three regions are close to or within genes previously shown to be important to the biologic characteristics of bone: the receptor activator of nuclear factor-kappaB ligand gene (RANKL) (chromosomal location, 13q14), the osteoprotegerin gene (OPG) (8q24), and the estrogen receptor 1 gene (ESR1) (6q25). The two other regions are close to the zinc finger and BTB domain containing 40 gene (ZBTB40) (1p36) and the major histocompatibility complex region (6p21). The 1p36, 8q24, and 6p21 loci were also associated with osteoporotic fractures, as were loci at 18q21, close to the receptor activator of the nuclear factor-kappaB gene (RANK), and loci at 2p16 and 11p11.

    Conclusions: We have discovered common sequence variants that are consistently associated with bone mineral density and with low-trauma fractures in three populations of European descent. Although these variants alone are not clinically useful in the prediction of risk to the individual person, they provide insight into the biochemical pathways underlying osteoporosis.

    The New England journal of medicine 2008;358;22;2355-65

  • Fusion of PRKG2 and SPTBN1 to the platelet-derived growth factor receptor beta gene (PDGFRB) in imatinib-responsive atypical myeloproliferative disorders.

    Gallagher G, Horsman DE, Tsang P and Forrest DL

    Leukemia/BMT Program of British Columbia, Division of Hematology, Vancouver General Hospital, British Columbia Cancer Agency and University of British Columbia, Gordon & Leslie Diamond Health Care Centre, Vancouver, BC V5Z 1M9, Canada.

    Chromosomal translocations involving the platelet-derived growth factor receptor beta gene (PDGFRB) have been reported in a subset of patients with atypical myeloproliferative disorders (MPDs). The fusion of the PDGFRB gene, which encodes a tyrosine kinase receptor, with different partner genes results in its constitutive activation. We present the cases of two patients with atypical MPD carrying t(4;5)(q21;q33) and t(2;5)(p21;q33), respectively. Fluorescence in situ hybridization demonstrated that PDGFRB was involved in both translocations. Further characterization of the 4q21 breakpoint using a bacterial artificial chromosome probe revealed PRKG2 as the likely gene partner to PDGFRB. Characterization of the 2p21 breakpoint identified a novel gene partner to PDGFRB, the SPTBN1 gene. Both patients achieved a complete molecular remission after introduction of imatinib mesylate therapy.

    Cancer genetics and cytogenetics 2008;181;1;46-51

  • Lipid-binding role of betaII-spectrin ankyrin-binding domain.

    Bok E, Plazuk E, Hryniewicz-Jankowska A, Chorzalska A, Szmaj A, Dubielecka PM, Stebelska K, Diakowski W, Lisowski M, Langner M and Sikorski AF

    Institute of Biochemistry and Molecular Biology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-149 Wrocław, Poland.

    It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing betaII(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including betaII and also betaI full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of betaII and betaI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution.

    Cell biology international 2007;31;12;1482-94

  • Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation.

    Kitisin K, Ganesan N, Tang Y, Jogunoori W, Volpe EA, Kim SS, Katuri V, Kallakury B, Pishvaian M, Albanese C, Mendelson J, Zasloff M, Rashid A, Fishbein T, Evans SR, Sidawy A, Reddy EP, Mishra B, Johnson LB, Shetty K and Mishra L

    Department of Surgical Sciences, School of Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA.

    Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

    Funded by: NCI NIH HHS: P01 CA095569, P01CA095569, R01 CA042857, R01 CA106614, R01 CA106614A, R01 CA4285718A; NIDDK NIH HHS: R01 DK056111, R01 DK56111, R01 DK58637

    Oncogene 2007;26;50;7103-10

  • Conformational change of erythroid alpha-spectrin at the tetramerization site upon binding beta-spectrin.

    Long F, McElheny D, Jiang S, Park S, Caffrey MS and Fung LW

    Department of Chemistry, University of Illinois at Chicago 60607, USA.

    We previously determined the solution structures of the first 156 residues of human erythroid alpha-spectrin (SpalphaI-1-156, or simply Spalpha). Spalpha consists of the tetramerization site of alpha-spectrin and associates with a model beta-spectrin protein (Spbeta) with an affinity similar to that of native alpha- and beta-spectrin. Upon alphabeta-complex formation, our previous results indicate that there is an increase in helicity in the complex, suggesting conformational change in either Spalpha or Spbeta or in both. We have now used isothermal titration calorimetry, circular dichroism, static and dynamic light scattering, and solution NMR methods to investigate properties of the complex as well as the conformation of Spalpha in the complex. The results reveal a highly asymmetric complex, with a Perrin shape parameter of 1.23, which could correspond to a prolate ellipsoid with a major axis of about five and a minor axis of about one. We identified 12 residues, five prior to and seven following the partial domain helix in Spalpha that moved freely relative to the structural domain in the absence of Spbeta but when in the complex moved with a mobility similar to that of the structural domain. Thus, it appears that the association with Spbeta induced an unstructured-to-helical conformational transition in these residues to produce a rigid and asymmetric complex. Our findings may provide insight toward understanding different association affinities of alphabeta-spectrin at the tetramerization site for erythroid and non-erythroid spectrin and a possible mechanism to understand some of the clinical mutations, such as L49F of alpha-spectrin, which occur outside the functional partial domain region.

    Funded by: NIGMS NIH HHS: GM68621, P41 GM068944, P41 GM68944, R01 GM068621, R01 GM068621-01, R01 GM068621-02, R01 GM068621-03, R01 GM068621-04

    Protein science : a publication of the Protein Society 2007;16;11;2519-30

  • Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos.

    Kizhatil K, Davis JQ, Davis L, Hoffman J, Hogan BL and Bennett V

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

    E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.

    The Journal of biological chemistry 2007;282;36;26552-61

  • Ankyrin-G and beta2-spectrin collaborate in biogenesis of lateral membrane of human bronchial epithelial cells.

    Kizhatil K, Yoon W, Mohler PJ, Davis LH, Hoffman JA and Bennett V

    Howard Hughes Medical Institute and Departments of Cell Biology, Biochemistry, and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of beta(2)-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and beta(2)-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells.

    The Journal of biological chemistry 2007;282;3;2029-37

  • Phosphorylation of a threonine unique to the short C-terminal isoform of betaII-spectrin links regulation of alpha-beta spectrin interaction to neuritogenesis.

    Bignone PA, King MD, Pinder JC and Baines AJ

    Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, Great Britain.

    Spectrin tetramers are cytoskeletal proteins required in the formation of complex animal tissues. Mammalian alphaII- and betaII-spectrin subunits form dimers that associate head to head with high affinity to form tetramers, but it is not known if this interaction is regulated. We show here that the short C-terminal splice variant of betaII-spectrin (betaIISigma2) is a substrate for phosphorylation. In vitro, protein kinase CK2 phosphorylates Ser-2110 and Thr-2159; protein kinase A phosphorylates Thr-2159. Antiphospho-Thr-2159 peptide antibody detected phosphorylated betaIISigma2 in Cos-1 cells. Immunoreactivity was increased in Cos-1 cells by treatment with forskolin, indicating that phosphorylation is promoted by elevated cAMP. The effect of forskolin was counteracted by the cAMP-dependent kinase inhibitor, H89. In vitro, protein kinase A phosphorylation of an active fragment of betaIISigma2 greatly reduced its interaction with alphaII-spectrin at the tetramerization site. Mutation of Thr-2159 to alanine eliminated inhibition by phosphorylation. Among the processes that require spectrin in mammals is the formation of neurites (incipient nerve axons). We tested the relationship of spectrin phosphorylation to neuritogenesis by transfecting the neuronal cell line, PC12, with enhanced green fluorescent protein-coupled fragments of betaIISigma2-spectrin predicted to act as inhibitors of spectrin tetramer formation. Both wild-type and T2159E mutant fragments allowed normal neuritogenesis in PC12 cells in response to nerve growth factor. The mutant T2159A inhibited neuritogenesis. Because the T2159A mutant represents a high affinity inhibitor of tetramer formation, we conclude that tetramers are requisite for neuritogenesis. Furthermore, because both the T2159E mutant and the wild-type allow neuritogenesis, we conclude that the short C-terminal betaII-spectrin is phosphorylated during this process.

    Funded by: Biotechnology and Biological Sciences Research Council: C18062

    The Journal of biological chemistry 2007;282;2;888-96

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Global phosphoproteome of HT-29 human colon adenocarcinoma cells.

    Kim JE, Tannenbaum SR and White FM

    Biological Engineering Division, Massachusetts Institute of Technology, 77 Massassachusetts Avenue, Cambridge, MA 02139, USA.

    Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.

    Funded by: NIEHS NIH HHS: P30 ES 002109, P30 ES002109; NIGMS NIH HHS: 1P50 GM 68762-01

    Journal of proteome research 2005;4;4;1339-46

  • Membrane localization of the U2 domain of Protein 4.1B is necessary and sufficient for meningioma growth suppression.

    Robb VA, Gerber MA, Hart-Mahon EK and Gutmann DH

    Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA.

    Meningiomas are common central nervous system tumors; however, the molecular mechanisms underlying their pathogenesis are largely undefined. Previous work has implicated Protein 4.1B as an important tumor suppressor involved in the development of these neoplasms. In this report, we demonstrate that the U2 domain is necessary and sufficient for the ability of Protein 4.1B to function as a meningioma growth suppressor. Using a series of truncation and deletion constructs of DAL-1 (a fragment of Protein 4.1B that retains all the growth suppressive properties), we narrowed the domain required for 4.1B growth suppression to a fragment containing a portion of the FERM domain and the U2 domain using clonogenic assays on meningioma cells. Deletion of the U2 domain in the context of the full-length DAL-1 molecule eliminated growth suppressor function, as measured by thymidine incorporation and caspase-3 activation. Moreover, targeting the U2 domain to the plasma membrane using a membrane localization signal (MLS) reduced cell proliferation, similar to wild-type DAL-1. Collectively, the data suggest that the U2 domain, when properly targeted to the plasma membrane, contains all the residues necessary for mediating Protein 4.1B growth suppression.

    Funded by: NCI NIH HHS: 1-F32-CA-097816-01; NINDS NIH HHS: NS41520

    Oncogene 2005;24;11;1946-57

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • Ankyrin-B targets beta2-spectrin to an intracellular compartment in neonatal cardiomyocytes.

    Mohler PJ, Yoon W and Bennett V

    Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. p.mohler@cellbio.duke.edu

    Ankyrin-B is a spectrin-binding protein that is required for localization of inositol 1,4,5-trisphosphate receptor and ryanodine receptor in neonatal cardiomyocytes. This work addresses the interaction between ankyrin-B and beta(2)-spectrin in these cells. Ankyrin-B and beta(2)-spectrin are colocalized in an intracellular striated compartment overlying the M-line and distinct from T-tubules, sarcoplasmic reticulum, Golgi, endoplasmic reticulum, lysosomes, and endosomes. Beta(2)-Spectrin is absent in ankyrin-B-null cardiomyocytes and is restored to a normal striated pattern by rescue with green fluorescent protein-220-kDa ankyrin-B. We identified two mutants (A1000P and DAR976AAA) located in the ZU5 domain which eliminate spectrin binding activity of ankyrin-B. Ankyrin-B mutants lacking spectrin binding activity are normally targeted but do not reestablish beta(2)-spectrin in ankyrin-B(+/-) cardiomyocytes. However, both mutant forms of ankyrin-B are still capable of restoring inositol 1,4,5-trisphosphate receptor localization and normal contraction frequency of cardiomyocytes. Therefore, direct binding of beta(2)-spectrin to ankyrin-B is required for the normal targeting of beta(2)-spectrin in neonatal cardiomyocytes. In contrast, ankyrin-B localization and function are independent of beta(2)-spectrin. In summary, this work demonstrates that interaction between members of the ankyrin and beta-spectrin families previously established in erythrocytes and axon initial segments also occurs in neonatal cardiomyocytes with ankyrin-B and beta(2)-spectrin. This work also establishes a functional hierarchy in which ankyrin-B determines the localization of beta(2)-spectrin and operates independently of beta(2)-spectrin in its role in organizing membrane-spanning proteins.

    The Journal of biological chemistry 2004;279;38;40185-93

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Analysis of erythrocyte and platelet membrane proteins in various forms of beta-thalassemia.

    Alekperova GA, Orudzhev AG and Javadov SA

    Azerbaijan Medical University, Baku 370022, Azerbaijan.

    Major membrane proteins have been quantitatively analyzed in erythrocytes and platelets from patients with homozygous (splenectomized and non-splenectomized) and heterozygous forms of beta-thalassemia depending on severity of clinical manifestation of this disease. Quantitative analysis of erythrocyte membrane proteins revealed increase in alpha- and beta-spectrin. (In non-splenectomized patients with homozygous beta-thalassemia the amount of this protein was lower than in corresponding controls.) Besides spectrin, the increase of 2.1-2.3 fractions of ankyrin, and the decrease of band 3 protein (anion-transport protein), 4.1, palladin, and glyceraldehyde-3-phosphate dehydrogenase were also found. Analysis of major platelet membrane proteins revealed significant increase in gelsolin. This increase was found in all forms of beta-thalassemia irrespective of gender. Significant changes in platelet membrane protein fractions were found in patients (especially non-splenectomized) with homozygous beta-thalassemia. These included significant decrease in myosin, profilin, and gamma-actin and increase in actin-binding protein in both male and female patients. The content of other protein fractions (alpha-actinin, tubulin, tropomyosin) remained unchanged. Changes in protein fractions of erythrocytes and platelets correlated with severity of clinical manifestation of the disease.

    Biochemistry. Biokhimiia 2004;69;7;748-53

  • Functional proteomics mapping of a human signaling pathway.

    Colland F, Jacq X, Trouplin V, Mougin C, Groizeleau C, Hamburger A, Meil A, Wojcik J, Legrain P and Gauthier JM

    Hybrigenics SA, 75014 Paris, France. fcolland@hybrigenics.fr

    Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

    Genome research 2004;14;7;1324-32

  • Disruption of 14-3-3 binding does not impair Protein 4.1B growth suppression.

    Robb VA, Li W and Gutmann DH

    Department of Neurology, Washington University School of Medicine, Box 8111, 660 S. Euclid Avenue, St Louis, MO 63110, USA.

    Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely unknown. Collaborative studies from our laboratory demonstrated a direct association of 14-3-3 with the meningioma tumor suppressor Protein 4.1B, which was not observed with other members of the Protein 4.1 family, including the NF2 meningioma tumor suppressor, merlin/schwannomin. Given the role of 14-3-3 in the regulation of cell proliferation and apoptosis, we sought to determine the functional significance of 14-3-3 binding to Protein 4.1B growth suppression. Based on comparative binding studies performed with additional members of the Protein 4.1 family, we generated specific missense mutations within the minimal growth suppressor fragment of Protein 4.1B (DAL-1, differentially expressed in adenocarcinoma of the lung). Complementary in vitro GST affinity chromatography and in vivo interaction experiments demonstrated that the F359Y mutation abrogated binding to 14-3-3, but did not impair DAL-1 binding to other known Protein 4.1B interacting proteins. Similar to wild-type DAL-1, the expression of the F359Y DAL-1 14-3-3-binding mutant resulted in reduced Protein 4.1B-deficient IOMM-Lee and CH157-MN meningioma cell line colony formation. Moreover, similar to wild-type DAL-1, the stable expression of the DAL-1 F359Y mutant significantly reduced cell proliferation in independently isolated IOMM-Lee clones, as assessed by thymidine incorporation. Collectively, these results suggest that binding to 14-3-3 is not essential for the growth suppressor function of Protein 4.1B in meningiomas.

    Funded by: NCI NIH HHS: 1-F32-CA-097816-01; NINDS NIH HHS: NS35848, NS41520

    Oncogene 2004;23;20;3589-96

  • A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

    Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B and Superti-Furga G

    Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. tewis.bouwmeester@cellzome.com

    Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

    Nature cell biology 2004;6;2;97-105

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Spectrin alpha II and beta II isoforms interact with high affinity at the tetramerization site.

    Bignone PA and Baines AJ

    Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK. paola.bignone@cancer.org.uk

    Spectrin tetramers form by the interaction of two alpha-beta dimers through two helices close to the C-terminus of a beta subunit and a single helix at the N-terminus of an alpha subunit. Early work on spectrin from solid tissues (typified by alphaII and betaII polypeptides) indicated that it forms a more stable tetramer than erythroid spectrin (alphaI-betaI). In the present study, we have probed the molecular basis of this phenomenon. We have quantified the interactions of N-terminal regions of two human alpha polypeptides (alphaI and alphaII) with the C-terminal regions of three beta isoforms (betaISigma1, betaIISigma1 and betaIISigma2). alphaII binds either betaII form with a much higher affinity than alphaI binds betaISigma1 ( K (d) values of 5-9 nM and 840 nM respectively at 25 degrees C). betaIISigma1 and betaIISigma2 are splice variants with different C-terminal extensions outside the tetramerization site: these extensions affect the rate rather than the affinity of alpha subunit interaction. alphaII spectrin interacts with each beta subunit with higher affinity than alphaI, and the betaII polypeptides have higher affinities for both alpha chains than betaISigma1. The first full repeat of the alpha subunit has a major role in determining affinity. Enthalpy changes in the alphaII-betaIISigma2 interaction are large, but the entropy change is comparatively small. The interaction is substantially reduced, but not eliminated, by concentrated salt solutions. The high affinity and slow overall kinetics of association and dissociation of alphaII-betaII spectrin may suit it well to a role in strengthening cell junctions and providing stable anchor points for transmembrane proteins at points specified by cell-adhesion molecules.

    Funded by: Biotechnology and Biological Sciences Research Council: C18062

    The Biochemical journal 2003;374;Pt 3;613-24

  • Identification of a third Protein 4.1 tumor suppressor, Protein 4.1R, in meningioma pathogenesis.

    Robb VA, Li W, Gascard P, Perry A, Mohandas N and Gutmann DH

    Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely undefined. In this report, we demonstrate that a third Protein 4.1 family member, Protein 4.1R, functions as a meningioma tumor suppressor. We observed loss of Protein 4.1R expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningiomas by immunohistochemistry and fluorescence in situ hybridization. In support of a meningioma tumor suppressor function, Protein 4.1R overexpression resulted in reduced IOMM-Lee and CH157-MN cell proliferation. Similar to the Protein 4.1B and merlin tumor suppressors, Protein 4.1R membrane localization increased significantly under conditions of growth arrest in vitro. Lastly, we show that Protein 4.1R interacted with a subset of merlin/Protein 4.1B interactors including CD44 and betaII-spectrin. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor in the molecular pathogenesis of meningioma.

    Funded by: NCI NIH HHS: F32-CA097816; NINDS NIH HHS: NS35848, NS41520

    Neurobiology of disease 2003;13;3;191-202

  • Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif.

    Tomsig JL, Snyder SL and Creutz CE

    Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.

    We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins.

    Funded by: NIGMS NIH HHS: GM53266

    The Journal of biological chemistry 2003;278;12;10048-54

  • Disruption of transforming growth factor-beta signaling in ELF beta-spectrin-deficient mice.

    Tang Y, Katuri V, Dillner A, Mishra B, Deng CX and Mishra L

    Laboratory of Developmental Biology, Department of Medicine, Georgetown University, Washington, DC 20007, USA.

    Disruption of the adaptor protein ELF, a beta-spectrin, leads to disruption of transforming growth factor-beta (TGF-beta) signaling by Smad proteins in mice. Elf-/- mice exhibit a phenotype similar to smad2+/-/smad3+/- mutant mice of midgestational death due to gastrointestinal, liver, neural, and heart defects. We show that TGF-beta triggers phosphorylation and association of ELF with Smad3 and Smad4, followed by nuclear translocation. ELF deficiency results in mislocalization of Smad3 and Smad4 and loss of the TGF-beta-dependent transcriptional response, which could be rescued by overexpression of the COOH-terminal region of ELF. This study reveals an unexpected molecular link between a major dynamic scaffolding protein and a key signaling pathway.

    Funded by: NIDDK NIH HHS: R01 DK56111, R01 DK58637, R03 DK53861

    Science (New York, N.Y.) 2003;299;5606;574-7

  • Organization of focal adhesion plaques is disrupted by action of the HIV-1 protease.

    Shoeman RL, Hartig R, Hauses C and Traub P

    Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany. rshoeman@zellbio.mpg.de

    Focal adhesion plaques were severely affected in human embryonic fibroblasts permeabilized with digitonin and incubated in buffer containing the human immunodeficiency virus type 1 protease (HIV-1 PR). A mutant HIV-1 PR (3271 HIV-1 PR) had no effect on focal adhesion plaques. Similar effects were seen with cells microinjected with either HIV-1 PR or 3271 HIV-1 PR. Immunoblots of the human embryonic fibroblasts demonstrated that a number of focal adhesion plaque proteins were specifically cleaved by HIV-1 PR. These included fimbrin, focal adhesion plaque kinase (FAK), talin, and, to a lesser extent, filamin, spectrin and fibronectin. Proteins detected by antibodies to beta 4 integrin and alpha 3 integrin were also cleaved by the HIV-1 PR. Control experiments demonstrated that the effect and protein cleavages described are due to action of the HIV-1 PR and not to the action of endogenous host cell proteases.

    Cell biology international 2002;26;6;529-39

  • Binding of the merlin-I product of the neurofibromatosis type 2 tumour suppressor gene to a novel site in beta-fodrin is regulated by association between merlin domains.

    Neill GW and Crompton MR

    Centre for Cutaneous Research, St Bartholomew's and the Royal London, Queen Mary and Westfield College, 2 Newark Street, London E1 2AT, UK.

    The mechanism underlying the tumour-suppressor activity of the neurofibromatosis type 2 (NF2) gene product, merlin, is largely undefined but there is evidence that the biological function of the protein might be mediated partly through interactions with the cytoskeleton. Merlin is expressed predominantly as two isoforms that differ at their C-termini owing to alternative splicing of exon 16. By expressing merlin isoform I as bait in a yeast two-hybrid screen, we isolated a clone encoding a region of the cytoskeletal protein beta-fodrin. Confirmation of the merlin-fodrin interaction was provided by using the mammalian two-hybrid system and binding assays in vitro. In addition, these assays and co-immunoprecipitation from mammalian cells revealed that the binding site for fodrin is located in the C-terminal half of merlin at a site that is masked in the native protein. Co-expression of the N-terminus of merlin decreased the interaction of its C-terminus with fodrin, implicating homophilic interactions of merlin isoform I in masking the fodrin-binding site. The effect of three disease-associated mutations on the merlin-fodrin interaction and merlin dimerization was also investigated. The mutation L535P, but not L360P or K413E, significantly decreased the merlin-fodrin interaction but not dimerization, indicating that the tumour suppressor ability of merlin might reside partly in its ability to interact with the cytoskeleton via fodrin.

    The Biochemical journal 2001;358;Pt 3;727-35

  • A novel isoform of beta-spectrin II localizes to cerebellar Purkinje-cell bodies and interacts with neurofibromatosis type 2 gene product schwannomin.

    Chen Y, Yu P, Lu D, Tagle DA and Cai T

    Department of Physiology, Georgetown University, Washington, DC 20016, USA. ywchen@fjmu.edu.cn

    We report the identification of a full-length novel beta-spectrin II gene (betaSpIIsigma2) in human brain. The betaSpIIsigma2 gene has 32 exons encoding an actin-binding domain, followed by 17-spectrin repeats, and a short COOH-terminal regulatory region that lacks the Pleckstrin homology (PH) domain. Pair-wise sequence analysis showed an additional 36 and 28 amino acids located at the NH2 and COOH-terminal regions of betaSpIIsigma2, respectively. Northern-blot analysis showed an abundant expression of betaSpIIsigma2 transcripts in brain, lung, and kidney. Western-blot analysis confirmed the predicted approximately 225 kD molecular size of betaSpIIsigma2 protein in these same tissues. In brain, immunofluorescent staining revealed that betaSpIIsigma2 was enriched in cerebellar neurons, with specific enrichment in Purkinje cell bodies, but not in dendrites. Of considerable interest, neurofibromatosis type 2 (NF2) gene product schwannomin was found to co-immunoprecipitate with betaSpIIsigma2 in cultured Purkinje cells. These results suggest that betaSpIIsigma2 may play an important role in the assembly of the specialized plasma membrane domain of Purkinje neurons and that schwannomin may be involved in actin-cytoskeleton organization by interacting with betaSpIIsigma2.

    Journal of molecular neuroscience : MN 2001;17;1;59-70

  • The prototypical 4.1R-10-kDa domain and the 4.1g-10-kDa paralog mediate fodrin-actin complex formation.

    Kontrogianni-Konstantopoulos A, Frye CS, Benz EJ and Huang SC

    Department of Medicine, The Johns Hopkins University School of Medicine, University, Baltimore, Maryland 21205, USA.

    A complex family of 4.1R isoforms has been identified in non-erythroid tissues. In this study we characterized the exonic composition of brain 4.1R-10-kDa or spectrin/actin binding (SAB) domain and identified the minimal sequences required to stimulate fodrin/F-actin association. Adult rat brain expresses predominantly 4.1R mRNAs that carry an extended SAB, consisting of the alternative exons 14/15/16 and part of the constitutive exon 17. Exon 16 along with sequences carried by exon 17 is necessary and sufficient to induce formation of fodrin-actin-4.1R ternary complexes. The ability of the respective SAB domains of 4.1 homologs to sediment fodrin/actin was also investigated. 4.1G-SAB stimulates association of fodrin/actin, although with an approximately 2-fold reduced efficiency compared with 4.1R-10-kDa, whereas 4.1N and 4.1B do not. Sequencing of the corresponding domains revealed that 4.1G-SAB carries a cassette that shares significant homology with 4.1R exon 16, whereas the respective sequence is divergent in 4.1N and absent from brain 4.1B. An approximately 150-kDa 4.1R and an approximately 160-kDa 4.1G isoforms are present in PC12 lysates that occur in vivo in a supramolecular complex with fodrin and F-actin. Moreover, proteins 4.1R and 4.1G are distributed underneath the plasma membrane in PC12 cells. Collectively, these observations suggest that brain 4.1R and 4.1G may modulate the membrane mechanical properties of neuronal cells by promoting fodrin/actin association.

    Funded by: NHLBI NIH HHS: HL 44985

    The Journal of biological chemistry 2001;276;23;20679-87

  • alpha -Catenin binds directly to spectrin and facilitates spectrin-membrane assembly in vivo.

    Pradhan D, Lombardo CR, Roe S, Rimm DL and Morrow JS

    Department of Pathology, Yale University, New Haven, Connecticut 06510, USA.

    The anchorage of spectrin to biological membranes is mediated by protein and phosphoinositol phospholipid interactions. In epithelial cells, a nascent spectrin skeleton assembles in regions of cadherin-mediated cell-cell contact, and conversely, cytoskeletal assembly is required to complete the cell-adhesion process. The molecular interactions guiding these processes remain incompletely understood. We have examined the interaction of spectrin with alpha-catenin, a component of the adhesion complex. Spectrin (alphaIIbetaII) and alpha-catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and alpha-catenin in vitro. By surface plasmon resonance and in vitro binding assays, we find that alpha-catenin binds alphaIIbetaII spectrin with an apparent K(d) of approximately 20-100 nm. By gel-overlay assay, alpha-catenin binds recombinant betaII-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of alpha-catenin is fully accounted for in recombinant peptides encompassing the NH(2)-terminal 228 amino acid region of alpha-catenin. An in vivo role for the interaction of spectrin with alpha-catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective alpha-catenin. Transfection of these cells with wild-type alpha-catenin reestablishes alpha-catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between alpha-catenin and the amino-terminal domain of beta-spectrin augment the interaction between alpha-catenin and actin, and together they provide a polyvalent linkage directing the topographic assembly of a nascent spectrin-actin skeleton to membrane regions enriched in E-cadherin.

    The Journal of biological chemistry 2001;276;6;4175-81

  • Identification of a novel C-terminal variant of beta II spectrin: two isoforms of beta II spectrin have distinct intracellular locations and activities.

    Hayes NV, Scott C, Heerkens E, Ohanian V, Maggs AM, Pinder JC, Kordeli E and Baines AJ

    Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, England.

    It is established that variations in the structure and activities of betaI spectrin are mediated by differential mRNA splicing. The two betaI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of betaII spectrins? Thus far, only a long form of betaII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of betaII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7. 0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of betaII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of betaII spectrin isoforms correlates with their differential intracellular targeting.

    Journal of cell science 2000;113 ( Pt 11);2023-34

  • Interaction of the C-terminal domain of delta glutamate receptor with spectrin in the dendritic spines of cultured Purkinje cells.

    Hirai H and Matsuda S

    Laboratory for Memory and Learning, RIKEN Brain Science Institute, Wako, Saitama, Japan. hirai@postman.riken.go.jp

    The interaction of neurotransmitter receptors with the underlying cytoskeleton via subsynaptic proteins is an important mechanism for the targeting of the receptors to synapses in the central nervous system. We show that delta glutamate receptors (delta receptors), expressed predominantly in the dendritic spines of cerebellar Purkinje cells, directly interact with spectrin, a member of the actin-binding family of proteins. Moreover, the interaction between spectrin and C-terminal domain of the delta receptor is 50% inhibited by 1 microM of Ca2+ in vitro, compared with that in the absence of Ca2+. These results suggest that delta receptors on the postsynaptic membrane of the dendritic spines of cerebellar Purkinje cells are anchored to the actin cytoskeleton via spectrin, and that Ca2+ elevation in the dendritic spines causes delta receptor declustering by dissociation of the receptors from spectrin. This mechanism for receptor anchoring at postsynaptic sites may regulate synaptogenesis and/or synaptic plasticity.

    Neuroscience research 1999;34;4;281-7

  • Calpain-induced proteolysis of beta-spectrins.

    Löfvenberg L and Backman L

    Department of Biochemistry, Umeå University, Sweden.

    The calcium-activated neutral protease calpain is activated in several pathological conditions. Calpain usually hydrolyses one or only a few peptide bonds in its substrate. One prominent substrate for calpain is spectrin and it has been shown that alpha-spectrin is the preferred substrate. We now show that the beta-chain of spectrin is also a substrate for calpain proteolysis, and that the cleavage site in each beta-subunit is located at the very C-terminal part of the molecule. Surprisingly, beta1sigma-spectrin is cleaved at a different site than betaIsigma2- and betaIIsigma1-spectrins despite their high degree of sequence identity.

    FEBS letters 1999;443;2;89-92

  • Structural comparisons of calponin homology domains: implications for actin binding.

    Bañuelos S, Saraste M and Djinović Carugo K

    European Molecular Biology Laboratory Postfach 10.2209 D-69012 Heidelberg, Germany.

    Background: The actin-binding site of several cytoskeletal proteins is comprised of two calponin homology (CH) domains in a tandem arrangement. As a single copy, the CH domain is also found in regulatory proteins in muscle and in signal-transduction proteins. The three-dimensional structures of three CH domains are known, but they have not yet clarified the molecular details of the interaction between actin filaments and proteins harbouring CH domains.

    Results: We have compared the crystal structure of a CH domain from beta-spectrin, which has been refined to 1.1 A resolution, with the two CH domains that constitute the actin-binding region of fimbrin. This analysis has allowed the construction of a structure-based sequence alignment of CH domains that can be used in further comparisons of members of the CH domain family. The study has also improved our understanding of the factors that determine domain architecture, and has led to discussion on the functional differences that seem to exist between subfamilies of CH domains, as regards binding to F-actin.

    Conclusions: Our analysis supports biochemical data that implicate a surface centered at the last helix of the N-terminal CH domain as the most probable actin-binding site in cytoskeletal proteins. It is not clear whether the C-terminal domains of the tandem arrangement or the single CH domains have this function alone. This may imply that although the CH domains are homologous and have a conserved structure, they may have evolved to perform different functions.

    Structure (London, England : 1993) 1998;6;11;1419-31

  • Brain beta-spectrin phosphorylation: phosphate analysis and identification of threonine-347 as a heparin-sensitive protein kinase phosphorylation site.

    Sihag RK

    Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4062, USA.

    Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain beta-spectrin yielded a stoichiometry of 3.2 +/- 0.18 mol of PO4/mol of beta-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO4/mol of spectrin heterodimer, respectively. The 32P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [32P]orthophosphate showed phosphorylation of only beta-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on beta-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated beta-spectrin showed that [32P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an alpha-chymotryptic 32P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338-348 on the beta1 repeat of beta-spectrinG (betaSPIIa) gene. These data suggest that phosphorylation of Thr347, which is localized on the presumptive synapsin I binding domain of beta-spectrinG, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.

    Journal of neurochemistry 1998;71;5;2220-8

  • Neurofibromatosis 2 tumour suppressor schwannomin interacts with betaII-spectrin.

    Scoles DR, Huynh DP, Morcos PA, Coulsell ER, Robinson NG, Tamanoi F and Pulst SM

    Division of Neurology, CSMC Burns and Allen Research Institute, Cedars-Sinai Medical Center, University of California School of Medicine at Los Angeles, 90048, USA.

    NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein. Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations. The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types.

    Funded by: NINDS NIH HHS: NS01428-01A1, NS10524-01

    Nature genetics 1998;18;4;354-9

  • Crystal structure of a calponin homology domain.

    Djinovic Carugo K, Bañuelos S and Saraste M

    The three-dimensional structure of the calponin homology domain present in many actin binding cytoskeletal and signal-transducing proteins has been determined at 2.0 A resolution.

    Nature structural biology 1997;4;3;175-9

  • Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.

    Holleran EA, Tokito MK, Karki S and Holzbaur EL

    Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia 19104, USA.

    Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.

    Funded by: NIGMS NIH HHS: GM48661

    The Journal of cell biology 1996;135;6 Pt 2;1815-29

  • Calcium-dependent peripheral localization of 4.1-like proteins and fodrin in cultured human keratinocytes.

    Shimizu T, Takakuwa Y, Koizumi H, Ishibashi T and Ohkawara A

    Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan.

    Recently, several proteins immunologically related to erythrocyte membrane skeletal proteins, such as protein 4.1 and fodrin (non-erythroid spectrin), have been found in keratinocytes. In the present study, in order to investigate the roles of these proteins in cell-cell contact, we analyzed the distribution of non-erythroid protein 4.1, beta-fodrin and actin in cultured human keratinocytes at low (0.15 mM) and standard (1.85 mM) Ca2+ concentrations. Immunofluorescence microscopy revealed that immunoreactive forms of protein 4.1, beta-fodrin and actin filaments were present in the cytoplasm of cells cultured in low Ca2+ medium, while in cells in the standard Ca2+ medium, these proteins were localized at the cell boundary and partially in the cytoplasm. When cells in the low-Ca2+ medium were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 1 h, these proteins were also present at the cell boundary. Increasing extracellular Ca2+ concentration from low to standard in the medium induces cell-cell contact among the cultured human keratinocytes, accompanied by the translocation of protein 4.1 and beta-fodrin from the cytoplasm to the membrane. On the basis of the present study, movement of membrane skeletal proteins from the cytosol to the membrane suggests that either these proteins or the membrane skeletal lattice plays an important role in the regulation of cell-cell intergigitations in response to changes in the Ca2+ concentrations in culture medium, and that phosphorylation of these skeletal proteins might be involved in the regulation of the membrane skeletal proteins of keratinocytes in response to Ca2+.

    Biology of the cell 1996;86;1;19-26

  • Cloning of a portion of the chromosomal gene and cDNA for human beta-fodrin, the nonerythroid form of beta-spectrin.

    Chang JG, Scarpa A, Eddy RL, Byers MG, Harris AS, Morrow JS, Watkins P, Shows TB and Forget BG

    Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.

    A 96-bp synthetic oligonucleotide corresponding to an amino acid sequence near the N-terminus of erythroid beta-spectrin was used to screen a human genomic library, and two overlapping recombinants were isolated. DNA sequence analysis established that the genomic fragment encoded beta-fodrin, the nonerythroid form of beta-spectrin, by correlation to a known amino acid sequence of human brain beta-fodrin. The genomic DNA contained regions that cross-hybridized with an erythroid beta-spectrin cDNA probe, and the DNA sequence of these regions revealed a high degree of identity with that of erythroid beta-spectrin and a similar exon/intron organization. A single-copy DNA fragment of the beta-fodrin genomic clone was used to screen a lymphoid cell cDNA library and two recombinants were isolated. The composite DNA sequence of these various genomic and cDNA clones encoded almost all of the first twelve 106 amino acid repeat segments of beta-fodrin that shared 58% identity and 75.5% similarity with the amino acid sequence of beta-spectrin and 66% identity with the nucleotide sequence of beta-spectrin cDNA. The chromosomal localization of the gene was determined to be chromosome 2 by hybridization of a single-copy probe derived from the cloned genomic DNA to DNA of a panel of somatic hybrid cell lines, and in situ hybridization localized the gene to band 2p21. beta-Fodrin was assigned the gene symbol SPTBN1.

    Genomics 1993;17;2;287-93

  • Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

    Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH and Bar-Sagi D

    Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France.

    A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

    Funded by: NCI NIH HHS: CA46370, CA55360

    Science (New York, N.Y.) 1993;260;5112;1338-43

  • Characterization of human brain cDNA encoding the general isoform of beta-spectrin.

    Hu RJ, Watanabe M and Bennett V

    Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.

    The complete primary structure of the general form of human beta-spectrin (beta G) has been deduced from cDNAs isolated from human brain. beta G-Spectrin is encoded by a gene located on human chromosome 2. beta G-Spectrin and erythrocyte beta-spectrin (beta R) share identical domain organization, with sequence identity of 60% and sequence similarity of 77%. beta-Spectrins have closely related N-terminal domains implicated in binding to actin, and 17 copies of a 106-residue repeat motif with consensus residues that are highly conserved between beta-spectrins as well as alpha-spectrins. C-terminal domains of beta G and the 270-kDa beta R-spectrins are candidate regions to associate with alpha-spectrin, and exhibit 75% similarity. beta G- and beta R-spectrins exhibit different patterns of expression in tissues and follow different developmental programs in those tissues where they are co-expressed. beta G-Spectrin is present in all tissues examined except for erythrocytes, while beta R-spectrin could be detected only in erythrocytes, brain, and heart. beta G- and beta R-Spectrins are both expressed in brain, but beta R appeared later in post-natal development and was highly enriched in cerebellum in contrast to the broad regional distribution of beta G-spectrin. beta-Spectrins are likely to perform related but distinct functions, with beta G in a general, constitutive role and beta R-spectrin involved in more specialized activities of differentiated cells.

    The Journal of biological chemistry 1992;267;26;18715-22

  • Presence of erythroid and nonerythroid spectrin transcripts in human lens and cerebellum.

    Yoon SH, Skalka H and Prchal JT

    Department of Ophthalmology, University of Alabama, Birmingham.

    Spectrin is a major protein of the red cell membrane, and is composed of alpha- and beta-subunits. While spectrin was initially thought to be specific for erythrocytes, similar, but nonidentical peptides have recently been identified in other tissues, including lens, suggesting the existence of a spectrin gene family. To study the nature of spectrin-like peptides in the lens, we examined the transcription of erythroid and nonerythroid spectrin in human lens and cerebellum by direct hybridization with known human alpha- and beta-spectrin cDNA (erythroid probes), as well as with a human alpha-fodrin cDNA (nonerythroid probe). Northern blots of poly(A)+ RNA from erythroid, as well as several nonerythroid cells, and the total RNA from lens, showed that the beta-spectrin cDNA probe hybridized to two distinct bands of 8.6 and 7.4 kb mRNA in human lens, and was present in abundance. The erythroid beta-spectrin 11 kb mRNA transcript was also found in the human cerebellum. The beta-spectrin transcripts from these nonerythroid tissues were found to have different sizes compared to the major erythroid 7.8 kb beta-spectrin mRNA. Human lens contained a 7.5 kb transcript of the erythroid alpha-spectrin subunit. In addition, the human lens was found to have a 7.2 kb alpha-fodrin transcript; while the cerebellum had an 8.5 kb alpha-fodrin mRNA, the same size as that found in other nonerythroid tissues. The abundance of erythroid and nonerythroid spectrin transcripts in lens was significantly greater than in any other tissues examined. These data show that the human lens has abundant erythroid alpha- and beta-spectrin transcripts, as well as a unique alpha-fodrin transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NHLBI NIH HHS: HL-37462

    Investigative ophthalmology & visual science 1989;30;8;1860-6

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
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
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
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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