G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
actinin, alpha 1
G00000542 (Mus musculus)

Databases (8)

ENSG00000072110 (Ensembl human gene)
87 (Entrez Gene)
964 (G2Cdb plasticity & disease)
ACTN1 (GeneCards)
102575 (OMIM)
Marker Symbol
Protein Expression
4303 (human protein atlas)
Protein Sequence
P12814 (UniProt)

Literature (95)

Pubmed - other

  • 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

  • Cathepsin X cleaves the beta2 cytoplasmic tail of LFA-1 inducing the intermediate affinity form of LFA-1 and alpha-actinin-1 binding.

    Jevnikar Z, Obermajer N, Pecar-Fonović U, Karaoglanovic-Carmona A and Kos J

    Faculty of Pharmacy, University of Ljubljana, Askerceva, Ljubljana, Slovenia. zala.Jevnikar@ffa.uni-lj.si

    The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the beta(2) chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S(769), E(768) and A(767) from the C-terminal of the beta(2) cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein alpha-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with alpha-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration.

    European journal of immunology 2009;39;11;3217-27

  • Expression and tissue localization of beta-catenin, alpha-actinin and chondroitin sulfate proteoglycan 6 is modulated during rat and human left ventricular hypertrophy.

    Ridinger H, Rutenberg C, Lutz D, Buness A, Petersen I, Amann K and Maercker C

    RZPD German Resource Center for Genome Research, 69120 Heidelberg, Germany.

    Left ventricular hypertrophy (LVH) correlates with chronic renal failure and is one of the most important causes of cardiac mortality. The understanding of the molecular complexity of the disease will help to find biomarkers that open new perspectives about early diagnosis and therapy. This work describes the identification of mediators during pathogenesis relevant for structural remodeling processes of cardiac tissue in uremic LVH. An established rat model of chronic renal failure allowed whole-genome transcriptome analyses as well as the investigation of differential expressed proteins in uremic LVH. The localization of potential biomarkers encoded by candidate genes was done by immunohistochemical analyses of cardiac tissue of the animal model as well as cardiac sections of LVH diseased patients. In addition, the induction of human cardiac fibroblasts (HCF) and human umbilical vein endothelial cells (HUVEC) with the LVH mediator angiotensin II enabled us to investigate uremic LVH progression in vitro. These results point to alterations of myocardial intercellular and cell-matrix contacts in hypertrophic cardiac tissue. Obviously, structural changes of the extracellular matrix are significantly modulated by beta-catenin associated signaling pathways. Interestingly, intracellular translocation of beta-catenin, alpha-actinin and chondroitin sulfate proteoglycan 6 (CSPG6/SMC3) was observed in the animal model and in LVH patients. Our results show that the parallel investigation of rat and human cardiac tissue as well as human cellular models in vitro represents a promising strategy to identify reliable biomarkers of LVH.

    Experimental and molecular pathology 2009;86;1;23-31

  • Genome-wide and candidate gene association study of cigarette smoking behaviors.

    Caporaso N, Gu F, Chatterjee N, Sheng-Chih J, Yu K, Yeager M, Chen C, Jacobs K, Wheeler W, Landi MT, Ziegler RG, Hunter DJ, Chanock S, Hankinson S, Kraft P and Bergen AW

    Division of Cancer Epidemiology and Genetics, NCI, Bethesda, Maryland, United States of America. caporaso@nih.gov

    The contribution of common genetic variation to one or more established smoking behaviors was investigated in a joint analysis of two genome wide association studies (GWAS) performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project in 2,329 men from the Prostate, Lung, Colon and Ovarian (PLCO) Trial, and 2,282 women from the Nurses' Health Study (NHS). We analyzed seven measures of smoking behavior, four continuous (cigarettes per day [CPD], age at initiation of smoking, duration of smoking, and pack years), and three binary (ever versus never smoking, < or = 10 versus > 10 cigarettes per day [CPDBI], and current versus former smoking). Association testing for each single nucleotide polymorphism (SNP) was conducted by study and adjusted for age, cohabitation/marital status, education, site, and principal components of population substructure. None of the SNPs achieved genome-wide significance (p<10(-7)) in any combined analysis pooling evidence for association across the two studies; we observed between two and seven SNPs with p<10(-5) for each of the seven measures. In the chr15q25.1 region spanning the nicotinic receptors CHRNA3 and CHRNA5, we identified multiple SNPs associated with CPD (p<10(-3)), including rs1051730, which has been associated with nicotine dependence, smoking intensity and lung cancer risk. In parallel, we selected 11,199 SNPs drawn from 359 a priori candidate genes and performed individual-gene and gene-group analyses. After adjusting for multiple tests conducted within each gene, we identified between two and five genes associated with each measure of smoking behavior. Besides CHRNA3 and CHRNA5, MAOA was associated with CPDBI (gene-level p<5.4x10(-5)), our analysis provides independent replication of the association between the chr15q25.1 region and smoking intensity and data for multiple other loci associated with smoking behavior that merit further follow-up.

    Funded by: Intramural NIH HHS; NCI NIH HHS: P01 CA087969, T32 CA009547; NIDA NIH HHS: U01 DA020830

    PloS one 2009;4;2;e4653

  • Smooth muscle titin Zq domain interaction with the smooth muscle alpha-actinin central rod.

    Chi RJ, Simon AR, Bienkiewicz EA, Felix A and Keller TC

    Department of Biological Science, College of Medicine, Florida State University, Tallahassee, FL 32306, USA.

    Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein alpha-actinin. In striated muscle Z-disks, alpha-actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in alpha-actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the alpha-actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to alpha-actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in alpha-actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for alpha-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the alpha-actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the alpha-actinin central rod.

    Funded by: NIBIB NIH HHS: R01 EB 006158

    The Journal of biological chemistry 2008;283;30;20959-67

  • Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis.

    Thorsen K, Sørensen KD, Brems-Eskildsen AS, Modin C, Gaustadnes M, Hein AM, Kruhøffer M, Laurberg S, Borre M, Wang K, Brunak S, Krainer AR, Tørring N, Dyrskjøt L, Andersen CL and Orntoft TF

    Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, DK-8200 Aarhus N, Denmark.

    Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications.

    Funded by: NCI NIH HHS: P01 CA013106

    Molecular & cellular proteomics : MCP 2008;7;7;1214-24

  • UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) binds to alpha-actinin 1: novel pathways in skeletal muscle?

    Amsili S, Zer H, Hinderlich S, Krause S, Becker-Cohen M, MacArthur DG, North KN and Mitrani-Rosenbaum S

    Goldyne Savad Institute for Gene Therapy, Hadassah Hebrew University Medical Center, Jerusalem, Israel.

    Background: Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle.

    We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed alpha-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line.

    The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of alpha-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM.

    PloS one 2008;3;6;e2477

  • Expression of alpha-actinin-1 in human glomerular mesangial cells in vivo and in vitro.

    Yang C and Glass WF

    8701 Watertown Plank Road, Milwaukee, WI 53226, USA. chenyang@mcw.edu

    Recent studies have demonstrated important roles of alpha-actinins in glomerular disease, while little information is known about the expression profile of alpha-actinins in human glomerular mesangial cells. Here, immunofluorescence and confocal microscopy showed that alpha-actinin-1 exclusively distributed along mesangial cells in human glomeruli of IgA nephropathy. RT-PCR and Western blot further confirmed the expression of alpha-actinin-1 in primary cultured human mesangial cells. We also found that transforming growth factor-beta 1 (TGF-beta 1) stimulated ACTN1 gene transcription and that transiently transfected alpha-actinin-1 significantly increased TGF-beta 1-induced plasminogen activator inhibitor-1 (PAI-1) promoter activity in human mesangial cells. These findings suggest that alpha-actinin-1 may play a role in human glomerular disease.

    Experimental biology and medicine (Maywood, N.J.) 2008;233;6;689-93

  • 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

  • ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma.

    Yoon KJ, Phelps DA, Bush RA, Remack JS, Billups CA and Khoury JD

    Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. karina.yoon@stjude.org

    The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.

    Funded by: NCI NIH HHS: CA21765, P30 CA021765

    PloS one 2008;3;11;e3629

  • Alpha-actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction.

    Craig DH, Haimovich B and Basson MD

    Department of Surgery, John D Dingell Veterans Affairs Medical Center, Wayne State University, and Karmanos Cancer Institute, Detroit, MI, USA.

    Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.

    Funded by: NIDDK NIH HHS: R01 DK06771

    American journal of physiology. Cell physiology 2007;293;6;C1862-74

  • Direct binding of alpha-actinin enhances TRPP3 channel activity.

    Li Q, Dai XQ, Shen PY, Wu Y, Long W, Chen CX, Hussain Z, Wang S and Chen XZ

    Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.

    Transient receptor potential (TRP) polycystin 2 and 3 (TRPP2 and 3) are homologous members of the TRP superfamily of cation channels but have different physiological functions. TRPP2 is part of a flow sensor, and is defective in autosomal dominant polycystic kidney disease and implicated in left-right asymmetry development. TRPP3 is reported to implicate in sour tasting in bipolar cells of taste buds of the tongue and in the regulation of pH-sensitive action potential in neurons surrounding the central canal of spinal cord. TRPP3 is present in both excitable and non-excitable cells in various tissues, such as retina, brain, heart, testis, and kidney, but its common and cell type-specific functional characteristics remain largely unknown. In this study, we investigated physical and functional interactions between TRPP3 and alpha-actinin, an actin-bundling protein known to regulate several types of ion channels. We employed planer lipid bilayer electrophysiology system to study the function of TRPP3 channel that was affinity-purified from Madin-Darby canine kidney cells. Upon reconstitution in bilayer, TRPP3 exhibited cation channel activities that were substantially augmented by alpha-actinin. The TRPP3-alpha-actinin association was documented by co-immunoprecipitation using native cells and tissues, yeast two-hybrid, and in vitro binding assays. Further, TRPP3 was abundantly present in mouse brain where it associates with alpha-actinin-2. Taken together, alpha-actinin not only attaches TRPP3 to the cytoskeleton but also up-regulates TRPP3 channel function. It remains to be determined whether the TRPP3-alpha-actinin interaction is relevant to acid sensing and other functions in neuronal and non-neuronal cells.

    Journal of neurochemistry 2007;103;6;2391-400

  • Phosphoinositide binding regulates alpha-actinin CH2 domain structure: analysis by hydrogen/deuterium exchange mass spectrometry.

    Full SJ, Deinzer ML, Ho PS and Greenwood JA

    Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.

    alpha-Actinin is an actin bundling protein that regulates cell adhesion by directly linking actin filaments to integrin adhesion receptors. Phosphatidylinositol (4,5)-diphosphate (PtdIns (4,5)-P(2)) and phosphatidylinositol (3,4,5)-triphosphate (PtdIns (3,4,5)-P(3)) bind to the calponin homology 2 domain of alpha-actinin, regulating its interactions with actin filaments and integrin receptors. In this study, we examine the mechanism by which phosphoinositide binding regulates alpha-actinin function using mass spectrometry to monitor hydrogen-deuterium (H/D) exchange within the calponin homology 2 domain. The overall level of H/D exchange for the entire protein showed that PtdIns (3,4,5)-P(3) binding alters the structure of the calponin homology 2 domain increasing deuterium incorporation, whereas PtdIns (4,5)-P(2) induces changes in the structure decreasing deuterium incorporation. Analysis of peptic fragments from the calponin homology 2 domain showed decreased local H/D exchange within the loop region preceding helix F with both phosphoinositides. However, the binding of PtdIns (3,4,5)-P(3) also induced increased exchange within helix E. This suggests that the phosphate groups on the fourth and fifth position of the inositol head group of the phosphoinositides constrict the calponin homology 2 domain, thereby altering the orientation of actin binding sequence 3 and decreasing the affinity of alpha-actinin for filamentous actin. In contrast, the phosphate group on the third position of the inositol head group of PtdIns (3,4,5)-P(3) perturbs the calponin homology 2 domain, altering the interaction between the N and C terminus of the full-length alpha-actinin antiparallel homodimer, thereby disrupting bundling activity and interaction with integrin receptors.

    Funded by: NIEHS NIH HHS: ES00040, ES00210, P01 ES000040, P30 ES000210; NIGMS NIH HHS: GM63711, R01 GM062957, R01 GM063711, R01 GM063711-05, R1GM62957A

    Protein science : a publication of the Protein Society 2007;16;12;2597-604

  • Mechanical response and conformational changes of alpha-actinin domains during unfolding: a molecular dynamics study.

    Soncini M, Vesentini S, Ruffoni D, Orsi M, Deriu MA and Redaelli A

    Department of Bioengineering, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133, Milan, Italy. monica.soncini@polimi.it

    Alpha-actinin is a cytoskeleton-binding protein involved in the assembly and regulation of the actin filaments. In this work molecular dynamics method was applied to investigate the mechanical behaviour of the human skeletal muscle alpha-actinin. Five configurations were unfolded at an elongation speed of 0.1 nm/ps in order to investigate the conformational changes occurring during the extension process. Moreover, a sensitivity analysis at different velocities was performed for one of the R2-R3 spectrin-like repeat configuration extracted in order to evaluate the effect of the pulling speed on the mechanical behaviour of the molecule. Two different behaviours were recognized with respect to the pulling speed. In particular, at speed higher than 0.025 nm/ps a continuous rearrangement without evident force peaks was obtained, on the contrary at lower speed evident peaks in the range 500-750 pN were detected. R3 repeat resulted more stable than R2 during mechanical unfolding, due to the lower hydrophobic surface available to the solvent. The characterization of the R2-R3 units can be useful for the development of cytoskeleton network models based on stiffness values obtained by analyses performed at the molecular level.

    Biomechanics and modeling in mechanobiology 2007;6;6;399-407

  • The cytoskeletal proteins alpha-actinin, Ezrin, and talin are De-expressed in endometriosis and endometrioid carcinoma compared with normal uterine epithelium.

    Slater M, Cooper M and Murphy CR

    Department of Anatomy and Histology, School of Biomedical Sciences, The University of Sydney, NSW, Australia. michaels@anatomy.usyd.edu.au

    In this retrospective study on banked tissue, we found that alpha-actinin and talin were completely de-expressed in both endometriosis and endometrioid carcinoma tissue. Some patchy, depolarized labeling for ezrin was noted in the endometrioid carcinoma but not in endometriosis. The loss of these proteins in both endometriosis and endometrioid carcinoma tissue indicates a significant change in the integrity of these tissues compared with normal and the possibility that individual cells may break away from the parent histology due to loss of cell adhesion. It also indicates a similarity between endometrioid cancer and endometriosis with respect to epithelial cell function and adhesion.

    Applied immunohistochemistry & molecular morphology : AIMM 2007;15;2;170-4

  • Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor.

    Cabello N, Remelli R, Canela L, Soriguera A, Mallol J, Canela EI, Robbins MJ, Lluis C, Franco R, McIlhinney RA and Ciruela F

    Institut d'Investigacions Biomèdiques August Pi i Sunyer and Department of Biochemistry and Molecular Biology, University of Barcelona, Facultat de Biologia, Avda. Diagonal 645, Barcelona 08028, Spain.

    Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

    Funded by: Medical Research Council: MC_U138162357

    The Journal of biological chemistry 2007;282;16;12143-53

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

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

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

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

    Cell 2006;127;3;635-48

  • 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

  • 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

  • alpha-Actinin-dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth.

    Nyman-Huttunen H, Tian L, Ning L and Gahmberg CG

    Division of Biochemistry, Faculty of Biosciences, PO Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland.

    Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendrite-expressed membrane glycoprotein of telencephalic neurons in the mammalian brain. By deletion of the cytoplasmic and membrane-spanning domains of ICAM-5, we observed that the membrane distribution of ICAM-5 was determined by the cytoplasmic portion. Therefore we have characterized the intracellular associations of ICAM-5 by using a bacterially expressed glutathione S-transferase (GST) fusion protein encompassing the cytoplasmic part of ICAM-5. One of the main proteins in the neuronal cell line Paju that bound to the ICAM-5 cytodomain was alpha-actinin. ICAM-5 expressed in transfected Paju cells was found in alpha-actinin immunoprecipitates, and ICAM-5 colocalized with alpha-actinin both in Paju cells and in dendritic filopodia and spines of primary hippocampal neurons. We were also able to coprecipitate alpha-actinin from rat brain homogenate. Binding to alpha-actinin appeared to be mediated mainly through the N-terminal region of the ICAM-5 cytodomain, as the ICAM-5(857-861) cytoplasmic peptide (KKGEY) mediated efficient binding to alpha-actinin. Surface plasmon resonance analysis showed that the turnover of the interaction was rapid. In a mutant cell line, Paju-ICAM-5-KK/AA, the distribution was altered, which implies the importance of the lysines in the interaction. Furthermore, we found that the ICAM-5/alpha-actinin interaction is involved in neuritic outgrowth and the ICAM-5(857-861) cytoplasmic peptide induced morphological changes in Paju-ICAM-5 cells. In summary, these results show that the interaction between ICAM-5 and alpha-actinin is mediated through binding of positively charged amino acids near the transmembrane domain of ICAM-5, and this interaction may play an important role in neuronal differentiation.

    Journal of cell science 2006;119;Pt 15;3057-66

  • 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

  • Structure of the alpha-actinin-vinculin head domain complex determined by cryo-electron microscopy.

    Kelly DF, Taylor DW, Bakolitsa C, Bobkov AA, Bankston L, Liddington RC and Taylor KA

    Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA.

    The vinculin binding site on alpha-actinin was determined by cryo-electron microscopy of 2D arrays formed on phospholipid monolayers doped with a nickel chelating lipid. Chicken smooth muscle alpha-actinin was cocrystallized with the beta1-integrin cytoplasmic domain and a vinculin fragment containing residues 1-258 (vinculin(D1)). Vinculin(D1) was located at a single site on alpha-actinin with 60-70% occupancy. In these arrays, alpha-actinin lacks molecular 2-fold symmetry and the two ends of the molecule, which contain the calmodulin-like and actin binding domains, are held in distinctly different environments. The vinculin(D1) difference density has a shape very suggestive of the atomic structure. The atomic model of the complex juxtaposes the alpha-actinin binding site on vinculin(D1) with the N-terminal lobe of the calmodulin-like domain on alpha-actinin. The results show that the interaction between two species with weak affinity can be visualized in a membrane-like environment.

    Funded by: NIGMS NIH HHS: GM64346, U54 GM064346

    Journal of molecular biology 2006;357;2;562-73

  • Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration.

    Zhang Z, Lin SY, Neel BG and Haimovich B

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

    The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.

    Funded by: NHLBI NIH HHS: HL54104

    The Journal of biological chemistry 2006;281;3;1746-54

  • 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

  • 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

  • Phosphorylation-dependent interactions of alpha-Actinin-1/IQGAP1 with the AMPA receptor subunit GluR4.

    Nuriya M, Oh S and Huganir RL

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

    AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors play key roles in excitatory synaptic transmission and synaptic plasticity in the CNS. Although a variety of proteins has been characterized to interact with AMPA receptors and regulate their function, little is known about the regulation of the AMPA receptor subunit GluR4. To understand the molecular mechanisms of GluR4 functional regulation, the yeast two-hybrid system was used to identify GluR4-interacting molecules. alpha-Actinin-1 and IQGAP1 were identified to be GluR4-specific binding partners. Both proteins interact specifically with GluR4 and co-cluster with GluR4 individually in neurons. Mapping experiments revealed that alpha-Actinin-1 and IQGAP1 bind to the same region within the C-terminus of GluR4 that contains a previously identified PKA phosphorylation site, Ser842, phosphorylation of which is regulated by synaptic activity. Interestingly, the phosphorylation of Ser842 differentially regulates interactions of GluR4 with alpha-Actinin-1 and IQGAP1; phosphorylation strongly inhibits interaction of GluR4 with alpha-Actinin-1 but has little effect on its interaction with IQGAP1. These results suggest that alpha-Actinin-1 and IQGAP1 regulate GluR4 functions via their specific associations with GluR4. In addition, our data indicate that activity-dependent phosphorylation of GluR4 may regulate its synaptic targeting through phosphorylation-dependent interactions with alpha-Actinin-1 and IQGAP1.

    Journal of neurochemistry 2005;95;2;544-52

  • Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin.

    Singleton PA, Dudek SM, Chiang ET and Garcia JG

    Division of Pulmonary and Critical Care Medicine, Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 microM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase alpha and beta catalytic subunits, the Rac1 GEF, Tiam1, and alpha-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, alpha-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and alpha-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both alpha-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for alpha-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;12;1646-56

  • Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC.

    Gevaert K, Staes A, Van Damme J, De Groot S, Hugelier K, Demol H, Martens L, Goethals M and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, Ghent, Belgium.

    We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+-immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O-18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190 phosphorylated peptides from 152 different proteins. This dataset includes 38 novel protein phosphorylation sites.

    Proteomics 2005;5;14;3589-99

  • Structural dynamics of alpha-actinin-vinculin interactions.

    Bois PR, Borgon RA, Vonrhein C and Izard T

    Department of Hematology-Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 38105, USA.

    Alpha-actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. alpha-Actinin interacts with vinculin through the binding of an alpha-helix (alphaVBS) present within the R4 spectrin repeat of its central rod domain to vinculin's N-terminal seven-helical bundle domain (Vh1). The Vh1:alphaVBS structure suggests that alphaVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. alphaVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculin's tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, alphaVBS binds to Vh1 in an inverted orientation compared to the binding of talin's VBSs to vinculin. Importantly, the binding of alphaVBS and talin's VBSs to vinculin's Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculin's Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and alpha-actinin binding in focal adhesions versus adherens junctions, respectively.

    Funded by: NCI NIH HHS: CA21765, P30 CA021765; NIGMS NIH HHS: GM071596, R01 GM071596

    Molecular and cellular biology 2005;25;14;6112-22

  • Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling.

    Fraley TS, Pereira CB, Tran TC, Singleton C and Greenwood JA

    Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.

    The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of alpha-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-alpha-actinin and a GFP-alpha-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039-24045). In fibroblasts, recovery of the mutant alpha-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, alpha-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of alpha-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant alpha-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the alpha-actinin mutant inhibited actin reorganization with the appearance of enlarged alpha-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of alpha-actinin with actin filaments and integrin adhesion receptors and that the dynamics of alpha-actinin is important for PI 3-kinase-induced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of alpha-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.

    Funded by: NCRR NIH HHS: 1S10RR107903-01; NIEHS NIH HHS: P30 ES00210; NIGMS NIH HHS: GM63711

    The Journal of biological chemistry 2005;280;15;15479-82

  • Automated yeast two-hybrid screening for nuclear receptor-interacting proteins.

    Albers M, Kranz H, Kober I, Kaiser C, Klink M, Suckow J, Kern R and Koegl M

    PheneX Pharmaceuticals AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany.

    High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.

    Molecular & cellular proteomics : MCP 2005;4;2;205-13

  • 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

  • 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

  • Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction.

    Yamaji S, Suzuki A, Kanamori H, Mishima W, Yoshimi R, Takasaki H, Takabayashi M, Fujimaki K, Fujisawa S, Ohno S and Ishigatsubo Y

    The First Dept. of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.

    The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)-binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with alpha-actinin in an ILK kinase activity-dependent manner, suggesting that integrin-ILK signaling evoked by substrate adhesion induces affixin-alpha-actinin interaction. The overexpression of a peptide corresponding to the alpha-actinin-binding site of affixin as well as the knockdown of endogenous affixin by small interference RNA resulted in the blockade of cell spreading. Time-lapse observation revealed that in both experiments cells were round with small peripheral blebs and failed to develop lamellipodia, suggesting that the ILK-affixin complex serves as an integrin-anchoring site for alpha-actinin and thereby mediates integrin signaling to alpha-actinin, which has been shown to play a critical role in actin polymerization at focal adhesions.

    The Journal of cell biology 2004;165;4;539-51

  • Molecular analysis of the interaction between palladin and alpha-actinin.

    Rönty M, Taivainen A, Moza M, Otey CA and Carpén O

    Biomedicum Helsinki, Department of Pathology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland. mikko.ronty@helsinki.fi

    Palladin is a novel component of stress fiber dense regions. Antisense and transient overexpression studies have indicated an important role for palladin in the regulation of actin cytoskeleton. Palladin colocalizes and coimmunoprecipitates with alpha-actinin, a dense region component, but the molecular details and functional significance of the interaction have not been studied. We show here a direct association between the two proteins and have mapped the binding site within a short sequence of palladin and in the carboxy-terminal calmodulin domain of alpha-actinin. Using transfection-based targeting assays, we show that palladin is involved in targeting of alpha-actinin to specific subcellular foci indicating a functional interplay between the two actin-associated proteins.

    Funded by: NIGMS NIH HHS: GM61743

    FEBS letters 2004;566;1-3;30-4

  • 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

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

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

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

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

    Nature genetics 2004;36;1;40-5

  • Direct interaction between alpha-actinin and hepatitis C virus NS5B.

    Lan S, Wang H, Jiang H, Mao H, Liu X, Zhang X, Hu Y, Xiang L and Yuan Z

    Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China.

    It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication. Our results suggest alpha-actinin may be a component of the HCV replication complex.

    FEBS letters 2003;554;3;289-94

  • 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

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions.

    Garvalov BK, Higgins TE, Sutherland JD, Zettl M, Scaplehorn N, Köcher T, Piddini E, Griffiths G and Way M

    European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

    The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.

    The Journal of cell biology 2003;161;1;33-9

  • The lipoma preferred partner LPP interacts with alpha-actinin.

    Li B, Zhuang L, Reinhard M and Trueb B

    ITI Research Institute, University of Bern, PO Box 54, CH-3010 Bern, Switzerland.

    The lipoma preferred partner LPP is a member of the zyxin family of proteins. In this paper, we demonstrate that the structural similarities observed between zyxin and LPP also extend to their interaction capabilities. Similar to zyxin, LPP was found to bind to alpha-actinin in vitro. This interaction was confirmed in yeast and mammalian cells. Studies utilizing the three-hybrid system further indicated that zyxin and LPP compete for the same binding site in alpha-actinin. This site was mapped to the central rod of alpha-actinin, which contains spectrin-like repeats 2 and 3. In the case of LPP, a conserved motif present at the N-terminus was shown to be responsible for the interaction. Constructs lacking this motif did not bind to alpha-actinin in the yeast two-hybrid system and were not able to recruit alpha-actinin to an ectopic site in mammalian cells. Quantitative data obtained with the two-hybrid and the three-hybrid system suggest that LPP has a lower affinity for alpha-actinin than zyxin. It is likely that this difference leads to slightly different roles played by LPP and zyxin during the assembly and disassembly of focal adhesions.

    Journal of cell science 2003;116;Pt 7;1359-66

  • Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy.

    Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Hoffmann K, Pilz B, Martiniak Y, Gehmlich K, van der Ven PF, Fürst DO, Vornwald A, von Hodenberg E, Nürnberg P, Scheffold T, Dietz R and Osterziel KJ

    Universitätsklinikum Charité/Kardiologie am Campus Buch und Virchow Klinikum, Humboldt-Universität zu Berlin, Berlin, Germany.

    Background: Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans.

    We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with alpha-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to alpha-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP.

    Conclusion: Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

    Circulation 2003;107;10;1390-5

  • Syndecan-4 associates with alpha-actinin.

    Greene DK, Tumova S, Couchman JR and Woods A

    Department of Cell Biology, University of Alabama, Birmingham, Alabama 35294-0006, USA.

    Cell adhesion to the extracellular matrix influences many cellular functions. The integrin family of matrix receptors plays major roles in the formation of adhesions, but other proteins modulate integrin signaling. Syndecan-4, a transmembrane proteoglycan, cooperatively signals with integrins during the formation of focal adhesions. To date, a direct link between syndecan-4 and the cytoskeleton has remained elusive. We now demonstrate by Triton X-100 extraction immunoprecipitation and in vitro binding assays that the focal adhesion component alpha-actinin interacts with syndecan-4 in a beta-integrin-independent manner.

    Funded by: NIGMS NIH HHS: GM50194

    The Journal of biological chemistry 2003;278;9;7617-23

  • ADIP, a novel Afadin- and alpha-actinin-binding protein localized at cell-cell adherens junctions.

    Asada M, Irie K, Morimoto K, Yamada A, Ikeda W, Takeuchi M and Takai Y

    Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/ Faculty of Medicine, Suita 565-0871, Japan.

    Afadin is an actin filament (F-actin)-binding protein that is associated with the cytoplasmic tail of nectin, a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule. Nectin and afadin strictly localize at cell-cell adherens junctions (AJs) undercoated with F-actin bundles and are involved in the formation of AJs in cooperation with E-cadherin in epithelial cells. In epithelial cells of afadin (-/-) mice and (-/-) embryoid bodies, the proper organization of AJs is markedly impaired. However, the molecular mechanism of how the nectin-afadin system is associated with the E-cadherin-catenin system or functions in the formation of AJs has not yet been fully understood. Here we identified a novel afadin-binding protein, named ADIP (afadin DIL domain-interacting protein). ADIP consists of 615 amino acids with a calculated M(r) of 70,954 and has three coiled-coil domains. Northern and Western blot analyses in mouse tissues indicated that ADIP was widely distributed. Immunofluorescence and immunoelectron microscopy revealed that ADIP strictly localized at cell-cell AJs undercoated with F-actin bundles in small intestine absorptive epithelial cells. This localization pattern was the same as those of afadin and nectin. ADIP was undetectable at cell-matrix AJs. ADIP furthermore bound alpha-actinin, an F-actin-bundling protein known to be indirectly associated with E-cadherin through its direct binding to alpha-catenin. These results indicate that ADIP is an afadin- and alpha-actinin-binding protein that localizes at cell-cell AJs and may have two functions. ADIP may connect the nectin-afadin and E-cadherin-catenin systems through alpha-actinin, and ADIP may be involved in organization of the actin cytoskeleton at AJs through afadin and alpha-actinin.

    The Journal of biological chemistry 2003;278;6;4103-11

  • Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly.

    Salmikangas P, van der Ven PF, Lalowski M, Taivainen A, Zhao F, Suila H, Schröder R, Lappalainen P, Fürst DO and Carpén O

    Department of Pathology and Neuroscience Program, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

    The assembly and maintenance of the muscle sarcomere requires a complex interplay of actin- and myosin-associated proteins. Myotilin is a thin filament-associated Z-disc protein that consists of two Ig-domains flanked by a unique serine-rich amino-terminus and a short carboxy-terminal tail. It binds to alpha-actinin and filamin c and is mutated in limb girdle muscular dystrophy 1A (LGMD1A). Here we show that myotilin also directly binds F-actin, efficiently cross-links actin filaments alone or in concert with alpha-actinin and prevents filament disassembly induced by Latrunculin A. Myotilin forms dimers via its carboxy-terminal half, which may be necessary for the actin-bundling activity. Overexpression of full-length myotilin but not the carboxy-terminal half induces formation of thick actin cables in non-muscle cells devoid of endogenous myotilin. The expression of myotilin in muscle cells is tightly regulated to the later stages of in vitro myofibrillogenesis, when preassembled myofibrils begin to align. Expression of either amino- or carboxy-terminally truncated myotilin fragments but not wild-type myotilin in differentiating myocytes leads to myofibril disarray. The disease association and functional characteristics indicate an indispensable role for myotilin in stabilization and anchorage of thin filaments, which may be a prerequisite for correct Z-disc organization.

    Human molecular genetics 2003;12;2;189-203

  • Immunohistochemical localization of human fructose-1,6-bisphosphatase in subcellular structures of myocytes.

    Gizak A, Rakus D and Dzugaj A

    Department of Animal Physiology, Institute of Zoology, Wroclaw Unviersity, Cybulskiego 30, 50-205 Wroclaw, Poland.

    The localization of fructose-1,6-bisphosphatase (FBPase) in human skeletal muscle was determined immunohistochemically using polyclonal antibodies. Light microscopy analysis, confirmed with the use of confocal microscopy, indicated that the enzyme is localized on both sides of the Z line of myocytes. The immunohistochemical investigation was confirmed by a co-sedimentation experiment which revealed that muscle FBPase binds strongly to alpha-actinin--a major structural protein of the Z line. This is the first report on localization of FBPase in skeletal muscle tissue.

    Histology and histopathology 2003;18;1;135-42

  • Prohibitin and prohibitone are contained in high-molecular weight complexes and interact with alpha-actinin and annexin A2.

    Bacher S, Achatz G, Schmitz ML and Lamers MC

    Max Planck Institute for Immunobiology, Stübeweg 51, 79108 Freiburg, Germany.

    The closely related proteins prohibitin (p32) and prohibitone (p37) are evolutionarily conserved with homologues found from cyanobacteria to man. They are thought to be exclusively mitochondrial and have been assigned many-rather different-functions, ranging from a role in lifespan, in mitochondrial inheritance and as chaperones of mitochondrial proteases in yeast. Evidence for a localisation outside of mitochondria has been brought forward in mammalian cells, where they influence cell-cycle progression and are found in association with cell surface receptors. We have employed a yeast two-hybrid screen to identify other interacting proteins and have identified alpha-actinin and annexin A2 as binding partners for prohibitin and prohibitone. Coprecipitation experiments supported the putative binding between prohibitin and prohibitone on the one hand and annexin A2 or alpha-actinin on the other hand in intact cells. Surface plasmon resonance analysis was used to determine relative affinities between prohibitin and alpha-actinin and between prohibitone and annexin A2 and alpha-actinin, respectively. We further show that prohibitin and prohibitone can also form homomeric (preferentially tetrameric) and heteromultimeric complexes, with significant affinities.

    Biochimie 2002;84;12;1207-20

  • Amorphin is phosphorylase; phosphorylase is an alpha-actinin-binding protein.

    Chowrashi P, Mittal B, Sanger JM and Sanger JW

    Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, 19104-6058, USA.

    In a study of myofibrillar proteins, Chowrashi and Pepe [1982: J. Cell Biol. 94:565-573] reported the isolation of a new, 85-kD Z-band protein that they named amorphin. We report that partial sequences of purified amorphin protein indicate that amorphin is identical to phosphorylase, an enzyme important in the metabolism of glycogen. Anti-amorphin antibodies also reacted with purified chicken and rabbit phosphorylase. To explore the basis for phosphorylase's (amorphin's) localization in the Z-bands of skeletal muscles, we reacted biotinylated alpha-actinin with purified amorphin and with purified phosphorylase and found that alpha-actinin bound to each. Radioimmune assays also indicated that phosphorylase (amorphin) bound to alpha-actinin, and, with lower affinity, to F-actin. Negative staining of actin filaments demonstrated that alpha-actinin mediates the binding of phosphorylase to actin filaments. There are several glycolytic enzymes that bind actin (e.g., aldolase, phosphofructokinase, and pyruvate kinase), but phosphorylase is the first one demonstrated to bind alpha-actinin. Localization of phosphorylase in live cells was assessed by transfecting cultures of quail embryonic myotubes with plasmids expressing phosphorylase fused to Green Fluorescent Protein (GFP). This resulted in targeting of the fusion protein to Z-bands accompanied by a diffuse pattern in the cytoplasm.

    Cell motility and the cytoskeleton 2002;53;2;125-35

  • The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.

    Dhavan R, Greer PL, Morabito MA, Orlando LR and Tsai LH

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

    Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;18;7879-91

  • Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase.

    Xu YC, Wu RF, Gu Y, Yang YS, Yang MC, Nwariaku FE and Terada LS

    Department of Internal Medicine, University of Texas Southwestern and The Dallas Veterans Affairs Medical Center, Dallas, Texas 75216, USA.

    We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.

    Funded by: NHLBI NIH HHS: R01 HL061897, R01-HL61897

    The Journal of biological chemistry 2002;277;31;28051-7

  • Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein.

    Vallenius T and Mäkelä TP

    Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, P.O. Box 63, 00014 University of Helsinki, Finland.

    In this report we have characterized a novel, ubiquitously expressed kinase, Clik1, that is predominantly nuclear and undergoes autophosphorylation. Yeast two-hybrid analysis indicated a highly specific association between Clik1 and CLP-36, which was identified in 36 out of 37 Clik1-interacting clones. CLP-36 is a PDZ-LIM protein that localizes to actin stress fibers in nonmuscle cells and associates with alpha-actinin via its PDZ-domain. The association of CLP-36 with Clik1, in turn, is mediated by the C-terminal part of CLP-36 containing the LIM domain, and association was not noted with the closely related ALP PDZ-LIM protein. Interestingly, the association with CLP-36 led to relocalization of the otherwise nuclear Clik1 kinase to actin stress fibers, where it disrupted the periodic staining pattern of CLP-36. Taken together these results establish the CLP-36 PDZ-LIM protein as an adapter, recruiting the Clik1 kinase to actin stress fibers in nonmuscle cells, and suggest that Clik1 represents a novel regulator of actin stress fibers.

    Journal of cell science 2002;115;Pt 10;2067-73

  • Functional consequences of caspase activation in cardiac myocytes.

    Communal C, Sumandea M, de Tombe P, Narula J, Solaro RJ and Hajjar RJ

    Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.

    Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac myofilament including alpha-actin, alpha-actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of alpha-actin and alpha-actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using beta-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca(2+)-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca(2+) relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.

    Funded by: NHLBI NIH HHS: F32 HL010409, F32HL10409-02, HL 57623, HL61557, P01 HL062426, P01-HL62426, R01 HL061557; PHS HHS: R01-63704

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;9;6252-6

  • Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins.

    Frey N and Olson EN

    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, USA.

    The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.

    The Journal of biological chemistry 2002;277;16;13998-4004

  • Intracellular domains of NR2 alter calcium-dependent inactivation of N-methyl-D-aspartate receptors.

    Vissel B, Krupp JJ, Heinemann SF and Westbrook GL

    Molecular Neurobiology Laboratory, the Salk Institute, La Jolla, California, USA.

    At central excitatory synapses, the transient elevation of intracellular calcium reduces N-methyl-D-aspartate (NMDA) receptor activity. Such 'calcium-dependent inactivation' is mediated by interactions of calcium/calmodulin and alpha-actinin with the C terminus of NMDA receptor 1 (NR1) subunit. However, inactivation is also NR2-subunit specific, because it occurs in NR2A- but not NR2C-containing receptors. We examined the molecular basis for NR2-subunit specificity using chimeric and mutated NMDA receptor subunits expressed in HEK293 cells. We report that the intracellular loop immediately distal to the pore-forming P-loop M2 (M2-3 loop), as well as a short region in the C terminus, are involved in NR2-subunit specificity. Within the M2-3 loop, substitution of residue 619 in NR2A (valine) for the corresponding NR2C residue (isoleucine) reduced inactivation without affecting calcium permeability of the channel. In contrast, a Q620E mutation in NR2A reduced the relative calcium permeability without altering inactivation. Mutation of three serine/threonine residues in the M2-3 loop also reduced inactivation, as did substitution of the intracellular C terminus of NR2A for NR2C. We speculate that the M2-3 loop of NR2 modulates calcium-dependent inactivation by interacting with the NR1 C terminus, a region known to be essential for inactivation.

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

    Molecular pharmacology 2002;61;3;595-605

  • Titin mutations as the molecular basis for dilated cardiomyopathy.

    Itoh-Satoh M, Hayashi T, Nishi H, Koga Y, Arimura T, Koyanagi T, Takahashi M, Hohda S, Ueda K, Nouchi T, Hiroe M, Marumo F, Imaizumi T, Yasunami M and Kimura A

    Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan.

    Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.

    Biochemical and biophysical research communications 2002;291;2;385-93

  • Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

    Feng S, Reséndiz JC, Christodoulides N, Lu X, Arboleda D, Berndt MC and Kroll MH

    Veterans' Affairs Medical Center, Baylor College of Medicine and Rice University, Houston, Texas 77030, USA.

    Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.

    Funded by: NHLBI NIH HHS: HL18454, HL65967

    Biochemistry 2002;41;4;1100-8

  • 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

  • Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex.

    Atkinson RA, Joseph C, Kelly G, Muskett FW, Frenkiel TA, Nietlispach D and Pastore A

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

    The interaction between alpha-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of alpha-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.

    Nature structural biology 2001;8;10;853-7

  • Analysis of the alpha-actinin/zyxin interaction.

    Li B and Trueb B

    M. E. Müller Institute, University of Bern, P. O. Box 30, CH-3010 Bern, Switzerland.

    The yeast two-hybrid system was used to search for interaction partners of human zyxin. Screening of two different cDNA libraries, one prepared from human placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, nebulette, and alpha-actinin. The zyxin/alpha-actinin interaction was analyzed in detail. By site-directed mutagenesis, a linear motif of 6 amino acids (Phe-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role. Replacement of a single amino acid within this motif abolished binding to alpha-actinin in blot overlays as well as in living cells. On the other hand, the interaction site in alpha-actinin was mapped to a conformational determinant present in the center of the protein as demonstrated by a fragment deletion analysis. This binding site involved a tandem array of two complete spectrin-like domains. Only fragments that were able to dimerize in yeast also bound to zyxin, suggesting that dimerization of alpha-actinin is essential for zyxin binding.

    The Journal of biological chemistry 2001;276;36;33328-35

  • The cytoskeletal/non-muscle isoform of alpha-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase.

    Izaguirre G, Aguirre L, Hu YP, Lee HY, Schlaepfer DD, Aneskievich BJ and Haimovich B

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

    alpha-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012--37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that alpha-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His(6) tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant alpha-actinin protein containing a Tyr --> Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored alpha-actinin phosphorylation. Purified wild type alpha-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of alpha-actinin that cosedimented with actin filaments. These results establish that alpha-actinin is a direct substrate for FAK and suggest that alpha-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton.

    Funded by: NCI NIH HHS: R29 CA75240; NHLBI NIH HHS: HL-541044

    The Journal of biological chemistry 2001;276;31;28676-85

  • Crystal structure of the alpha-actinin rod reveals an extensive torsional twist.

    Ylänne J, Scheffzek K, Young P and Saraste M

    European Molecular Biology Laboratory, EMBL, Structural and Computational Biology Programme, Meyerhofstrasse 1, D-69117, Heidelberg, Germany. ylanne@embl-heidelberg.de

    Background: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins.

    Results: We report here the crystal structure of the alpha-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface.

    Conclusions: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.

    Structure (London, England : 1993) 2001;9;7;597-604

  • A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain.

    Joseph C, Stier G, O'Brien R, Politou AS, Atkinson RA, Bianco A, Ladbury JE, Martin SR and Pastore A

    NIMR, The Ridgeway, London NW7 1AA, UK.

    Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.

    Funded by: Medical Research Council: MC_U117584256

    Biochemistry 2001;40;16;4957-65

  • Calsarcins, a novel family of sarcomeric calcineurin-binding proteins.

    Frey N, Richardson JA and Olson EN

    Departments of Molecular Biology and Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

    The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;26;14632-7

  • Human CLP36, a PDZ-domain and LIM-domain protein, binds to alpha-actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells.

    Bauer K, Kratzer M, Otte M, de Quintana KL, Hagmann J, Arnold GJ, Eckerskorn C, Lottspeich F and Siess W

    Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, München, Germany.

    A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36. The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells. CLP36 binds to alpha-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with alpha-actinin-1 along endothelial actin stress fibers. In contrast to alpha-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells. The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent protein fusion proteins to stress fibers in endothelial cells. Yeast 2-hybrid analysis demonstrated that the intervening sequence, but not the PDZ or the LIM domain of CLP36, binds to the spectrinlike repeats 2 and 3 of alpha-actinin-1. The study further shows that CLP36 binds to alpha-actinin in resting platelets and translocates as a CLP36/alpha-actinin complex to the newly formed actin cytoskeleton in activated platelets. The results indicate that CLP36 binds via alpha-actinin-1 to actin filaments and stress fibers in activated human platelets and endothelial cells. The study suggests that CLP36 may direct alpha-actinin-1 to specific actin structures and at this position might modulate the function of alpha-actinin-1. (Blood. 2000;96:4236-4245)

    Blood 2000;96;13;4236-45

  • Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation.

    Geng L, Burrow CR, Li HP and Wilson PD

    Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, Box 1243, 1 Gustave L. Levy Place, 10029, New York, NY 10029, USA.

    Mutations in the PKD1 gene are responsible for >85% of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1, polycystin-1, is a large, modular membrane protein, with putative ligand-binding motifs in the extracelluar N-terminal portion, 9-11 transmembrane domains and an intracellular C-terminal portion with phosphorylation sites. A role for polycystin-1 as a cell surface receptor involved in cell-matrix and cell-cell interactions has been proposed. In this study, we have analyzed polycystin-1 and associated protein distribution in normal human epithelial cells and examined the role of cell-matrix versus cell-cell interactions in regulation of the assembly of polycystin-1 multiprotein complexes. Immunocytochemistry, sucrose density gradient sedimentation, co-immunoprecipitation analyses and in vitro binding assays have shown that polycystin-1 associates with the focal adhesion proteins talin, vinculin, p130Cas, FAK, alpha-actinin, paxillin and pp60c-src in subconfluent normal human fetal collecting tubule (HFCT) epithelia when cell-matrix interactions predominate. Polycystin-1 also forms higher S value complexes with the cell-cell adherens junction proteins E-cadherin, beta- and gamma-catenins in confluent cultures when cell-cell interactions are predominant. Polycystin-1 multiprotein complexes can be disrupted by cytochalasin D but not by colchicine, suggesting involvement of the actin cytoskeleton. Although inhibition of tyrosine phosphorylation by tyrphostin inhibits polycystin-1-FAK interactions, E-cadherin interactions are enhanced. High calcium treatment also increases polycystin-1-E-cadherin interactions.

    Funded by: NIDDK NIH HHS: F32 DK 09778-01, R01 DK 40698, R01 DK 44833

    Biochimica et biophysica acta 2000;1535;1;21-35

  • Fine mapping of the alpha-actinin binding site within cysteine-rich protein.

    Harper BD, Beckerle MC and Pomiès P

    Department of Biology and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112-5550, USA.

    The cysteine-rich proteins (CRPs) are a family of highly conserved LIM (an acronym derived from the three gene products lin-11, isl-1 and mec-3) domain proteins that have been implicated in muscle differentiation. All CRP family members characterized so far have been shown to interact with the filamentous actin cross-linker alpha-actinin. The region of CRP required for this interaction has previously been broadly mapped to the molecule's N-terminal half. Here we report that the alpha-actinin-binding region of CRP, which we have mapped by using a combination of blot overlay and Western immunoblot techniques, is confined to an 18-residue sequence occurring within the protein's N-terminal glycine-rich repeat. A site-directed mutagenesis analysis of the binding region has revealed the critical importance of a single lysine residue (lysine 65 in human CRP1). Alterations at this site lead to a 10-fold decrease in alpha-actinin binding in comparison with wild-type CRP. The critical lysine residue localizes within a short alpha-helix, raising the possibility that mutagenesis-induced alterations in alpha-actinin-binding capacity might be attributed to the disruption of a key structural element.

    Funded by: NHLBI NIH HHS: HL60591, R01 HL060591

    The Biochemical journal 2000;350 Pt 1;269-74

  • Cardiac phospholipase D2 localizes to sarcolemmal membranes and is inhibited by alpha-actinin in an ADP-ribosylation factor-reversible manner.

    Park JB, Kim JH, Kim Y, Ha SH, Yoo JS, Du G, Frohman MA, Suh PG and Ryu SH

    Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.

    Myocardial phospholipase D (PLD) has been implicated in the regulation of Ca(2+) mobilization and contractile performance in the heart. However, the molecular identity of this myocardial PLD and the mechanisms that regulate it are not well understood. Using subcellular fractionation and Western blot analysis, we found that PLD2 is the major myocardial PLD and that it localizes primarily to sarcolemmal membranes. A 100-kDa PLD2-interacting cardiac protein was detected using a protein overlay assay employing purified PLD2 and then identified as alpha-actinin using peptide-mass fingerprinting with matrix-assisted laser desorption/ionization mass spectroscopy. The direct association between PLD2 and alpha-actinin was confirmed using an in vitro binding assay and localized to PLD2's N-terminal 185 amino acids. Purified alpha-actinin potently inhibits PLD2 activity (IC(50) = 80 nm) in an interaction-dependent and ADP-ribosylation factor-reversible manner. Finally, alpha-actinin co-localizes with actin and with PLD2 in the detergent-insoluble fraction from sarcolemmal membranes. These results suggest that PLD2 is reciprocally regulated in sarcolemmal membranes by alpha-actinin and ARF1 and accordingly that a major role for PLD2 in cardiac function may involve reorganization of the actin cytoskeleton.

    Funded by: NIGMS NIH HHS: GM54813

    The Journal of biological chemistry 2000;275;28;21295-301

  • ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain.

    Nakagawa N, Hoshijima M, Oyasu M, Saito N, Tanizawa K and Kuroda S

    Department of Structural Molecular Biology, Osaka University, Japan.

    The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

    Biochemical and biophysical research communications 2000;272;2;505-12

  • Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli.

    Berryman M and Bretscher A

    Department of Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, Ohio 45701, USA. berryman@ohiou.edu

    The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.

    Funded by: NIGMS NIH HHS: GM14352, GM36652, R01 GM036652

    Molecular biology of the cell 2000;11;5;1509-21

  • 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

  • Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.

    Salmikangas P, Mykkänen OM, Grönholm M, Heiska L, Kere J and Carpén O

    Department of Pathology, University of Helsinki, Haartman Institute, Helsinki, Finland.

    The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.

    Human molecular genetics 1999;8;7;1329-36

  • An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment.

    Reinhard M, Zumbrunn J, Jaquemar D, Kuhn M, Walter U and Trueb B

    Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Versbacher Strasse 5, D-97078 Würzburg, Germany. Matthias.Reinhard@mail.uniwuerzburg.de

    The LIM domain protein zyxin is a component of adherens type junctions, stress fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we demonstrate that human zyxin, like the avian protein, specifically interacts with alpha-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin. This motif is both necessary and sufficient for alpha-actinin binding, whereas a downstream region, which is related in sequence, appears to be dispensable. A synthetic peptide comprising human zyxin residues 21-42 specifically binds to alpha-actinin in solid phase binding assays. In contrast to full-length zyxin, constructs lacking this motif do not interact with alpha-actinin in blot overlays and fail to recruit alpha-actinin in living cells. When zyxin lacking the alpha-actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting to focal adhesions is grossly impaired. Our results suggest a crucial role for the alpha-actinin-zyxin interaction in subcellular zyxin localization and microfilament organization.

    The Journal of biological chemistry 1999;274;19;13410-8

  • Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton.

    Bunn RC, Jensen MA and Reed BC

    The Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, Shreveport, Louisiana 71130-3932, USA.

    Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin-Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, alpha-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

    Molecular biology of the cell 1999;10;4;819-32

  • Functional dissection of nebulette demonstrates actin binding of nebulin-like repeats and Z-line targeting of SH3 and linker domains.

    Moncman CL and Wang K

    Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, USA.

    Nebulette, a 107 kDa protein associated with the I-Z-I complex of cardiac myofibrils, may play an important role in the assembly of the Z-line. Determination of the complete primary structure of 1011 residue human fetal nebulette reveals a four-domain layout similar to skeletal muscle nebulin: a short N-terminal domain, followed by 22 nebulin-like repeats that are linked to a C-terminal Src homology 3 (SH3) domain via a short linker domain. To elucidate the mechanisms of assembly for nebulette in the Z-line, the complete coding sequence or fusions of nebulette domains with green fluorescent protein (GFP) were expressed in cardiomyocytes and fibroblasts. The complete protein localized to Z-lines in cardiac cells and to dense bodies in nonmuscle cells. The GFP-repeat domain forms bundles that are associated with actin filaments in both cell types and disrupts the microfilament network. In contrast, the GFP-repeat plus linker shows limited interaction with dense bodies in nonmuscle cells and the Z-lines of cardiomyocytes. Interestingly, the tagged linker or SH3 is diffusely distributed in nonmuscle cells, but localizes to the Z-lines in cardiomyocytes. Supporting the cellular localization work, recombinant nebulette fragments bind to actin, tropomyosin, and alpha-actinin in in vitro binding assays. These results suggest the repeat domain contains actin binding functions and that the linker domain may target this interaction to Z-lines and dense bodies. Our data also indicate that the linker and SH3 domains can distinguish between dense bodies and Z-lines, suggesting that the ligands for their interactions are specific to these muscular substructures.

    Funded by: NIAMS NIH HHS: AR43514

    Cell motility and the cytoskeleton 1999;44;1;1-22

  • Integrins and development: how might these receptors regulate differentiation of the lens.

    Menko S, Philp N, Veneziale B and Walker J

    Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Integrins transduce both internal signals and signals from the matrix. These interactions between integrins, their extracellular matrix ligands, and their cytoskeletal partners play an important role in the regulation of cellular differentiation. We have shown them to be important in lens cell differentiation. In the lens capsule there is a compartmentalization of matrix components with fibronectin, primarily localized to the anterior capsule, and tenascin in the posterior capsule. Integrins are developmentally regulated in the lens. alpha 5 beta 1 integrin, like fibronectin, is primarily associated with the lens epithelial cells, where together they are likely to be important in regulation of adhesion and proliferation. alpha 6A beta 1, the integrin laminin receptor, is expressed at its highest levels in the equatorial epithelium and the peripheral fiber cells, both migratory populations. Because laminin is uniformly distributed in the lens capsule, such changes in alpha 6A integrin expression are likely critical to the cell's ability to regulate its response to laminin in the matrix. The organization of cytoskeletal molecules associated with the integrin cytoplasmic face also changes with development. In the epithelial regions of the lens, where the initiation of lens cell differentiation occurs, expression of the cytoskeletal proteins involved in cell-substrate interactions, talin, alpha-actinin, and the signaling proteins, are high. In the fiber cell region of the lens, where the cells establish stable cell-cell contacts, vinculin predominates and becomes highly associated with the cytoskeletal fraction. The role of integrins in lens development is not only regulated by changes in the expression of different integrin receptors but is also closely correlated with the expression and organization of the molecules with which they associate.

    Annals of the New York Academy of Sciences 1998;842;36-41

  • The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton.

    Hazan RB and Norton L

    Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. rhazan@smtplink.mssm.edu

    Alterations in the expression or function of molecules that affect cellular adhesion and proliferation are thought to be critical events for tumor progression. Loss of expression of the cell adhesion molecule E-cadherin and increased expression of the epidermal growth factor receptor are two prominent molecular events that are associated with tumorigenesis. The regulation of E-cadherin-dependent cell adhesion by epidermal growth factor (EGF) was therefore examined in the human breast cancer cell line, MDA-MB-468. In this study, changes were observed in the subcellular distribution of components that mediate the cytoplasmic connection between E-cadherin and the actin-based cytoskeleton in response to activation of the EGF receptor. Serum withdrawal activated E-cadherin-dependent cell-cell aggregation in MDA-MB-468 cells, and this treatment stimulated the interaction of actin, alpha-actinin, and vinculin with E-cadherin complexes, despite the absence of alpha-catenin in these cells. By contrast, the co-precipitation of actin with E-cadherin was not detected in several alpha-catenin positive epithelial cell lines. Treatment with EGF inhibited cellular aggregation but did not affect either the levels of E-cadherin or catenin expression nor the association of catenins (beta-catenin, plakoglobin/gamma-catenin, or p120(cas)) with E-cadherin. However, EGF treatment of the MDA-MB-468 cell line dissociated actin, alpha-actinin, and vinculin from the E-cadherin-catenin complex, and this coincided with a robust phosphorylation of beta-catenin, plakoglobin/gamma-catenin, and p120(cas) on tyrosine residues. Furthermore, inactivation of the EGF receptor in serum-treated MDA-MB-468 cells with either a function-blocking antibody or EGF receptor kinase inhibitors mimicked the effects of serum starvation by stimulating both cellular aggregation and assembly of E-cadherin complexes with vinculin and actin. These results demonstrate that the EGF receptor directly regulates cell-cell adhesion through modulation of the interaction of E-cadherin with the actin cytoskeleton and thus substantiates the coordinate role of both of these molecules in tumor progression and metastasis.

    Funded by: NCI NIH HHS: 1P50CA68425

    The Journal of biological chemistry 1998;273;15;9078-84

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Interaction of PKN with alpha-actinin.

    Mukai H, Toshimori M, Shibata H, Takanaga H, Kitagawa M, Miyahara M, Shimakawa M and Ono Y

    Radioisotope Research Center, Kobe University, Kobe 657, Japan.

    PKN is a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein alpha-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and alpha-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoA-binding domain was sufficiently shown to associate with alpha-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type alpha-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type alpha-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type alpha-actinin in a Ca2+-insensitive manner. alpha-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and alpha-actinin lacking the actin-binding domain. In vitro translated full-length alpha-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4, 5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length alpha-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and alpha-actinin.

    The Journal of biological chemistry 1997;272;8;4740-6

  • Identification of the 70kD heat shock cognate protein (Hsc70) and alpha-actinin-1 as novel phosphotyrosine-containing proteins in T lymphocytes.

    Egerton M, Moritz RL, Druker B, Kelso A and Simpson RJ

    Transplantation Biology Unit, Queensland Institute for Medical Research, Brisbane, Australia. markE@qimr.edu.au

    T cell antigen receptor (TCR) ligation results in the tyrosine phosphorylation of numerous intracellular protein substrates, and the identification of these substrates has been a major undertaking by several groups. We have used pervanadate treatment to artificially increase cellular phosphotyrosine levels and immobilized anti-phosphotyrosine monoclonal antibodies to partially purify tyrosine phosphorylated proteins in quantities suitable for amino acid sequencing. This strategy was used to identify three phosphotyrosine containing proteins, with relative molecular masses of 105, 81, and 76 kD by amino acid sequencing. Here we report the identification of pp105 as alpha-actinin-1, pp81 as the murine equivalent of the HS1 gene product, and pp76 as Hsc70. This is the first report that alpha-actinin-1 and Hsc70 are targets of activated tyrosine kinases. Furthermore, we show that Hsc70 is tyrosine phosphorylated in response to TCR ligation, which constitutes the first evidence that Hsc70 might be subject to regulation by tyrosine kinase signaling pathways.

    Biochemical and biophysical research communications 1996;224;3;666-74

  • The cleavage of host cell proteins by HIV-1 protease.

    Snásel J and Pichová I

    Department of Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

    Folia biologica 1996;42;5;227-30

  • Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin.

    Knudsen KA, Soler AP, Johnson KR and Wheelock MJ

    Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096, USA.

    Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E-, P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta-catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha-actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin-independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin.

    Funded by: NIGMS NIH HHS: GM 51188

    The Journal of cell biology 1995;130;1;67-77

  • Identification of the cytoskeletal protein alpha-actinin as a platelet thrombospondin-binding protein.

    Dubernard V, Faucher D, Launay JM and Legrand C

    Unité INSERM 353, Hôpital Saint-Louis, Paris, France.

    Binding of the alpha-granular thrombospondin (TSP) to the plasma membrane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not been elucidated. Using a ligand blot binding assay where electrophoretically separated platelet proteins were incubated with purified 125I-labeled TSP, we observed a strong interaction of [125I]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, the 100 kDa protein was predominantly localized in the cytosol from which it was purified by preparative electrophoresis and was identified by amino acid sequencing to the cytoskeletal protein, alpha-actinin. We further demonstrated that [125I]TSP interacts with alpha-actinin in a specific manner and with a high affinity (Kd = 6.6 nM) in a solid-phase binding assay.

    FEBS letters 1995;364;2;109-14

  • Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening.

    Robertson NG, Khetarpal U, Gutiérrez-Espeleta GA, Bieber FR and Morton CC

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

    We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with 32P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than with brain cDNA probes revealed some previously characterized genes, including mitochondrial sequences, collagen type I alpha-2 (COL1A2), collagen type II alpha-1 (COL2A1), collagen type III alpha-1 (COL3A1), spermidine/spermine N1-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, showing differential expression of this novel gene in the cochlea.

    Funded by: NHLBI NIH HHS: T32 HL07627; NIDCD NIH HHS: DC00871

    Genomics 1994;23;1;42-50

  • Modulation of alpha-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells.

    Glück U and Ben-Ze'ev A

    Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.

    alpha-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, alpha-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of beta 1-integrin. The expression of alpha-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Glück, U., Kwiatkowski, D. J. and Ben-Ze'ev, A. Proc. Nat. Acad. Sci. USA 90, 383-387, 1993). To study the effect of changes in alpha-actinin levels on cell behavior, alpha-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle alpha-actinin cDNA. To suppress alpha-actinin levels, 3T3 cells were transfected with an antisense alpha-actinin cDNA construct. Cells overexpressing alpha-actinin by 40-60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of alpha-actinin was reduced to 25-60% of control levels, after antisense alpha-actinin transfection, had an increased cell motility. Moreover, such alpha-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in alpha-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased alpha-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.

    Journal of cell science 1994;107 ( Pt 7);1773-82

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • 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

  • Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments.

    Tokuue Y, Goto S, Imamura M, Obinata T, Masaki T and Endo T

    Department of Biology, Chiba University, Japan.

    alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments.

    Experimental cell research 1991;197;2;158-67

  • Non-viral cellular substrates for human immunodeficiency virus type 1 protease.

    Shoeman RL, Kesselmier C, Mothes E, Höner B and Traub P

    Max-Planck-Institut für Zellbiologie, Ladenburg/Heidelberg, Germany.

    A computer search revealed 10 proteins with homology to the sequence we originally identified in vimentin as the site of cleavage by human immunodeficiency virus type 1 (HIV-1) protease. Of these 10 proteins (actin, alpha-actinin, spectrin, tropomyosins, vinculin, dystrophin, MAP-2, villin, TRK-1 and Ig mu-chain), we show that 4 of the first 5 were cleaved in vitro by this protease, as are MAP-1 and -2 [(1990) J. Gen. Virol. 71, 1985-1991]. In these proteins, cleavage is not restricted to a single motif, but occurs at many sites. However, cleavage is not random, since 9 other proteins including the cytoskeletal proteins filamin and band 4.1 are not cleaved in the in vitro assay. Thus, the ability of HIV-1 protease to cleave specific components of the cytoskeleton may be an important, although as yet unevaluated aspect of the life cycle of this retrovirus and/or may directly contribute to the pathogenesis observed during infection.

    FEBS letters 1991;278;2;199-203

  • Expression of human alpha-actinin in human hepatocellular carcinoma.

    Nishiyama M, Ozturk M, Frohlich M, Mafune K, Steele G and Wands JR

    Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Charlestown 02129.

    Little is known regarding gene expression during hepatocyte transformation. We have isolated an alpha-actinin complementary DNA from a human hepatocellular carcinoma library. This partial 2.4-kilobase complementary DNA has high homology with human placental and chicken nonmuscle alpha-actinins; our isolate contains the entire 3' noncoding region and it is within these sequences where the major differences between the vertebrate alpha-actinin complementary DNAs arise. Northern analysis revealed a 3.5-kilobase transcript in nonmuscle and a smaller 3.0-kilobase species in muscle tissue. Levels of alpha-actinin expression were low in normal liver and we investigated its expression during both hepatocyte proliferation and transformation. We found no increase during rat hepatocyte regeneration up to 24 h following two-thirds hepatectomy. However, high levels of alpha-actinin transcripts were observed in human hepatocellular carcinoma compared to noninvolved adjacent liver. We conclude that the alpha-actinin gene is highly expressed when hepatocytes have assumed the malignant phenotype.

    Funded by: NCI NIH HHS: CA-35711, CA-49832; NIAAA NIH HHS: AA-02666; ...

    Cancer research 1990;50;19;6291-4

  • 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

  • Cloning and chromosomal localization of the human cytoskeletal alpha-actinin gene reveals linkage to the beta-spectrin gene.

    Youssoufian H, McAfee M and Kwiatkowski DJ

    Whitehead Institute for Biomedical Research, Cambridge, MA.

    We report the cloning and characterization of a full-length cDNA encoding the human cytoskeletal isoform of alpha-actinin (alpha A), a ubiquitous actin-binding protein that shares structural homology with spectrin and dystrophin. The gene encodes 891 amino acids with 96%-98% sequence identity at the amino acid level to chicken nonskeletal muscle alpha A. Transient expression in COS cells produces a protein of approximately 104 kD that comigrates on SDS-PAGE with native alpha A. This alpha A gene is localized to chromosome 14q22-q24 by somatic cell hybrid and in situ hybridization analyses. Pulsed-field gel analysis of human genomic DNA revealed identically sized fragments when cDNA probes for alpha A and erythroid beta-spectrin were used; the latter gene has been previously localized to chromosome 14, band q22. These observations indicate that the genes for cytoskeletal alpha A and beta-spectrin are, in all likelihood, closely physically linked and that, in accordance with their similar structural features, they arose by partial duplication of an ancestral gene.

    Funded by: NHLBI NIH HHS: HL01582, HL02277

    American journal of human genetics 1990;47;1;62-72

  • The cDNA sequence of a human placental alpha-actinin.

    Millake DB, Blanchard AD, Patel B and Critchley DR

    Department of Biochemistry, University of Leicester, UK.

    Nucleic acids research 1989;17;16;6725

Gene lists (6)

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
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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