G2Cdb::Gene report

Gene id
G00001793
Gene symbol
ACTN4 (HGNC)
Species
Homo sapiens
Description
actinin, alpha 4
Orthologue
G00000544 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000070478 (Vega human gene)
Gene
ENSG00000130402 (Ensembl human gene)
81 (Entrez Gene)
170 (G2Cdb plasticity & disease)
ACTN4 (GeneCards)
Literature
604638 (OMIM)
Marker Symbol
HGNC:166 (HGNC)
Protein Expression
1873 (human protein atlas)
Protein Sequence
O43707 (UniProt)

Diseases (2)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000267: Focal segmental glomerulosclerosis N Y (16251236) Single nucleotide polymorphism (SNP) Y
D00000267: Focal segmental glomerulosclerosis N Y (16251236) Single nucleotide deletion (SND) Y
D00000269: Congenital nephrotic syndrome N Y (15780077) No mutation found (N) N
D00000267: Focal segmental glomerulosclerosis N Y (15627790) Single nucleotide polymorphism (SNP) N

References

  • Mutational and Biological Analysis of alpha-actinin-4 in focal segmental glomerulosclerosis.

    Weins A, Kenlan P, Herbert S, Le TC, Villegas I, Kaplan BS, Appel GB and Pollak MR

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

    Mutations in the alpha-actinin-4 gene (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS). A mutational analysis was performed of ACTN4 in DNA from probands with a family history of FSGS as well as in individuals with nonfamilial FSGS. The possible contribution of noncoding variation in ACTN4 to the development of FSGS also was assessed. Multiple nucleotide variants were identified in coding and noncoding sequence. The segregation of nonsynonymous coding sequence variants was examined in the relevant families. Only a small number of nucleotide changes that seemed likely to be causing (or contributing to) disease were identified. Sequence changes that predicted I149del, W59R, V801M, R348Q, R837Q, and R310Q changes were identified. For studying their biologic relevance and their potential roles in the pathogenesis of FSGS, these variants were expressed as GFP-fusion proteins in cultured podocytes. F-actin binding assays also were performed. Three of these variants (W59R, I149del, and V801M) showed clear cellular mislocalization in the form of aggregates adjacent to the nucleus. Two of these mislocalized variants (W59R and I149del) also showed an increased actin-binding activity. The I149del mutation segregated with disease; W59R was found to be a de novo mutation in the proband. A total of five ACTN4 mutations that are believed to be disease causing (three reported previously and two novel) as well as a number of variants with unclear contribution to disease now have been identified. The possibility that some of these other variants increase the susceptibility to FSGS cannot be excluded. ACTN4 mutations seem to account for approximately 4% of familial FSGS.

    Funded by: NIDDK NIH HHS: DK54931, DK59588

    Journal of the American Society of Nephrology : JASN 2005;16;12;3694-701

  • Analysis of NPHS1, NPHS2, ACTN4, and WT1 in Japanese patients with congenital nephrotic syndrome.

    Sako M, Nakanishi K, Obana M, Yata N, Hoshii S, Takahashi S, Wada N, Takahashi Y, Kaku Y, Satomura K, Ikeda M, Honda M, Iijima K and Yoshikawa N

    Department of Pediatrics, Wakayama Medical University, Wakayama City, Wakayma, Japan.

    Background: Congenital nephrotic syndrome (CNS) causes significant renal failure, and is classified into two types: (1) Finnish type; and (2) other, including diffuse mesangial sclerosis. Mutations of NPHS1 and NPHS2, which encode the slit diaphragm components nephrin and podocin, cause CNS and autosomal-recessive familial steroid-resistant nephrotic syndrome, respectively. Most patients with Finnish-type CNS in Europe and the United States have NPHS1 mutations. However, NPHS2 mutations have been detected in some cases. Mutations in ACTN4, encoding alpha-actinin-4, cause an autosomal-dominant focal segmental glomerulosclerosis. alpha-actinin-4 stabilizes the podocyte cytoskeleton structure, connecting with actin filaments. WT1 mutations, causing Wilm's tumor, have been demonstrated in some CNS patients with diffuse mesangial sclerosis. Systematic investigation of genes for CNS in Japan has never been performed.

    Methods: To clarify the role of mutations in these four genes, we used polymerase chain reaction (PCR) and direct sequencing to investigate all exons and exon-intron boundaries for these genes in 13 unrelated CNS patients from regional pediatric kidney disease centers in Japan.

    Results: A novel homozygous nonsense mutation of NPHS1, E246X in exon 7, and a novel homozygous deletion mutation of NPHS1, 2156_2163del in exon 16 were detected in one patient each. A novel homozygous nonsense mutation of NPHS2, R196X in exon 5, was found in one patient, and the same heterozygous nonsense mutation was detected in another. No ACTN4 or WT1 mutations were detected.

    Conclusion: These studies demonstrate that mutation of NPHS1 is not a major cause of CNS in Japanese patients, and that mutation of NPHS2 can be responsible for CNS in this population.

    Kidney international 2005;67;4;1248-55

  • Molecular analysis of NPHS2 and ACTN4 genes in a series of 33 Italian patients affected by adult-onset nonfamilial focal segmental glomerulosclerosis.

    Aucella F, De Bonis P, Gatta G, Muscarella LA, Vigilante M, di Giorgio G, D'Errico M, Zelante L, Stallone C and Bisceglia L

    Department of Nephrology and Dialysis, Casa Sollievo della Sofferenza Hospital, IRCCS, San Giovanni Rotondo, Italy.

    Background: Mutations in the NPHS2 gene, encoding podocin, and in the ACTN4 gene, encoding alpha-actinin-4, have been identified in familial childhood-onset forms of focal and segmental glomerulosclerosis (FSGS). NPHS2 may be also responsible for some sporadic cases. The role of NPHS2 and ACTN4 in the adult sporadic form of the disease is being clarifying.

    Methods: Thirty-three adult subjects affected by sporadic FSGS were studied at molecular level. At biopsy, 12 patients had nephrotic syndrome, 5 patients had isolated proteinuria and 16 patients showed proteinuria and hematuria. Glomerular filtration rate (GFR) was in the normal range in 19 subjects and 14 patients had a variable degree of renal failure. Multiplex families presenting with a clear familial inheritance for proteinuria or other congenital nephrotic syndrome were excluded. The whole coding region, all intron/exon boundaries and flanking intronic regions of NPHS2 gene and the exon 8, i.e. hot-spot mutations of the ACTN4 gene, were analyzed in all patients by denaturing high-performance liquid chromatography (DHPLC) to search disease-causing defects.

    Results: The analysis identified four already described and two new polymorphisms, IVS3-21C>T and IVS3-46C>T, on the NPHS2 gene. Moreover, the R229Q allele was identified in 3/33 patients and in 7/124 controls, accounting for an allelic frequency of 0.045 and 0.028, respectively. The new intronic polymorphism IVS7-54C>T was also found in the exon 8 of the ACTN4 gene.

    Conclusions: In this study, we exhaustively analyzed the NPHS2 and the exon 8 of the ACTN4 genes in a series of sporadic 'adult-onset' FSGS patients. No causative mutations were found while the R229Q allele was identified in 3 patients confirming its possible role as a 'disease-associated NPHS2 allele' although its pathogenetic involvement needs to be further clarified. Moreover, the description of new intronic polymorphisms in both genes is reported.

    Nephron. Clinical practice 2005;99;2;c31-6

Literature (73)

Pubmed - human_disease

  • Molecular analysis of NPHS2 and ACTN4 genes in a series of 33 Italian patients affected by adult-onset nonfamilial focal segmental glomerulosclerosis.

    Aucella F, De Bonis P, Gatta G, Muscarella LA, Vigilante M, di Giorgio G, D'Errico M, Zelante L, Stallone C and Bisceglia L

    Department of Nephrology and Dialysis, Casa Sollievo della Sofferenza Hospital, IRCCS, San Giovanni Rotondo, Italy.

    Background: Mutations in the NPHS2 gene, encoding podocin, and in the ACTN4 gene, encoding alpha-actinin-4, have been identified in familial childhood-onset forms of focal and segmental glomerulosclerosis (FSGS). NPHS2 may be also responsible for some sporadic cases. The role of NPHS2 and ACTN4 in the adult sporadic form of the disease is being clarifying.

    Methods: Thirty-three adult subjects affected by sporadic FSGS were studied at molecular level. At biopsy, 12 patients had nephrotic syndrome, 5 patients had isolated proteinuria and 16 patients showed proteinuria and hematuria. Glomerular filtration rate (GFR) was in the normal range in 19 subjects and 14 patients had a variable degree of renal failure. Multiplex families presenting with a clear familial inheritance for proteinuria or other congenital nephrotic syndrome were excluded. The whole coding region, all intron/exon boundaries and flanking intronic regions of NPHS2 gene and the exon 8, i.e. hot-spot mutations of the ACTN4 gene, were analyzed in all patients by denaturing high-performance liquid chromatography (DHPLC) to search disease-causing defects.

    Results: The analysis identified four already described and two new polymorphisms, IVS3-21C>T and IVS3-46C>T, on the NPHS2 gene. Moreover, the R229Q allele was identified in 3/33 patients and in 7/124 controls, accounting for an allelic frequency of 0.045 and 0.028, respectively. The new intronic polymorphism IVS7-54C>T was also found in the exon 8 of the ACTN4 gene.

    Conclusions: In this study, we exhaustively analyzed the NPHS2 and the exon 8 of the ACTN4 genes in a series of sporadic 'adult-onset' FSGS patients. No causative mutations were found while the R229Q allele was identified in 3 patients confirming its possible role as a 'disease-associated NPHS2 allele' although its pathogenetic involvement needs to be further clarified. Moreover, the description of new intronic polymorphisms in both genes is reported.

    Nephron. Clinical practice 2005;99;2;c31-6

Pubmed - other

  • Phosphorylation of alpha-actinin 4 upon epidermal growth factor exposure regulates its interaction with actin.

    Shao H, Wu C and Wells A

    Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    The ubiquitously expressed family of alpha-actinins bridges actin filaments to stabilize adhesions, a process disrupted during growth factor-induced migration of cells. During the dissolution of the actin cytoskeleton, actinins are phosphorylated on tyrosines, although the consequences of this are unknown. We expressed the two isoforms of human alpha-actinin in murine fibroblasts that express human epidermal growth factor receptor (EGFR) and found that both alpha-actinin 1 (ACTN1) and alpha-actinin 4 (ACTN4) were phosphorylated on tyrosine residues after stimulation with EGF, although ACTN4 was phosphorylated to the greater extent. This required the activation of Src protein-tyrosine kinase and p38-MAPK (and phosphoinositide trisphosphate kinase in part) but not MEK/ERK or Rac1, as determined by inhibitors. The EGF-induced phosphorylation sites of ACTN4 were mapped to tyrosine 4, the major site, and tyrosine 31, the minor one. Truncation mutagenesis showed that the C-terminal domains of ACTN4 (amino acids 300-911), which cross-link the actin binding head domains, act as an inhibitory domain for both actin binding and EGF-mediated phosphorylation. These two properties were mutually exclusive; removal of the C terminus enhanced actin binding of ACTN4 mutants while limiting EGF-induced phosphorylation, and conversely EGF-stimulated phosphorylation of ACTN4 decreased its affinity to actin. Interestingly, a phosphomimetic of tyrosine 265 (which can be found in carcinoma cells and lies near the K255E mutation that causes focal segmental glomerulosclerosis) demonstrated increased actin binding activity and susceptibility of ACTN4 to calpain-mediated cleavage; this variant also retarded cell spreading. Remarkably, either treatment of cells with low concentrations of latrunculin A, which has been shown to depolymerize F-actin, or the deletion of the actin binding domain (100-252 amino acids) of ACTN4Y265E restored EGF-induced phosphorylation. An F-actin binding assay in vitro showed that Y4E/Y31E, a mimetic of diphosphorylated ACTN4, bound F-actin slightly compared with wild type (WT). Importantly, the EGF-mediated phosphorylation of ACTN4 at tyrosine 4 and 31 significantly inhibited multinucleation of proliferating NR6WT fibroblasts that overexpress ACTN4. These results suggest that EGF regulates the actin binding activity of ACTN4 by inducing tyrosyl-directed phosphorylation.

    Funded by: NIDDK NIH HHS: R01 DK054639, R01 DK054639-13; NIGMS NIH HHS: R01 GM065188, R01 GM065188-08, R01 GM069668

    The Journal of biological chemistry 2010;285;4;2591-600

  • Isoform-specific contributions of alpha-actinin to glioma cell mechanobiology.

    Sen S, Dong M and Kumar S

    Department of Bioengineering, University of California, Berkeley, California, USA.

    Glioblastoma Multiforme (GBM) is a malignant astrocytic tumor associated with low survival rates because of aggressive infiltration of tumor cells into the brain parenchyma. Expression of the actin binding protein alpha-actinin has been strongly correlated with the invasive phenotype of GBM in vivo. To probe the cellular basis of this correlation, we have suppressed expression of the nonmuscle isoforms alpha-actinin-1 and alpha-actinin-4 and examined the contribution of each isoform to the structure, mechanics, and motility of human glioma tumor cells in culture. While subcellular localization of each isoform is distinct, suppression of either isoform yields a phenotype that includes dramatically reduced motility, compensatory upregulation and redistribution of vinculin, reduced cortical elasticity, and reduced ability to adapt to changes in the elasticity of the extracellular matrix (ECM). Mechanistic studies reveal a relationship between alpha-actinin and non-muscle myosin II in which depletion of either alpha-actinin isoform reduces myosin expression and maximal cell-ECM tractional forces. Our results demonstrate that both alpha-actinin-1 and alpha-actinin-4 make critical and distinct contributions to cytoskeletal organization, rigidity-sensing, and motility of glioma cells, thereby yielding mechanistic insight into the observed correlation between alpha-actinin expression and GBM invasiveness in vivo.

    Funded by: NIH HHS: 1DP2OD004213, DP2 OD004213

    PloS one 2009;4;12;e8427

  • Actinin-4 expression in primary and metastasized pancreatic ductal adenocarcinoma.

    Welsch T, Keleg S, Bergmann F, Bauer S, Hinz U and Schmidt J

    Department of General, Visceral, and Transplant Surgery, University of Heidelberg, Heidelberg, Germany. thilo.welsch@med.uni-heidelberg.de

    Objectives: Actinin-4 is an actin-bundling protein that probably has a tumor-promoting potential in several solid tumors. The present study analyzed the expression of actinin-4 in the pancreas, in localized and metastasized pancreatic ductal adenocarcinoma (PDAC), and the correlation with clinical outcome.

    Methods: Pancreatic ductal adenocarcinoma tissue from 38 patients, 15 lymph node and 10 liver metastases, normal pancreas, and 4 PDAC cell lines, were examined by immunohistochemistry, and actinin-4 expression was quantified by immunofluorescence analysis.

    Results: In the normal pancreas, actinin-4 was most prominently expressed in ductal cells. In PDAC, tumor cells exhibited strong but differential cytoplasmic immunoreactivity for actinin-4. A multivariate analysis revealed actinin-4 immunoreactivity, advanced age, and undifferentiated grade as significant prognostic factors associated with worse survival after PDAC resection. Cells metastasized to lymph nodes or to the liver exhibited no significant increase of actinin-4 compared with the primary tumors. A nuclear staining was observed neither in any of the PDAC samples nor in the 4 cell lines. In PDAC cells, actinin-4 localized to dynamic actin structures and to invadopodia.

    Conclusions: Actinin-4 expression levels significantly correlate with worse survival after PDAC resection. Although actinin-4 has been reported to promote lymph node metastases, there was no enhanced expression in PDAC metastases.

    Pancreas 2009;38;8;968-76

  • Shifted Transversal Design smart-pooling for high coverage interactome mapping.

    Xin X, Rual JF, Hirozane-Kishikawa T, Hill DE, Vidal M, Boone C and Thierry-Mieg N

    Banting and Best Department of Medical Research and Department of Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

    "Smart-pooling," in which test reagents are multiplexed in a highly redundant manner, is a promising strategy for achieving high efficiency, sensitivity, and specificity in systems-level projects. However, previous applications relied on low redundancy designs that do not leverage the full potential of smart-pooling, and more powerful theoretical constructions, such as the Shifted Transversal Design (STD), lack experimental validation. Here we evaluate STD smart-pooling in yeast two-hybrid (Y2H) interactome mapping. We employed two STD designs and two established methods to perform ORFeome-wide Y2H screens with 12 baits. We found that STD pooling achieves similar levels of sensitivity and specificity as one-on-one array-based Y2H, while the costs and workloads are divided by three. The screening-sequencing approach is the most cost- and labor-efficient, yet STD identifies about twofold more interactions. Screening-sequencing remains an appropriate method for quickly producing low-coverage interactomes, while STD pooling appears as the method of choice for obtaining maps with higher coverage.

    Funded by: NHGRI NIH HHS: R01 HG001715, R01-HG001715

    Genome research 2009;19;7;1262-9

  • Patients with ACTN4 mutations demonstrate distinctive features of glomerular injury.

    Henderson JM, Alexander MP and Pollak MR

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

    Mutations in ACTN4, the gene encoding the actin-binding protein alpha-actinin-4, are a cause of familial FSGS. We examined kidney biopsies from patients with ACTN4 mutations to characterize systematically the histopathology of kidney damage in these patients and to determine whether distinctive morphologic changes are associated with mutations in this gene. The changes observed with light microscopy were typical of FSGS and were morphologically heterogeneous, similar to other inherited podocytopathies. The ultrastructural characteristics, however, were distinctive: Most notably, the presence of cytoplasmic electron-dense aggregates in podocytes. Indirect immunofluorescence using antibodies to a conserved domain of alpha-actinin-4 (present in both wild-type and mutant proteins) revealed a segmental and irregular granular staining pattern in the capillary walls of preserved glomeruli of ACTN4 mutants, whereas preserved glomeruli of patients with other podocyte diseases retained a global linear staining pattern for alpha-actinin-4. These characteristics resemble features observed in mouse models of this disease and may aid in the identification of patients and families who harbor ACTN4 mutations.

    Funded by: NIDDK NIH HHS: K08 DK073091, K08DK073091, R01 DK054931, R01 DK054931-11, R01DK054931, R37 DK059588

    Journal of the American Society of Nephrology : JASN 2009;20;5;961-8

  • Actinin-4 gene amplification in ovarian cancer: a candidate oncogene associated with poor patient prognosis and tumor chemoresistance.

    Yamamoto S, Tsuda H, Honda K, Onozato K, Takano M, Tamai S, Imoto I, Inazawa J, Yamada T and Matsubara O

    Department of Basic Pathology, National Defense Medical College, Tokorozawa, Japan.

    Actinin-4, an isoform of non-muscular alpha-actinin, enhances cell motility by bundling the actin cytoskeleton. We previously reported a prognostic implication of high immunohistochemical expression of actinin-4 protein in ovarian cancers. Chromosomal gain or amplification of the 19q12-q13 region has been reported in ovarian cancer. We hypothesized that the actinin-4 (ACTN4) gene might be a target of the 19q12-q13 amplicon and play an essential role of ovarian cancer progression. In total, 136 advanced-stage ovarian cancers were investigated for the copy number of the ACTN4 gene on chromosome 19q13, using fluorescence in situ hybridization, and the correlation of the ACTN4 copy number with actinin-4 protein immunoreactivity and major clinicopathological factors was investigated. A higher copy number (> or =4 copies) of the ACTN4 gene was detected in 29 (21%) cases and was highly associated with the intensity of actinin-4 immunoreactivity (P<0.0001), a high histological tumor grade (P=0.030), a clear-cell adenocarcinoma histology (P=0.012), resistance to first-line chemotherapies (P=0.028), and poor patient outcome (P=0.0011). Univariate analyses using the Cox regression model showed that a higher ACTN4 gene copy number was able to predict patient outcome more accurately than high actinin-4 immunoreactivity (relative risk: 2.48 vs 1.55). Multivariate analysis showed that a higher copy number of the ACTN4 gene and the degree of residual disease were independent prognostic factors for overall patient survival. The actinin-4 gene may be a target of the 19q amplicon, acting as a candidate oncogene, and serve as a predictor of poor outcome and tumor chemoresistance in patients with advanced-stage ovarian cancers.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2009;22;4;499-507

  • ACTN4 gene mutations and single nucleotide polymorphisms in idiopathic focal segmental glomerulosclerosis.

    Dai S, Wang Z, Pan X, Chen X, Wang W, Ren H, Feng Q, He JC, Han B and Chen N

    Department of Nephrology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.

    Aim: To investigate the association between mutations or single nucleotide polymorphisms (SNPs) of the gene ACTN4 in Chinese patients with idiopathic focal segmental glomerulosclerosis (FSGS).

    Genomic DNA of 82 Chinese idiopathic FSGS patients and 70 healthy people were used to analyze ACTN4 gene mutations by polymerase chain reaction, direct sequencing and GenBank matching. Hair follicle DNA of novel mutated patients' parents were sequenced and alpha-actinin-4 expression in patients' kidney was examined by immunofluorescence. For SNPs, after the Hardy-Weinberg equilibrium test, allele association and the frequencies of genotypes were analyzed, followed by association analysis between genotypes and clinical diagnosis.

    Results: We found a heterozygous candidate mutation 184T>A (S62T) in 1 patient and a 5' UTR candidate mutation 1-34C>T in another patient. Both patients had non-nephrotic syndrome FSGS with reduced kidney alpha-actinin-4 expression. Promoter activity analysis suggests that the 1-34C>T candidate mutation may affect the transcriptional regulation of ACTN4 gene. Additionally, 6 novel silent variants and 2 novel SNPs were also found in this study. Novel SNP 484 + 87C>G had a significant association with the level of urine protein excretion in these idiopathic FSGS patients.

    Conclusions: Our data suggest that mutations and SNP of ACTN4 gene may contribute to be associated with Chinese idiopathic FSGS.

    Nephron. Clinical practice 2009;111;2;c87-94

  • The R229Q mutation in NPHS2 may predispose to proteinuria in thin-basement-membrane nephropathy.

    Tonna S, Wang YY, Wilson D, Rigby L, Tabone T, Cotton R and Savige J

    Department of Medicine, The Northern Hospital, The University of Melbourne, Epping, VIC 3076, Australia.

    Thin-basement-membrane nephropathy (TBMN) is characterized by persistent dysmorphic hematuria, and the presence of proteinuria is a risk factor for renal impairment. TBMN is often due to mutations in the COL4A3 and COL4A4 genes, and this study determined whether additional mutations in genes encoding other structures in the glomerular filtration barrier contributed to the development of proteinuria. Fifty-six unrelated individuals with TBMN including 18 (32%) with proteinuria > or = 300 mg/L and ten (18%) with proteinuria > or = 500 mg/L were studied. Deoxyribonucleic acid (DNA) was screened for NPHS2 mutations and variants (R138Q and P375L) using single-stranded conformational analysis (SSCA) and for the R229Q mutation by sequencing. DNA was also screened for ACTN4 mutations. R229Q was more common in patients with TBMN and proteinuria > or = 500 mg/L (p < 0.05), and a possible NPHS2 mutation (671G>A, R224H) was identified in one patient with proteinuria 700 mg/L. No other NPHS2 variants correlated with proteinuria, and no ACTN4 mutations were found. Individuals with TBMN and R229Q are carriers of the autosomal recessive forms of both Alport syndrome and familial focal segmental glomerulosclerosis (FSGS). The early demonstration of R229Q in individuals with TBMN may indicate those at increased risk of proteinuria and renal impairment.

    Pediatric nephrology (Berlin, Germany) 2008;23;12;2201-7

  • Expression and gene amplification of actinin-4 in invasive ductal carcinoma of the pancreas.

    Kikuchi S, Honda K, Tsuda H, Hiraoka N, Imoto I, Kosuge T, Umaki T, Onozato K, Shitashige M, Yamaguchi U, Ono M, Tsuchida A, Aoki T, Inazawa J, Hirohashi S and Yamada T

    Chemotherapy Division, Cancer Proteomics Project, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.

    Purpose: An invasive growth pattern is one of the hallmarks of pancreatic ductal carcinoma. Actinin-4 is an actin-binding protein associated with enhanced cell motility, invasive growth, and lymph node metastasis. Actinin-4 might play an important role in the development and progression of pancreatic cancer.

    The expression of actinin-4 was examined immunohistochemically in 173 cases of invasive pancreatic ductal carcinoma. The copy number of the actinin-4 (ACTN4) gene was calculated by fluorescence in situ hybridization. The expression of actinin-4 was stably knocked down by short hairpin RNA, and tumorigenicity was evaluated by orthotopic implantation into mice with severe combined immunodeficiency.

    Results: The expression level of actinin-4 was increased in 109 (63.0%) of 173 cases of pancreatic cancer. Kaplan-Meier survival curves revealed that patients with increased expression of actinin-4 had a significantly poorer outcome (P=0.00001, log-rank test). Multivariate analysis by the Cox proportional hazard model showed that high expression of actinin-4 was the most significant independent negative predictor of survival (hazard ratio, 2.33; P=0.000009). Amplification (defined as more than four copies per interphase nucleus) of the ACTN4 gene was detected in 11 (37.9%) of 29 cases showing increased expression of actinin-4. Knockdown of actinin-4 expression inhibited the destructive growth of cancer cells in the pancreatic parenchyma.

    Conclusion: Recurrent amplification of chromosome 19q13.1-2 has been reported in pancreatic cancer, but the exact target gene has not been identified. Actinin-4 contributes to the invasive growth of pancreatic ductal carcinoma, and ACTN4 is one of the candidate oncogenes in this chromosome locus.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;17;5348-56

  • Motility-related actinin alpha-4 is associated with advanced and metastatic ovarian carcinoma.

    Barbolina MV, Adley BP, Kelly DL, Fought AJ, Scholtens DM, Shea LD and Stack MS

    Department of Chemical and Biochemical Engineering, Northwestern University, Chicago, IL, USA.

    Advanced and metastatic ovarian cancer is a leading cause of death from gynecologic malignancies. A more detailed understanding of the factors controlling invasion and metastasis may lead to novel anti-metastatic therapies. To model cellular interactions that occur during intraperitoneal metastasis, comparative cDNA microarray analysis and confirmatory real-time reverse transcription PCR (RT-PCR) were employed to uncover changes in gene expression that may occur in late stage ovarian cancer in response to microenvironmental cues, particularly native three-dimensional collagen I. Gene expression in human ovarian carcinoma tissues was evaluated on the RNA and protein level using real-time RT-PCR and immunohistochemistry. Cell invasion and migration were evaluated in a collagen invasion assay and a scratch wound assay. Three-dimensional collagen I culture led to differential expression of several genes. The role of actinin alpha-4 (ACTN4), a cytoskeleton-associated protein implicated in the regulation of cell motility, was examined in detail. ACTN4 RNA and protein expression were associated with advanced and metastatic human ovarian carcinoma. This report demonstrates that a cytoskeletal-associated protein ACTN4 is upregulated by three-dimensional collagen culture conditions, leading to increased invasion and motility of ovarian cancer cells. Expression of ACTN4 in human ovarian tumors was found to be associated with advanced-stage disease and peritoneal metastases.

    Funded by: NCI NIH HHS: R01 CA086984, R01 CA086984-07, R01 CA086984-08, R01 CA086984-09, R01 CA109545, R01 CA109545-03, R01 CA109545-04, R01 CA86984

    Laboratory investigation; a journal of technical methods and pathology 2008;88;6;602-14

  • Dynamic regulation of endothelial NOS mediated by competitive interaction with alpha-actinin-4 and calmodulin.

    Hiroi Y, Guo Z, Li Y, Beggs AH and Liao JK

    Vascular Medicine Research, Brigham & Women's Hospital, 65 Landsdowne Street, Boston, MA 02139, USA.

    Alpha-actinins are critical components of the actin cytoskeleton. Here we show that alpha-actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). Alpha-actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express alpha-actinin-1 and -4, alpha-actinin-4 interacted and colocalized with eNOS. Addition of alpha-actinin-4 directly inhibited eNOS recombinant protein, and overexpression of alpha-actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of alpha-actinin-4 by siRNA increased eNOS activity in BAECs. The alpha-actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on alpha-actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased alpha-actinin-4-eNOS interaction, leading to translocation of alpha-actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced alpha-actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with alpha-actinin-4 and calmodulin.

    Funded by: NHLBI NIH HHS: HL052233, R01 HL052233, R01 HL052233-10, R01 HL052233-11, R01 HL052233-12, R01 HL070274, R01 HL070274-03, R01 HL070274-04, R01 HL070274-05, R01 HL080187, R01 HL080187-01A1, R01 HL080187-02, R01 HL080187-03; NIAMS NIH HHS: AR044345, R01 AR044345, R01 AR044345-10, R01 AR044345-11; NIDDK NIH HHS: R01 DK062729, R01 DK062729-04, R01 DK062729-05; NINDS NIH HHS: P50 NS010828, P50 NS010828-300036, P50 NS010828-310036, P50 NS010828-320036

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;5;1450-7

  • Familial focal segmental glomerulosclerosis associated with an ACTN4 mutation and paternal germline mosaicism.

    Choi HJ, Lee BH, Cho HY, Moon KC, Ha IS, Nagata M, Choi Y and Cheong HI

    Department of Pediatrics, Seoul National University Children's Hospital, Seoul, Korea.

    Mutations in the ACTN4 gene cause focal segmental glomerulosclerosis (FSGS), which shows autosomal dominant inheritance (Online Mendelian Inheritance in Man No. 603278, FSGS1). Most patients with a diagnosis of FSGS1 show a mild to moderate degree of proteinuria during adolescence or later, and some patients gradually progress to end-stage renal disease. Here, we report a familial case of FSGS1 in which 2 affected siblings showed unusual clinical, pathological, and genetic features. Both patients presented with full-blown rapidly progressing nephrotic syndrome in early childhood. Renal pathological findings were of an FSGS collapsing variant and FSGS not otherwise specified. A novel ACTN4 mutation, p.Ser262Phe, was detected in the patients, and their father was found to have a germline mosaicism for the mutation. In addition, these siblings also had a heterozygous p.Thr5Met substitution in NPHS1, which encodes nephrin, although the functional significance of this substitution is unclear. This is the third clinical report of FSGS1 and the first case report of germline mosaicism confirmed in patients with hereditary podocyte disorders. FSGS1 may have widely variable clinical and pathological phenotypes and therefore should be considered in young children with full-blown and rapidly progressing nephrotic syndrome. The possibility of germline mosaicism makes interpretation of molecular diagnoses and genetic counseling more difficult.

    American journal of kidney diseases : the official journal of the National Kidney Foundation 2008;51;5;834-8

  • RelA/NF-kappaB transcription factor associates with alpha-actinin-4.

    Babakov VN, Petukhova OA, Turoverova LV, Kropacheva IV, Tentler DG, Bolshakova AV, Podolskaya EP, Magnusson KE and Pinaev GP

    Department of Cell Cultures, Institute of Cytology, St Petersburg, Russian Federation. vbabakov@gmail.com

    The NF-kappaB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-kappaB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-kappaB is associated with the actin-binding protein alpha-actinin-4. NF-kappaB and alpha-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-alpha, alpha-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-alpha led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-kappaB co-immunoprecipitated alpha-actinin-4 from A431 cell lysates and nuclear extracts, but alpha-actinin-1 and beta-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-kappaB can bind to matrix-bound chicken gizzard alpha-actinin. We suggest that the alpha-actinin-4 is important for the NF-kappaB nuclear translocation and its functions inside the nucleus.

    Experimental cell research 2008;314;5;1030-8

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Crystal structure of the actin-binding domain of alpha-actinin-4 Lys255Glu mutant implicated in focal segmental glomerulosclerosis.

    Lee SH, Weins A, Hayes DB, Pollak MR and Dominguez R

    University of Pennsylvania School of Medicine, Department of Physiology, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.

    Mutations in alpha-actinin-4 have been linked to familial focal segmental glomerulosclerosis (FSGS), a common renal disorder in humans, and produce an apparent increase in the actin-binding affinity of alpha-actinin-4 in vitro. One of the mutations, in particular, Lys255Glu, falls in the middle of the actin-binding interface of the actin-binding domain (ABD). The ABD consists of tandem calponin homology (CH) domains (CH1 and CH2). The crystal structures of most ABDs display a compact conformation, characterized by extensive inter-CH interactions. However, the conformation of F-actin-bound ABDs is unsettled. Some electron microscopy studies find that the compact conformation is preserved upon binding to F-actin, whereas other studies suggest that the CHs separate and the ABD becomes extended. The Lys255Glu mutation in CH2 is significant in this regard since it removes a crucial inter-CH interaction with Trp147 of CH1, thought to stabilize the compact conformation. Together, the increased actin-binding affinity and the removal of this important inter-CH contact suggested that the Lys255Glu mutation might facilitate the transition toward the open ABD conformation proposed by some of the electron microscopy studies. However, the crystal structure of the ABD of alpha-actinin-4 Lys255Glu mutant described here displays the canonical compact conformation. Furthermore, the sedimentation coefficients by analytical ultracentrifugation of wild-type and FSGS mutant ABDs (Lys255Glu, Ser262Pro, and Thr259Ile) are nearly identical (2.50+/-0.03 S) and are in good agreement with the theoretical value calculated from the crystal structure (2.382 S), implying that the compact conformation is retained in solution. The absence of a structural change suggests that the compact ABD conformation observed in the majority of the structures is highly stable and is preserved in solution, even in FSGS mutant ABDs.

    Funded by: NCRR NIH HHS: P41 RR001646, RR-01646; NHLBI NIH HHS: HL086655, P01 HL086655, P01 HL086655-01A10004; NIDDK NIH HHS: DK59588, R01 DK059588, R01 DK059588-09, R37 DK059588; NIGMS NIH HHS: DMR0225180

    Journal of molecular biology 2008;376;2;317-24

  • Association analysis of podocyte slit diaphragm genes as candidates for diabetic nephropathy.

    Ihalmo P, Wessman M, Kaunisto MA, Kilpikari R, Parkkonen M, Forsblom C, Holthöfer H, Groop PH and FinnDiane Study Group

    Folkhälsan Institute of Genetics, Folkhälsan Research Center, University of Helsinki, Helsinki, Finland.

    The slit diaphragm is an adhesion and signalling protein complex linking the interdigitating podocyte foot processes in the kidney glomerulus, and mutations in slit diaphragm-associated genes result in severe proteinuria. Here we report a genetic association analysis of four slit diaphragm genes, LRRC7, KIRREL, NPHS2 and ACTN4, in a Finnish diabetic nephropathy cohort.

    A total of 40 single nucleotide polymorphisms (SNPs) were genotyped in 1103 patients with type 1 diabetes. The patients were classified according to their renal status, and the genotype data were analysed in a cross-sectional case-control setting. To confirm positive associations, four SNPs were genotyped in 1,025 additional patients with type 1 diabetes.

    Results: No associations with diabetic nephropathy were observed for any of the analysed SNPs. The SNPs were not associated with the time from the onset of diabetes to the diagnosis of nephropathy or with glomerular filtration rate or AER as quantitative variables. In a sex-specific sub-analysis, the variants rs979972 and rs749701 in the first intron of ACTN4 were nominally associated with diabetic nephropathy in females, with odds ratios of 1.81 (95% CI 1.18-2.79, p = 0.007) and 1.93 (95% CI 1.26-2.96, p = 0.003) respectively.

    Our study has not found any evidence that common variants in LRRC7, KIRREL, NPHS2 and ACTN4 contribute to susceptibility to diabetic nephropathy in Finnish patients with type 1 diabetes.

    Diabetologia 2008;51;1;86-90

  • Expression of alpha-actinin-4 in human diabetic nephropathy.

    Kimura M, Toyoda M, Kato M, Kobayashi K, Abe M, Kobayashi T, Miyauchi M, Yamamoto N, Umezono T and Suzuki D

    Division of Nephrology and Metabolism, Department of Internal Medicine, Tokai University School of Medicine, Isehara.

    Background: Alpha-actinin-4 is an actin filament crosslinking protein that interacts with intercellular adhesion molecules. Recent animal studies suggested that alpha-actinin-4 is an essential component of the glomerular filtration barrier. However, little is known about its expression in human diabetic nephropathy (DN).

    Methods: Renal biopsy tissues were obtained from 17 patients with DN. We determined the mRNA and protein expression levels of alpha-actinin-4 by in situ hybridization and immunohistochemistry. The histopathological severity of DN was classified into two groups: mild and moderate mesangial expansion groups. We also measured urinary protein excretion and creatinine clearance.

    Results: Podocytes were positively stained for alpha-actinin-4 mRNA and protein. In the glomeruli, the percentage of cells positive for alpha-actinin-4 mRNA was significantly lower in moderate mesangial expansion group than in mild mesangial expansion group and control. The percentage of immunohistochemically positive area for alpha-actinin-4 protein in moderate mesangial expansion group was significantly lower than in mild mesangial expansion group and control. The percentage of cells positive for alpha-actinin-4 mRNA and area positive for the protein correlated inversely with severity of proteinuria.

    Conclusion: Our results suggest that low expression levels of alpha-actinin-4 mRNA and protein are linked to the progression of glomerulopathy and proteinuria in human DN.

    Internal medicine (Tokyo, Japan) 2008;47;12;1099-106

  • Actinin-4 expression in ovarian cancer: a novel prognostic indicator independent of clinical stage and histological type.

    Yamamoto S, Tsuda H, Honda K, Kita T, Takano M, Tamai S, Inazawa J, Yamada T and Matsubara O

    Department of Basic Pathology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, Japan.

    Actinin-4 is an isoform of non-muscular alpha-actinin and actin-bundling protein. By enhancing cell motility, actinin-4 shows different biological properties from another isoform of non-muscular actinin 'actinin-1' and variable clinicopathological implications of actinin-4 have been demonstrated in some human malignancies such as breast cancers, lung cancers, and colorectal cancers. We herein described the clinicopathological and prognostic significance of actinin-4 expression in ovarian cancers. Actinin-4 expression was analyzed immunohistochemically in 265 primary ovarian carcinomas: 116 serous, 71 clear cell, 43 endometrioid, and 35 mucinous adenocarcinomas. With reference to endothelial immunoreactivity, cytoplasmic expression of actinin-4 was classified as either low (including negative) or high. Then, various parameters such as patients' characteristics, histopathological findings including E-cadherin and beta-catenin immunoreactivity, and clinical outcome, were compared between groups showing differences in the intensity or intracellular distribution of actinin-4 immunoreactivity. High expression of actinin-4 was demonstrated in 137 (57%) cases and was associated with serous histology (P=0.0075), high histological grade (P<0.0001), an advanced disease stage (P=0.036), a high degree of residual disease after initial surgery (P=0.0047), poor patient outcome (5-year survival: 52.4% in the high-expression group vs 71.9% in the low expression group, P=0.0043 by log-rank test), and also with reduced E-cadherin and preserved beta-catenin expressions (P=0.0097 and 0.017, respectively). Nuclear immunoreactivity for actinin-4 was detected in 20 (7.5%) cases and was associated with low histological grade (P=0.0079) but not with other variables. Multivariate analysis showed that high actinin-4 expression was an independent prognostic factor for overall survival, as well as a high degree of residual disease and clear-cell histology. Accumulation of actinin-4 in the cytoplasm may be related to a higher propensity for tumor invasiveness and metastasis, probably by enhancing cell motility, and could be a novel prognostic indicator for patients with ovarian carcinomas.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2007;20;12;1278-85

  • Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.

    Weins A, Schlondorff JS, Nakamura F, Denker BM, Hartwig JH, Stossel TP and Pollak MR

    Renal and Translational Medicine Divisions, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

    Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.

    Funded by: NHLBI NIH HHS: HL19429, R01 HL019429; NIDDK NIH HHS: DK59588, F32 DK074308, F32-DK074308-01, R01 DK059588, R01 DK059588-09, R37 DK059588, T32 DK007527, T32-DK007527-20

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;41;16080-5

  • Cytokine activation of p38 mitogen-activated protein kinase and apoptosis is opposed by alpha-4 targeting of protein phosphatase 2A for site-specific dephosphorylation of MEK3.

    Prickett TD and Brautigan DL

    Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Jefferson Park Avenue, Hospital West Complex, Room 7225, Charlottesville, VA 22908-1400, USA.

    alpha-4 is an essential gene and is a dominant antiapoptotic factor in various tissues that is a regulatory subunit for type 2A protein phosphatases. A multiplexed phosphorylation site screen revealed that knockdown of alpha-4 by small interfering RNA (siRNA) increased p38 mitogen-activated protein kinase (MAPK) and c-Jun phosphorylation without changes in JNK or ERK. FLAG-alpha-4 coprecipitated hemagglutinin-MEK3 plus endogenous protein phosphatase 2A (PP2A) and selectively enhanced dephosphorylation of Thr193, but not Ser189, in the activation loop of MEK3. Overexpression of alpha-4 suppressed p38 MAPK activation in response to tumor necrosis factor alpha (TNF-alpha). The alpha-4 dominant-negative domain (DND) (residues 220 to 340) associated with MEK3, but not PP2A, and its overexpression sensitized cells to activation of p38 MAPK by TNF-alpha and interleukin-1beta, but not by ansiomycin or sorbitol. The response was diminished by nocodazole or by siRNA knockdown of the Opitz syndrome protein Mid1 that binds alpha-4 to microtubules. Interference by alpha-4 DND or alpha-4 siRNA increased caspase 3/7 activation in response to TNF-alpha. Growth of transformed cells in soft agar was enhanced by alpha-4 and suppressed by alpha-4 DND. The results show that alpha-4 targets PP2A activity to MEK3 to suppress p38 MAPK activation by cytokines, thereby inhibiting apoptosis and anoikis.

    Funded by: NCI NIH HHS: CA77584, R01 CA077584

    Molecular and cellular biology 2007;27;12;4217-27

  • Molecular composition of IMP1 ribonucleoprotein granules.

    Jønson L, Vikesaa J, Krogh A, Nielsen LK, Hansen Tv, Borup R, Johnsen AH, Christiansen J and Nielsen FC

    Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark.

    Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100-300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons.

    Molecular & cellular proteomics : MCP 2007;6;5;798-811

  • Alpha-actinin 4 potentiates myocyte enhancer factor-2 transcription activity by antagonizing histone deacetylase 7.

    Chakraborty S, Reineke EL, Lam M, Li X, Liu Y, Gao C, Khurana S and Kao HY

    Department of Biochemistry, School of Medicine, Case Western Reserve University, and the Research Institute of University Hospitals of Cleveland, OH 44106, USA.

    Histone deacetylase 7 (HDAC7) is a member of class IIa HDACs that regulate myocyte enhancer factor-2 (MEF2)-mediated transcription and participate in multiple cellular processes such as T cell apoptosis. We have identified alpha-actinin 1 and 4 as class IIa HDAC-interacting proteins. The interaction domains are mapped to C terminus of alpha-actinin 4 and amino acids 72-172 of HDAC7. A point mutation in HDAC7 that disrupts its association with MEF2A also disrupts its association with alpha-actinin 4, indicating that MEF2A and alpha-actinin 4 binding sites largely overlap. We have also isolated a novel splice variant of alpha-actinin 4 that is predominantly localized in the nucleus, a pattern distinct from the full-length alpha-actinin 4, which is primarily distributed in the cytoplasm and plasma membrane. Using small interfering RNA, chromatin immunoprecipitation, and transient transfection assays, we show that alpha-actinin 4 potentiates expression of TAF55, a putative MEF2 target gene. Loss of MEF2A interaction correlates with loss of the ability of alpha-actinin 4 to potentiate TAF55 promoter activity. Ectopic expression of alpha-actinin 4, but not the mutant defective in MEF2A association, leads to disruption of HDAC7.MEF2A association and enhancement of MEF2-mediated transcription. Taken together, we have identified a novel mechanism by which HDAC7 activity is negatively regulated and uncovered a previously unknown function of alpha-actinin 4.

    Funded by: NCI NIH HHS: P30 CA43703-12, T32 CA059366-11; NIDDK NIH HHS: R01 DK62985

    The Journal of biological chemistry 2006;281;46;35070-80

  • Disruption of alpha-actinin-integrin interactions at focal adhesions renders osteoblasts susceptible to apoptosis.

    Triplett JW and Pavalko FM

    Dept. of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

    Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of alpha-actinin (ROD-GFP) that competitively displaces endogenous alpha-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas alpha-actinin-GFP expression protected, cells from TNF-alpha-induced apoptosis. Further investigation revealed that activation of TNF-alpha-induced survival signals, specifically Akt phosphorylation and NF-kappaB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-alpha-induced apoptosis. Thus we conclude that alpha-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-alpha-induced survival signaling.

    Funded by: NIAMS NIH HHS: R01 AR-49728, R01 AR052682, R01 AR052682-01A1

    American journal of physiology. Cell physiology 2006;291;5;C909-21

  • A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners.

    Ding Z, Liang J, Lu Y, Yu Q, Songyang Z, Lin SY and Mills GB

    Department of Molecular Therapeutics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 950, Houston, TX 77030, USA.

    We developed a retrovirus-based protein-fragment complementation assay (RePCA) screen to identify protein-protein interactions in mammalian cells. In RePCA, bait protein is fused to one fragment of a rationally dissected fluorescent protein, such as GFP, intensely fluorescent protein, or red fluorescent protein. The second, complementary fragment of the fluorescent protein is fused to an endogenous protein by in-frame exon traps in the enhanced retroviral mutagen vector. An interaction between bait and host protein (prey) places the two parts of the fluorescent molecule in proximity, resulting in reconstitution of fluorescence. By using RePCA, we identified a series of 24 potential interaction partners or substrates of the serine/threonine protein kinase AKT1. We confirm that alpha-actinin 4 (ACTN4) interacts physically and functionally with AKT1. siRNA-mediated ACTN4 silencing down-regulates AKT phosphorylation, blocks AKT translocation to the membrane, increases p27(Kip1) levels, and inhibits cell proliferation. Thus, ACTN4 is a critical regulator of AKT1 localization and function.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;41;15014-9

  • Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis.

    Celli L, Ryckewaert JJ, Delachanal E and Duperray A

    Institut National de la Santé et de la Recherche Médicale, Unité 578, Grenoble, France, and Université Grenoble I, Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, Grenoble, France.

    ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle alpha-actinin (i.e., alpha-actinin 1 and alpha-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of alpha-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically alpha-actinin 1 or alpha-actinin 4 on endothelial cells indicated that alpha-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic alpha-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.

    Journal of immunology (Baltimore, Md. : 1950) 2006;177;6;4113-21

  • Interaction of zonula occludens-1 (ZO-1) with alpha-actinin-4: application of functional proteomics for identification of PDZ domain-associated proteins.

    Chen VC, Li X, Perreault H and Nagy JI

    Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.

    The use of recombinant "bait" proteins to capture protein-binding partners, followed by identification of protein interaction networks by mass spectrometry (MS), has gained popularity and widespread acceptance. We have developed an approach using recombinant PDZ protein interaction modules of the membrane-associated guanylate kinase (MAGUK) protein zonula occludens-1 (ZO-1) to pull-down and screen for proteins that interact with these modules via their PDZ domain binding motifs. Identification of proteins by MS of pull-down material was achieved using a vacuum-based chromatography sample preparation device designed for matrix-assisted laser desorption/ionization (MALDI) MS. MS analysis of tryptic fragments in pull-down material revealed a number of potential ZO-1 interacting candidates, including the presence of peptides corresponding to the cortical membrane scaffolding protein alpha-actinin-4. Interaction of alpha-actinin-4 with ZO-1 was confirmed by coimmunoprecipitation of these two proteins from cultured cells, as well as from brain, liver, and heart, and by immunoblot detection of alpha-actinin-4 after pull-down with the first PDZ domain of ZO-1. In contrast, the highly homologous alpha-actinin family member, alpha-actinin-1, displayed no association with ZO-1. Immunofluorescence showed colocalization of alpha-actinin-4 with ZO-1 in cultured HeLa and C6 glioma cells, as well as in a variety of tissues in vivo, including brain, heart, liver, and lung. This study demonstrates the utility of MS-based functional proteomics for identifying cellular components of the ZO-1 scaffolding network. Our finding of the interaction of ZO-1 with alpha-actinin-4 provides a mechanism for linking the known protein recruitment and signaling activities of ZO-1 with alpha-actinin-4-associated plasma membrane proteins that have regulatory activities at cell-cell and cell-extracellular matrix contacts.

    Journal of proteome research 2006;5;9;2123-34

  • A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.

    Lim J, Hao T, Shaw C, Patel AJ, Szabó G, Rual JF, Fisk CJ, Li N, Smolyar A, Hill DE, Barabási AL, Vidal M and Zoghbi HY

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

    Many human inherited neurodegenerative disorders are characterized by loss of balance due to cerebellar Purkinje cell (PC) degeneration. Although the disease-causing mutations have been identified for a number of these disorders, the normal functions of the proteins involved remain, in many cases, unknown. To gain insight into the function of proteins involved in PC degeneration, we developed an interaction network for 54 proteins involved in 23 inherited ataxias and expanded the network by incorporating literature-curated and evolutionarily conserved interactions. We identified 770 mostly novel protein-protein interactions using a stringent yeast two-hybrid screen; of 75 pairs tested, 83% of the interactions were verified in mammalian cells. Many ataxia-causing proteins share interacting partners, a subset of which have been found to modify neurodegeneration in animal models. This interactome thus provides a tool for understanding pathogenic mechanisms common for this class of neurodegenerative disorders and for identifying candidate genes for inherited ataxias.

    Funded by: NICHD NIH HHS: HD24064; NINDS NIH HHS: NS27699

    Cell 2006;125;4;801-14

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

    Kimura K, Wakamatsu A, 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 5a8 , 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

  • Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC).

    Foster LJ, Rudich A, Talior I, Patel N, Huang X, Furtado LM, Bilan PJ, Mann M and Klip A

    Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

    The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role for the actin cytoskeleton and for protein-protein interactions in regulating GLUT4 localization by insulin. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas components of the ubiquitin-proteasome degradation system were generally reduced. Proteins participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related proteins, alpha-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 +/- 0.5-fold by SILAC and 2.9 +/- 0.8-fold by immunoblotting. Insulin caused GLUT4 and alpha-actinin-4 co-localization as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably alpha-actinin-4, associate with the transporter, potentially to facilitate its routing to the plasma membrane.

    Journal of proteome research 2006;5;1;64-75

  • Mutational and Biological Analysis of alpha-actinin-4 in focal segmental glomerulosclerosis.

    Weins A, Kenlan P, Herbert S, Le TC, Villegas I, Kaplan BS, Appel GB and Pollak MR

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

    Mutations in the alpha-actinin-4 gene (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS). A mutational analysis was performed of ACTN4 in DNA from probands with a family history of FSGS as well as in individuals with nonfamilial FSGS. The possible contribution of noncoding variation in ACTN4 to the development of FSGS also was assessed. Multiple nucleotide variants were identified in coding and noncoding sequence. The segregation of nonsynonymous coding sequence variants was examined in the relevant families. Only a small number of nucleotide changes that seemed likely to be causing (or contributing to) disease were identified. Sequence changes that predicted I149del, W59R, V801M, R348Q, R837Q, and R310Q changes were identified. For studying their biologic relevance and their potential roles in the pathogenesis of FSGS, these variants were expressed as GFP-fusion proteins in cultured podocytes. F-actin binding assays also were performed. Three of these variants (W59R, I149del, and V801M) showed clear cellular mislocalization in the form of aggregates adjacent to the nucleus. Two of these mislocalized variants (W59R and I149del) also showed an increased actin-binding activity. The I149del mutation segregated with disease; W59R was found to be a de novo mutation in the proband. A total of five ACTN4 mutations that are believed to be disease causing (three reported previously and two novel) as well as a number of variants with unclear contribution to disease now have been identified. The possibility that some of these other variants increase the susceptibility to FSGS cannot be excluded. ACTN4 mutations seem to account for approximately 4% of familial FSGS.

    Funded by: NIDDK NIH HHS: DK54931, DK59588

    Journal of the American Society of Nephrology : JASN 2005;16;12;3694-701

  • 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

  • E-cadherin regulates the association between beta-catenin and actinin-4.

    Hayashida Y, Honda K, Idogawa M, Ino Y, Ono M, Tsuchida A, Aoki T, Hirohashi S and Yamada T

    Chemotherapy Division and Cancer Proteomics Project, National Cancer Center Research Institute, Tokyo, Japan.

    The E-cadherin/catenin system acts as an invasion suppressor of epithelial malignancies. This invasion suppressive activity seems be mediated not only by the cell adhesive activity of E-cadherin but by other undetermined signaling pathways elicited by beta-catenin. In fact, cancer cells that have infiltrated the stroma reduce the expression of E-cadherin and accumulate beta-catenin. We attempted to identify the alternative partner proteins that make complexes with beta-catenin in the absence of E-cadherin. An approximately 100-kDa protein was constantly coimmunoprecipitated with beta-catenin from SW480 colorectal cancer cells, which lack the expression of E-cadherin, and was identified as actinin-4 by mass spectrometry. Transfection of E-cadherin cDNA suppressed the association between beta-catenin and actinin-4. Inhibition of E-cadherin by RNA interference transferred the beta-catenin and actinin-4 proteins into the membrane protrusions of DLD-1 cells. Immunofluorescence histochemistry of clinical colorectal cancer specimens showed that the beta-catenin and actinin-4 proteins were colocalized in colorectal cancer cells infiltrating the stroma. We reported previously that overexpression of actinin-4 induces cell motility and specifically promotes lymph node metastasis by colorectal cancer. The association between beta-catenin and actinin-4 and its regulation by E-cadherin may represent a novel molecular link connecting cell adhesion and motility. Shutting down the signals mediating this association may be worth considering as a therapeutic approach to cancer invasion and metastasis.

    Cancer research 2005;65;19;8836-45

  • CART: an Hrs/actinin-4/BERP/myosin V protein complex required for efficient receptor recycling.

    Yan Q, Sun W, Kujala P, Lotfi Y, Vida TA and Bean AJ

    Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, TX 77030, USA.

    Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. Here, we report that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors.

    Funded by: NIGMS NIH HHS: GM-052092, R01 GM052092; NIMH NIH HHS: MH-58920, R01 MH058920

    Molecular biology of the cell 2005;16;5;2470-82

  • Synaptopodin regulates the actin-bundling activity of alpha-actinin in an isoform-specific manner.

    Asanuma K, Kim K, Oh J, Giardino L, Chabanis S, Faul C, Reiser J and Mundel P

    Department of Medicine, Albert Einstein College of Medicine, New York, New York, USA.

    Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. Synaptopodin-deficient (synpo(-/-)) mice lack the dendritic spine apparatus and display impaired activity-dependent long-term synaptic plasticity. In contrast, the ultrastructure of podocytes in synpo(-/-) mice is normal. Here we show that synpo(-/-) mice display impaired recovery from protamine sulfate-induced podocyte foot process (FP) effacement and LPS-induced nephrotic syndrome. Similarly, synpo(-/-) podocytes show impaired actin filament reformation in vitro. We further demonstrate that synaptopodin exists in 3 isoforms, neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). The C terminus of Synpo-long is identical to that of Synpo-T. All 3 isoforms specifically interact with alpha-actinin and elongate alpha-actinin-induced actin filaments. synpo(-/-) mice lack Synpo-short and Synpo-long expression but show an upregulation of Synpo-T protein expression in podocytes, though not in the brain. Gene silencing of Synpo-T abrogates stress-fiber formation in synpo(-/-) podocytes, demonstrating that Synpo-T serves as a backup for Synpo-long in synpo(-/-) podocytes. In concert, synaptopodin regulates the actin-bundling activity of alpha-actinin in highly dynamic cell compartments, such as podocyte FPs and the dendritic spine apparatus.

    Funded by: NIDA NIH HHS: DA18886, R01 DA018886; NIDDK NIH HHS: DK062472, DK064236, DK57683, P50 DK064236, R01 DK057683, R01 DK062472

    The Journal of clinical investigation 2005;115;5;1188-98

  • Analysis of NPHS1, NPHS2, ACTN4, and WT1 in Japanese patients with congenital nephrotic syndrome.

    Sako M, Nakanishi K, Obana M, Yata N, Hoshii S, Takahashi S, Wada N, Takahashi Y, Kaku Y, Satomura K, Ikeda M, Honda M, Iijima K and Yoshikawa N

    Department of Pediatrics, Wakayama Medical University, Wakayama City, Wakayma, Japan.

    Background: Congenital nephrotic syndrome (CNS) causes significant renal failure, and is classified into two types: (1) Finnish type; and (2) other, including diffuse mesangial sclerosis. Mutations of NPHS1 and NPHS2, which encode the slit diaphragm components nephrin and podocin, cause CNS and autosomal-recessive familial steroid-resistant nephrotic syndrome, respectively. Most patients with Finnish-type CNS in Europe and the United States have NPHS1 mutations. However, NPHS2 mutations have been detected in some cases. Mutations in ACTN4, encoding alpha-actinin-4, cause an autosomal-dominant focal segmental glomerulosclerosis. alpha-actinin-4 stabilizes the podocyte cytoskeleton structure, connecting with actin filaments. WT1 mutations, causing Wilm's tumor, have been demonstrated in some CNS patients with diffuse mesangial sclerosis. Systematic investigation of genes for CNS in Japan has never been performed.

    Methods: To clarify the role of mutations in these four genes, we used polymerase chain reaction (PCR) and direct sequencing to investigate all exons and exon-intron boundaries for these genes in 13 unrelated CNS patients from regional pediatric kidney disease centers in Japan.

    Results: A novel homozygous nonsense mutation of NPHS1, E246X in exon 7, and a novel homozygous deletion mutation of NPHS1, 2156_2163del in exon 16 were detected in one patient each. A novel homozygous nonsense mutation of NPHS2, R196X in exon 5, was found in one patient, and the same heterozygous nonsense mutation was detected in another. No ACTN4 or WT1 mutations were detected.

    Conclusion: These studies demonstrate that mutation of NPHS1 is not a major cause of CNS in Japanese patients, and that mutation of NPHS2 can be responsible for CNS in this population.

    Kidney international 2005;67;4;1248-55

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • Substrate-trapping techniques in the identification of cellular PTP targets.

    Blanchetot C, Chagnon M, Dubé N, Hallé M and Tremblay ML

    McGill Cancer Centre, McGill University, 3655 Sir William-Osler, Room 715, Montreal, Quebec, H3G1Y6 Canada. christophe.blanchetot@mcgill.ca

    Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP "substrate-trapping" mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.

    Methods (San Diego, Calif.) 2005;35;1;44-53

  • Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide.

    Kigawa A, Wakui H, Maki N, Okuyama S, Masai R, Ohtani H, Komatsuda A, Suzuki D, Toyoda M, Kobayashi R and Sawada K

    Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.

    Background: Podocyte alpha-actinin-4 (actinin-4) is an essential component of the glomerular filtration barrier. We recently reported that the central rod spectrin-like repeats (R1-R4) of actinin-4 have a high affinity to puromycin aminonucleoside (PAN), which can induce nephro-sis in animals. The aim of this study was to identify endogenous molecules that interact with the actinin-4 R1-R4 domain.

    Methods: To identify such molecules, we performed a bacterial two-hybrid screening of a human kidney cDNA library using as a bait human actinin-4 R1-R4. We further verified the identified interactions by in vitro affinity assays and immunofluorescent studies of cultured human embryonic kidney HEK293 cells. To investigate the expression of the identified molecules in podocytes, in situ hybridization, and immunohistochemical studies were performed.

    Results: One isolated cDNA from the library encoded humanin, a recently identified antiapoptotic peptide. In vitro affinity assays showed specific interactions of recombinant actinin-4 R1-R4, R1, R2, R3, and R4 proteins with humanin-Sepharose. PAN had no effect on these interactions. Green fluorescent protein-fused humanin and endogenous actinin colocalized mainly in the perinuclear cytoplasm of HEK293 cells. Altered colocalization was not observed by the addition of PAN. In situ hybridization and immunohistochemistry showed the expression of humanin in podocytes.

    Conclusions: Our results suggest that humanin is a novel binding partner of the actinin-4 R1-R4 domain in podocytes. Humanin and PAN are unlikely to compete for the same binding surface in actinin-4.

    Clinical and experimental nephrology 2004;8;4;331-8

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1).

    Magdolen U, Schroeck F, Creutzburg S, Schmitt M and Magdolen V

    Klinische Forschergruppe der Frauenklinik der Technischen Universität München, Klinikum rechts der Isar, Ismaninger str. 22, D-81675 München, Germany.

    PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated. An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.

    Biological chemistry 2004;385;9;801-8

  • The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin.

    Lin SY, Raval S, Zhang Z, Deverill M, Siminovitch KA, Branch DR and Haimovich B

    Department of Surgery, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey and the Cancer Institute of New Jersey, New Brunswick, New Jersey 08903, USA.

    Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.

    Funded by: NHLBI NIH HHS: HL54104

    The Journal of biological chemistry 2004;279;24;25755-64

  • Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases.

    Lanzetti L, Palamidessi A, Areces L, Scita G and Di Fiore PP

    Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milan, Italy.

    Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.

    Nature 2004;429;6989;309-14

  • Mutant alpha-actinin-4 promotes tumorigenicity and regulates cell motility of a human lung carcinoma.

    Menez J, Le Maux Chansac B, Dorothée G, Vergnon I, Jalil A, Carlier MF, Chouaib S and Mami-Chouaib F

    Laboratoire Cytokines et Immunologie des tumeurs Humaines, U487 INSERM, Institut Fédératif de Recherche 54, Institut Gustave Roussy, F-94805 Villejuif Cedex, France.

    The precise role of alpha-actinin-4 encoding gene (ACTN4) is not very well understood. It has been reported to elicit tumor suppressor activity and to regulate cellular motility. To further assess the function of human ACTN4, we studied a lung carcinoma cell line expressing a mutated alpha-actinin-4, which is recognized as a tumor antigen by autologous CD8(+) cytotoxic T lymphocytes (CTL). Confocal immunofluorescence microscopy indicated that, while wild-type (WT) alpha-actinin-4 stains into actin cytoskeleton and cell surface ruffles, the mutated protein is only dispersed in the cytoplasm of the lung carcinoma cells. This loss of association with the cell surface did not appear to correlate with a decrease in in vitro alpha-actinin-4 crosslinking to filamentous (F)-actin. Interestingly, experiments using cell lines stably expressing ACTN4 demonstrated that as opposed to WT gene, mutant ACTN4 was unable to inhibit tumor cell growth in vitro and in vivo. Moreover, the expression of mutant alpha-actinin-4 resulted in the loss of tumor cell capacity to migrate. The identification of an inactivating mutation in ACTN4 emphasizes its role as a tumor suppressor gene and underlines the involvement of cytoskeleton alteration in tumor development and metastasis.

    Oncogene 2004;23;15;2630-9

  • Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions.

    Coppinger JA, Cagney G, Toomey S, Kislinger T, Belton O, McRedmond JP, Cahill DJ, Emili A, Fitzgerald DJ and Maguire PB

    Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland.

    Proteins secreted by activated platelets can adhere to the vessel wall and promote the development of atherosclerosis and thrombosis. Despite this biologic significance, however, the complement of proteins comprising the platelet releasate is largely unknown. Using a proteomics approach, we have identified more than 300 proteins released by human platelets following thrombin activation. Many of the proteins identified were not previously attributed to platelets, including secretogranin III, a potential monocyte chemoattractant precursor; cyclophilin A, a vascular smooth muscle cell growth factor; calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction, as well as proteins of unknown function that map to expressed sequence tags. Secretogranin III, cyclophilin A, and calumenin were confirmed to localize in platelets and to be released upon activation. Furthermore, while absent in normal vasculature, they were identified in human atherosclerotic lesions. Therefore, these and other proteins released from platelets may contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, as soluble extracellular proteins, they may prove suitable as novel therapeutic targets.

    Blood 2004;103;6;2096-104

  • The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin.

    Burgueño J, Blake DJ, Benson MA, Tinsley CL, Esapa CT, Canela EI, Penela P, Mallol J, Mayor F, Lluis C, Franco R and Ciruela F

    Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain.

    Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.

    The Journal of biological chemistry 2003;278;39;37545-52

  • Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4.

    Daniliuc S, Bitterman H, Rahat MA, Kinarty A, Rosenzweig D, Lahat N and Nitza L

    Immunology Research Unit and Ischemia-Shock Research Laboratory, Carmel Medical Center, Rappaport Family Institute for Research in the Medical Sciences, and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.

    Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-gamma induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 +/- 14 micro M, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 +/- 1.7 micro M, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and alpha-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.

    Journal of immunology (Baltimore, Md. : 1950) 2003;171;6;3225-32

  • Analysis of mutations in alpha-actinin 4 and podocin genes of patients with chronic renal failure due to sporadic focal segmental glomerulosclerosis.

    Komatsuda A, Wakui H, Maki N, Kigawa A, Goto H, Ohtani H, Hamai K, Oyama Y, Makoto H, Sawada K and Imai H

    Third Department of Internal Medicine, Akita University School of Medicine, Akita City, Akita, Japan. komatsud@med.akita-u.ac.jp

    Although the pathogenesis of idiopathic focal segmental glomerulosclerosis (FSGS) may be heterogeneous, autosomal dominant and recessive forms of FSGS are recognized. Recently, mutations in alpha-actinin 4 (ACTN4) and podocin genes were reported in patients with such familial FSGS. However, whether mutations in ACTN4 and podocin genes are associated with sporadic FSGS has not been determined. In the present study, we clarified the relation between mutations in ACTN4 and podocin genes and sporadic FSGS. We analyzed these reported mutations in ACTN4 and podocin in five patients with chronic renal failure due to therapy-resistant FSGS by direct sequencing of polymerase chain reaction products of ACTN4 and podocin. We found a C to T transition at nucleotide 465 in the ACTN4 gene in all of patients, and a T to C transition at nucleotide 954 in exon eight of podocin gene in two of five patients, resulting in no amino acid substitutions. Other mutations were not found in ACTN4 and podocin genes. Our findings suggest that sporadic FSGS is a heterogeneous disease, since ACTN4 and podocin genes are not found in our patients with sporadic FSGS.

    Renal failure 2003;25;1;87-93

  • Renal alpha-actinin-4: purification and puromycin aminonucleoside-binding property.

    Goto H, Wakui H, Komatsuda A, Ohtani H, Imai H, Sawada K and Kobayashi R

    Third Department of Internal Medicine, Akita University School of Medicine, Akita, Japan.

    Mutations in the gene encoding nonmuscle alpha-actinin-4 (actinin-4), an actin cross-linking protein, lead to congenital nephrosis. This suggests that actinin-4 is an essential component of the glomerular filtration barrier. In the present study, we attempted to purify actinin-4 from the mammalian kidney. We also examined an interaction of the protein with puromycin aminonucleoside (PAN), which can induce nephrosis in animals. A 100-kD protein reactive with antibody against muscle alpha-actinin was purified from the Triton-insoluble cytoskeleton of porcine kidney, by MgCl2 treatment, ammonium sulfate fractionation, and subsequent DEAE-cellulose chromatography and hydroxyapatite chromatography. Its partial amino acid sequence was then determined. A filamentous actin (F-actin)-binding activity of the purified protein was examined by a cosedimentation assay. Interactions of the purified protein and its fragments with PAN were analyzed by an affinity assay using PAN-Sepharose. Determined 134 amino acid sequences of the purified porcine renal 100-kD protein were completely identical with those deduced from nucleotide sequence of the cDNA encoding human actinin-4. The purified protein possessed the known function of alpha-actinin, the F-actin-binding activity, and was tightly bound to PAN. The PAN-binding site was mapped within a central rod domain of the protein, which is a possible interaction site for various cytoskeletal and transmembrane proteins. We have established an efficient purification method for renal actinin-4. Moreover, our findings indicate that the central rod domain of actinin-4 has a high affinity to PAN. In the PAN nephrosis animal model, actinin-4 might be a target protein from PAN nephrotoxicity.

    Nephron. Experimental nephrology 2003;93;1;e27-35

  • Interaction of two actin-binding proteins, synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1.

    Patrie KM, Drescher AJ, Welihinda A, Mundel P and Margolis B

    Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, 48109-0650, USA.

    In an attempt to find podocyte-expressed proteins that may interact with the tight junction protein MAGI-1, we screened a glomerulus-enriched cDNA library with a probe consisting of both WW domains of MAGI-1. One of the isolated clones contained two WW domain-binding motifs and was identified as a portion of the actin-bundling protein synaptopodin. In vitro binding assays confirmed this interaction between MAGI-1 and synaptopodin and identified the second WW domain of MAGI-1 to be responsible for the interaction. MAGI-1 and synaptopodin can also interact in vivo, as they can be immunoprecipitated together from HEK293 cell lysates. Another actin-bundling protein that is found in glomerular podocytes and shown to be mutated in an inheritable form of glomerulosclerosis is alpha-actinin-4. We show that alpha-actinin-4 is also capable of binding to MAGI-1 in in vitro binding assays and that this interaction is mediated by the fifth PDZ domain of MAGI-1 binding to the C terminus of alpha-actinin-4. Exogenously expressed synaptopodin and alpha-actinin-4 were found to colocalize along with endogenous MAGI-1 at the tight junction of Madin-Darby canine kidney cells. The interaction and colocalization of MAGI-1 with two actin-bundling proteins suggest that MAGI-1 may play a role in actin cytoskeleton dynamics within polarized epithelial cells.

    Funded by: NIDDK NIH HHS: 2P50DK39255, 5F32DK09912-02, DK57683-01

    The Journal of biological chemistry 2002;277;33;30183-90

  • Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution.

    Echchakir H, Dorothée G, Vergnon I, Menez J, Chouaib S and Mami-Chouaib F

    Laboratoire Cytokines et Immunologie des Tumeurs Humaines, U487 Institut National de la Santé et de la Recherche Médicale, Institut Gustave Roussy, Institut Fédératif de Recherche-54, F-94805 Villejuif, Cedex, France.

    We have previously identified an antigen (Ag) recognized on a human large cell carcinoma of the lung by a tumor-specific cytotoxic T lymphocyte clone derived from autologous tumor infiltrating lymphocytes (TILs). The antigenic peptide is presented by HLA-A2 molecules and is encoded by a mutated alpha-actinin-4 (ACTN4) gene. In the present report, we have isolated two anti-alpha-actinin-4 T cell clones from the same patient TIL and from his peripheral blood lymphocytes (PBLs) by using tetramers of soluble HLA-A2 molecules loaded with the mutated peptide. Although all of the clones displayed similar tetramer labeling, those isolated from PBL showed lower avidity of Ag recognition and killed the specific target much less efficiently, indicating that tetramer staining does not correlate with clone avidity/tumor reactivity. T cell receptor (TCR) analysis revealed that alpha-actinin-4-reactive clones used distinct alpha and beta chain rearrangements, demonstrating TCR repertoire diversity. Interestingly, TCR beta chain gene usage indicated that only Ag-specific clones with high functional avidity were expanded at the tumor site, whereas a low-avidity clone was exclusively amplified in patient peripheral blood. Our results point to the existence of distinct but overlapping antitumor TCR repertoires in TIL and PBL and suggest a selective in situ expansion of tumor-specific cytotoxic T lymphocyte with high avidity/tumor reactivity.

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;14;9358-63

  • Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.

    Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CH and Donowitz M

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

    Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.

    Funded by: NIDDK NIH HHS: P01 DK44484, R01 DK26523

    The Journal of biological chemistry 2002;277;26;23714-24

  • Each actin subunit has three nebulin binding sites: implications for steric blocking.

    Lukoyanova N, VanLoock MS, Orlova A, Galkin VE, Wang K and Egelman EH

    Department of Biochemistry, University of Virginia, Charlottesville 22908-0733, USA.

    Nebulin is a giant protein that spans most of the muscle thin filament. Mutations in nebulin result in myopathies and dystrophies. Nebulin contains approximately 200 copies of approximately 35 residue modules, each believed to contain an actin binding site, organized into seven-module superrepeats. The strong correlation between the number of nebulin modules and the length of skeletal muscle thin filaments in different species suggests that nebulin determines thin filament length. Little information exists about the interactions between intact nebulin and F-actin. More insight has come from working with fragments of nebulin, containing from one to hundreds of actin binding modules. However, the observed stoichiometry of binding between these fragments and actin has ranged from 0.4 to 13 modules per actin subunit. We have used electron microscopy and a novel method of helical image analysis to characterize complexes of F-actin with a nebulin fragment. The fragment binds as an extended structure spanning three actin subunits and binding to different sites on each actin. Muscle regulation involves tropomyosin movement on the surface of actin, with binding in three states. Our results suggest the intriguing possibility that intact nebulin may also be able to occupy three different sites on F-actin.

    Current biology : CB 2002;12;5;383-8

  • Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

    Renoult C, Blondin L, Fattoum A, Ternent D, Maciver SK, Raynaud F, Benyamin Y and Roustan C

    UMR 5539 (CNRS) Laboratoire de Motilité Cellulaire (Ecole Pratique des Hautes Etudes), Université de Montpellier, France.

    It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.

    European journal of biochemistry 2001;268;23;6165-75

  • Interactions of a hemidesmosome component and actinin family members.

    Gonzalez AM, Otey C, Edlund M and Jones JC

    Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611, USA.

    Hemidesmosomes are multimeric protein complexes that attach epithelial cells to their underlying matrix and serve as cell surface anchorage sites for the keratin cytoskeleton. Two hemidesmosome components, the alpha6beta4 integrin heterodimer and a human autoantigen termed BP180, are transmembrane proteins that link the extracellular matrix to the keratin network in cells. Here, we report that actinin-4, an actin-bundling protein, is a potential binding partner for BP180. Using yeast two-hybrid, we have mapped the binding site for BP180 to the C-terminal region of actinin-4. This site contains two EF-hand, Ca2+ regulation domains and shares 87% sequence homology with the same region in actinin-1. Consistent with this, BP180 can bind actinin-1 in both the yeast two-hybrid assay and in immunoprecipitation assays. To determine whether the EF-hand domain is a consensus binding sequence for BP180, we tested whether other proteins with this domain bind BP180. None of the proteins tested including calmodulin, with 4 EF-hand domains, and myosin regulatory light chain, with 1 EF-hand domain, interacts with BP180 in yeast two-hybrid system and immunoprecipitation studies, suggesting that the interaction between BP180 and actinin family members is specific. We have compared the distribution of actinin-1 and actinin-4 with that of BP180 in MCF-10A and pp126 cells. Surprisingly, BP180 localizes not only to sites of cell-substratum interaction, but is also present at sites of cell-cell contacts where it co-distributes with both actinin-1 and actinin-4 as well as other adherens junction proteins. In oral tissues, BP180 is present along the basement membrane and at cell-cell contact sites in basal epithelial cells where it co-distributes with adherens junction proteins. Since BP180 antibodies inhibit association of junction proteins at sites of cell-cell contact in oral keratinocytes, these results suggest that BP180 may play a role in establishing cell-cell interactions. We discuss a role for BP180 in crosstalk between cell-matrix and cell-cell junctions.

    Funded by: NIDCR NIH HHS: DE12328; NIDDK NIH HHS: DK60589

    Journal of cell science 2001;114;Pt 23;4197-206

  • Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

    Hüttelmaier S, Illenberger S, Grosheva I, Rüdiger M, Singer RH and Jockusch BM

    Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.

    By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

    The Journal of cell biology 2001;155;5;775-86

  • Proteomic and functional evidence for a P2X7 receptor signalling complex.

    Kim M, Jiang LH, Wilson HL, North RA and Surprenant A

    Institute of Molecular Physiology, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield S10 2TN, UK.

    P2X receptors are ATP-gated ion channels in the plasma membrane, but activation of the P2X7 receptor also leads to rapid cytoskeletal re-arrangements such as membrane blebbing. We identified 11 proteins in human embryonic kidney cells that interact with the rat P2X7 receptor, by affinity purification followed by mass spectroscopy and immunoblotting [laminin alpha3, integrin beta2, beta-actin, alpha-actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase and receptor protein tyrosine phosphatase-beta (RPTPbeta)]. Activation of the P2X7 receptor resulted in its dephosphorylation. Whole-cell recordings from cells expressing P2X7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identified Tyr(343) in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identified a P2X7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPbeta, might exert feedback control of the channel itself through its dephosphorylation.

    The EMBO journal 2001;20;22;6347-58

  • Association of tyrosine phosphatase SHP-2 with F-actin at low cell densities.

    Xu F, Zhao R, Peng Y, Guerrah A and Zhao ZJ

    Department of Medicine/Hematology-Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6305, USA.

    SHP-2 is an intracellular SH2 domain-containing protein-tyrosine phosphatase with an essential role in cell signaling. Here we demonstrate that localization of SHP-2 is regulated by cell density in a cell adhesion-dependent manner. When cells were plated at low densities, SHP-2 was distributed in Triton X-100-insoluble fractions, whereas it was totally soluble when cells were plated at high densities or when low density cells approached confluency. In all cases, the total protein level of SHP-2 was not changed. Fluorescent cell staining revealed that SHP-2 was co-localized with actin stress fibers to the cell peripheral at low cell densities but was diffused in the entire cytoplasm at high cell densities. Transient transfection of cells with truncated forms of SHP-2 demonstrated that the catalytic domain of the enzyme was responsible for the density-regulated distribution of SHP-2, but the catalytic activity was not required. An in vitro co-sedimentation study demonstrated direct binding of full-length and SH2 domain-truncated forms of SHP-2 to F-actin. The data indicate that SHP-2 is regulated by cell density and that it may have a role in assembling and disassembling of the actin network.

    Funded by: NCI NIH HHS: CA-68485, CA75218; NHLBI NIH HHS: HL-57393; NIDDK NIH HHS: DK-15555, T32-DK07186

    The Journal of biological chemistry 2001;276;31;29479-84

  • A point mutation in the alpha-actinin-4 gene generates an antigenic peptide recognized by autologous cytolytic T lymphocytes on a human lung carcinoma.

    Echchakir H, Mami-Chouaib F, Vergnon I, Baurain JF, Karanikas V, Chouaib S and Coulie PG

    Laboratoire Cytokines et Immunologie des tumeurs Humaines, U487 Institut National de la Santé et de la Recherche Médicale, Institut Gustave Roussy, F-94805 Villejuif, Cedex, France.

    We have identified an antigen recognized on a human large cell carcinoma by an autologous tumor-specific CTL clone that was derived from mononuclear cells infiltrating the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and is encoded by the alpha-actinin-4 gene, which is expressed ubiquitously. In the tumor cells, a point mutation generates an amino-acid change that is essential for recognition by the CTLS: The mutation was not found in alpha-actinin-4 cDNA sequences from about 50 lung carcinoma cell lines, suggesting that it is unique to this patient. Although he did not receive chemotherapy or radiotherapy, the patient has been without evidence of tumor since the resection of the primary lesion in 1996. Using tetramers of soluble HLA-A2 molecules loaded with the mutated antigenic peptide, anti-alpha-actinin-4 CTLs could be derived from blood samples collected from the patient in 1998 and 2000. It is possible that these CTLs, recognizing a truly tumor-specific antigen, play a role in the clinical evolution of this lung cancer patient.

    Cancer research 2001;61;10;4078-83

  • Densin-180 forms a ternary complex with the (alpha)-subunit of Ca2+/calmodulin-dependent protein kinase II and (alpha)-actinin.

    Walikonis RS, Oguni A, Khorosheva EM, Jeng CJ, Asuncion FJ and Kennedy MB

    Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.

    Densin-180 is a transmembrane protein that is tightly associated with the postsynaptic density in CNS neurons and is postulated to function as a synaptic adhesion molecule. Here we report the identification of the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alpha-actinin-4 as potential binding partners for the densin-180 intracellular segment. We demonstrate by yeast two-hybrid and biochemical assays that the intracellular portion of densin-180, the alpha-subunit of CaMKII (CaMKIIalpha), and alpha-actinin interact with each other at distinct binding sites and can form a ternary complex stabilized by multiple interactions. Densin-180 binds specifically to the association domain of CaMKIIalpha and does not bind with high affinity to holoenzymes of CaMKII that contain beta-subunit. The PDZ (PSD-95, DIg, Z0-1) domain of densin contributes to its binding to alpha-actinin. A distinct domain of alpha-actinin interacts with the kinase domains of both alpha- and beta-subunits of CaMKII. Autophosphorylation of CaMKII increases its affinity for densin-180 from an EC(50) of >1 micrometer to an EC(50) of <75-150 nM. In contrast, phosphorylation of densin-180 by CaMKII at serine-1397 only slightly decreases its affinity for CaMKII. The specific interaction of densin-180 with holoenzymes of CaMKII containing only alpha-subunit and the increased affinity of CaMKII for densin-180 after autophosphorylation suggest that densin-180 may be involved in localization of activated CaMKII synthesized in dendrites.

    Funded by: NINDS NIH HHS: NS17660, NS28710

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;2;423-33

  • The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site.

    Holliday LS, Lu M, Lee BS, Nelson RD, Solivan S, Zhang L and Gluck SL

    Departments of Medicine and Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA. hollils@medicine.ufl.edu

    Vacuolar H(+)-ATPase (V-ATPase) binds actin filaments with high affinity (K(d) = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.

    Funded by: NIDDK NIH HHS: R01 DK38848, R01 DK52131

    The Journal of biological chemistry 2000;275;41;32331-7

  • CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4.

    Vallenius T, Luukko K and Mäkelä TP

    Haartman Institute & Biocentrum Helsinki, University of Helsinki, 00014 Helsinki, Finland.

    The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of alpha-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicate that both nonmuscle alpha-actinin-1 and alpha-actinin-4 form complexes with CLP-36. The high expression of alpha-actinin-4 in the colon, together with these results, suggests a specific function for the alpha-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells.

    The Journal of biological chemistry 2000;275;15;11100-5

  • Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.

    Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ, Mathis BJ, Rodríguez-Pérez JC, Allen PG, Beggs AH and Pollak MR

    Renal and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding alpha-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant alpha-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type alpha-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.

    Funded by: NIAMS NIH HHS: AR02026, AR44345; NIDDK NIH HHS: DK54931; ...

    Nature genetics 2000;24;3;251-6

  • BERP, a novel ring finger protein, binds to alpha-actinin-4.

    El-Husseini AE, Kwasnicka D, Yamada T, Hirohashi S and Vincent SR

    Graduate Program in Neuroscience, Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.

    We recently identified BERP as a novel RING finger protein belonging to the RBCC protein family. It contains an N-terminal RING finger, followed by a B-box zinc finger and a coiled-coil domain. BERP interacts with the tail domain of the class V myosins through a beta-propeller structure in the BERP C-terminal. To identify other proteins interacting with BERP, the yeast two-hybrid strategy was employed, using the RBCC domain as bait. Screening of a rat brain cDNA library identified alpha-actinin-4 as a specific binding partner for the N-terminus of BERP. This actinin isoform could be immunoprecipitated together with BERP from HEK 293 cells transfected with expression constructs for BERP and alpha-actinin-4. These proteins could also be colocalized immunohistochemically in the cytoplasm of differentiated PC12 cells. We suggest that BERP may anchor class V myosins to particular cell domains via its interaction with alpha-actinin-4.

    Biochemical and biophysical research communications 2000;267;3;906-11

  • The human non-muscle alpha-actinin protein encoded by the ACTN4 gene suppresses tumorigenicity of human neuroblastoma cells.

    Nikolopoulos SN, Spengler BA, Kisselbach K, Evans AE, Biedler JL and Ross RA

    Department of Biological Sciences, Fordham University, Bronx, New York, NY 10458 USA.

    alpha-Actinins are actin-binding proteins important in organization of the cytoskeleton and in cell adhesion. We have cloned and characterized a cDNA from human neuroblastoma cell variants which encodes the second non-muscle alpha-actinin isoform designated ACTN4 (actinin-4). mRNA encoded by the ACTN4 gene, mapped to chromosome 4, is abundant in non-tumorigenic, substrate-adherent human neuroblastoma cell variants but absent or only weakly expressed in malignant, poorly substrate-adherent neuroblasts. It is also present in many adherent tumor cell lines of diverse tissue origins. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle alpha-actinin gene. Expression is correlated with substrate adhesivity. Analysis of deduced amino acid sequences suggests that the two isoforms may differ in function and in regulation by calcium. Moreover, ACTN4 exhibits tumor suppressor activity. Stable clones containing increased levels of alpha-actinin, isolated from highly malignant neuroblastoma stem cells [BE(2)-C] after transfection with a full-length ACTN4 cDNA, show decreased anchorage-independent growth ability, loss of tumorigenicity in nude mice, and decreased expression of the N-myc proto-oncogene.

    Oncogene 2000;19;3;380-6

  • Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

    Zhang H, Wang L, Kao S, Whitehead IP, Hart MJ, Liu B, Duus K, Burridge K, Der CJ and Su L

    The Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.

    Funded by: NCI NIH HHS: CA63071, R01 CA063071; NIAID NIH HHS: AI07273, AI41356, R01 AI041356, R01 AI048407, T32 AI007273; NIGMS NIH HHS: R01 GM029860

    Current biology : CB 1999;9;21;1271-4

  • Actinin-4, a novel actin-bundling protein associated with cell motility and cancer invasion.

    Honda K, Yamada T, Endo R, Ino Y, Gotoh M, Tsuda H, Yamada Y, Chiba H and Hirohashi S

    Pathology Division, National Cancer Center Research Institute, Tokyo 104, Japan.

    Regulation of the actin cytoskeleton may play a crucial role in cell motility and cancer invasion. We have produced a monoclonal antibody (NCC- Lu-632, IgM, k) reactive with an antigenic protein that is upregulated upon enhanced cell movement. The cDNA for the antigen molecule was found to encode a novel isoform of nonmuscle alpha-actinin. This isoform (designated actinin-4) was concentrated in the cytoplasm where cells were sharply extended and in cells migrating and located at the edge of cell clusters, but was absent from focal adhesion plaques or adherens junctions, where the classic isoform (actinin-1) was concentrated. Actinin-4 shifted steadily from the cytoplasm to the nucleus upon inhibition of phosphatidylinositol 3 kinase or actin depolymerization. The cytoplasmic localization of actinin-4 was closely associated with an infiltrative histological phenotype and correlated significantly with a poorer prognosis in 61 cases of breast cancer. These findings suggest that cytoplasmic actinin-4 regulates the actin cytoskeleton and increases cellular motility and that its inactivation by transfer to the nucleus abolishes the metastatic potential of human cancers.

    The Journal of cell biology 1998;140;6;1383-93

  • A locus for inherited focal segmental glomerulosclerosis maps to chromosome 19q13.

    Mathis BJ, Kim SH, Calabrese K, Haas M, Seidman JG, Seidman CE and Pollak MR

    Department of Medicine, Oklahoma State University College of Osteopathic Medicine, Tulsa, USA.

    We performed a genome-wide linkage analysis search for a genetic locus responsible for kidney dysfunction in a large family. This inherited condition, characterized by proteinuria, progressive renal insufficiency, and focal segmental glomerulosclerosis, follows autosomal dominant inheritance. We show with a high degree of certainty (maximum 2-point lod score 12.28) that the gene responsible for this condition is located on chromosome 19q13.

    Kidney international 1998;53;2;282-6

  • Activation of human neutrophils induces an interaction between the integrin beta 2-subunit (CD18) and the actin binding protein alpha-actinin.

    Pavalko FM and LaRoche SM

    Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.

    Mac-1 and LFA-1, members of the leukocyte or CD18 integrin subfamily of adhesion molecules, rapidly change from a low avidity to a high avidity state on activation of neutrophils by various agonists. The control of CD18 integrin-dependent neutrophil adhesion and the mechanisms that regulate integrin avidity are poorly understood. Cytoplasmic domain deletion experiments indicate that the cytoplasmic domains of integrins are necessary for proper integrin function and suggest that interactions with intracellular proteins are involved. We have focused on identifying cytoskeletal proteins that interact with the cytoplasmic domain of the beta-subunit (beta 2 or CD18) common to the leukocyte subfamily of integrins, which include LFA-1, Mac-1, and p150,95. The actin binding protein alpha-actinin associates in vitro with a peptide corresponding to a portion of the CD18 cytoplasmic domain in solid phase binding assays and affinity chromatography experiments. The peptide sequence within the CD18 cytoplasmic domain that binds alpha-actinin is homologous with a region in the cytoplasmic domain of the integrin beta 1-subunit, which also binds alpha-actinin. We demonstrate that the association of alpha-actinin with CD18 is physiologically relevant by coimmunoprecipitating CD18 with alpha-actinin from stimulated human neutrophils under nondenaturing conditions. Using a mAb against CD18 to probe Western blots of immunoprecipitated complexes, CD18 was found to coprecipitate with alpha-actinin when cells were activated with the chemotactic peptide FMLP or with the cytokines leukotriene B4 or TNF-alpha. Very little CD18 coprecipitates with alpha-actinin from unactivated cells. FMLP concentrations as low as 10 nM were sufficient to induce the association of CD18 with alpha-actinin; very little association was detected in cells activated with 1 nM FMLP. The association between alpha-actinin and CD18 was transient, peaking 5-10 min after activation and decreasing to near resting levels by 20 min. CD18 did not coimmunoprecipitate with talin or vinculin in vivo. We conclude that activation of neutrophils results in an alpha-actinin-mediated association between CD18 integrins and actin filaments. In addition to its actin bundling activity, alpha-actinin has a major function as an actin membrane linker molecule, and integrin avidity may be affected by an association with the actin cytoskeleton involving alpha-actinin.

    Journal of immunology (Baltimore, Md. : 1950) 1993;151;7;3795-807

  • Treatment of Haemophilus aphrophilus endocarditis with ciprofloxacin.

    Dawson SJ and White LA

    Department of Microbiology, Southampton General Hospital, U.K.

    A patient with Haemophilus aphrophilus endocarditis was successfully treated with ciprofloxacin. The response to treatment with cefotaxime and netilmicin for 12 days was poor but was satisfactory to a 6 weeks' course of ciprofloxacin.

    The Journal of infection 1992;24;3;317-20

  • Alpha-actinin and vinculin in human neutrophils: reorganization during adhesion and relation to the actin network.

    Yürüker B and Niggli V

    Department of Pathology, University of Bern, Switzerland.

    We have studied the reorganization of vinculin and alpha-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twelve minutes after addition of cells to glass, vinculin was found to become concentrated in small patches at the cell periphery, partially colocalizing with the black IRM areas and with small F-actin-containing adherent protrusions. In contrast, vinculin was not significantly enriched in the less adherent F-actin-rich large pseudopods. alpha-Actinin became enriched during cell adhesion in retraction fibers and, in 40-50% of the inspected cells, also in large less adherent pseudopods where it colocalized with F-actin. The latter finding suggests a continuous dynamic reorganization of pseudopods, with incorporation of alpha-actinin at a certain stage. Disruption of the actin network with cytochalasin D revealed a differential interaction of alpha-actinin and vinculin with the actin network. alpha-Actinin was strongly influenced by cytochalasin D, comparable to F-actin, and both proteins formed colocalizing peripheral caps in 10(-5) M of the drug. Vinculin organization in contrast was not affected by up to 10(-6) M cytochalasin. At 10(-5) M of the drug, however, the patches disappeared completely, vinculin now assuming a diffuse cytoplasmic location. Our results suggest a specialized function of vinculin in adhesion sites of human neutrophils, whereas alpha-actinin may structure the actin network in retraction fibers and in less adherent pseudopods.

    Journal of cell science 1992;101 ( Pt 2);403-14

  • An interaction between alpha-actinin and the beta 1 integrin subunit in vitro.

    Otey CA, Pavalko FM and Burridge K

    Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599.

    A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.

    Funded by: NCI NIH HHS: CA-08493; NIGMS NIH HHS: GM-29860

    The Journal of cell biology 1990;111;2;721-9

Gene lists (7)

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
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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