Differential VASP phosphorylation controls remodeling of the actin cytoskeleton

Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family link signal transduction pathways to actin cytoskeleton dynamics. VASP is substrate of cAMP-dependent, cGMP-dependent and AMP-activated protein kinases that primarily phosphorylate the sites S157, S239 and T278, respectively. Here, we systematically analyzed functions of VASP phosphorylation patterns for actin assembly and subcellular targeting in vivo and compared the phosphorylation effects of Ena/VASP family members. Methods used were the reconstitution of VASP-null cells with `locked' phosphomimetic VASP mutants, actin polymerization of VASP mutants in vitro and in living cells, site-specific kinase-mediated VASP phosphorylation, and analysis of the endogenous protein with phosphorylation-status-specific antibodies. Phosphorylation at S157 influenced VASP localization, but had a minor impact on F-actin assembly. Phosphorylation of the S157-equivalent site in the Ena/VASP family members Mena and EVL had no effect on the ratio of cellular F-actin to G-actin. By contrast, VASP phosphorylation at S239 (and the equivalent site in Mena) or T278 impaired VASP-driven actin filament formation. The data show that VASP functions are precisely regulated by differential phosphorylation and provide new insights into cytoskeletal control by serine/threonine kinase-dependent signaling pathways.

[1]  Frank B. Gertler,et al.  Ena/VASP: towards resolving a pointed controversy at the barbed end , 2009, Journal of Cell Science.

[2]  A. Ham,et al.  VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis , 2009, Journal of Cell Science.

[3]  S. Elledge,et al.  A quantitative atlas of mitotic phosphorylation , 2008, Proceedings of the National Academy of Sciences.

[4]  L. Pasic,et al.  Ena/VASP Proteins Capture Actin Filament Barbed Ends* , 2008, Journal of Biological Chemistry.

[5]  Léa Trichet,et al.  Relaxing the actin cytoskeleton for adhesion and movement with Ena/VASP , 2008, The Journal of cell biology.

[6]  T. Renné,et al.  Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function. , 2008, American journal of physiology. Lung cellular and molecular physiology.

[7]  U. Walter,et al.  Phosphoproteome of resting human platelets. , 2008, Journal of proteome research.

[8]  T. Renné,et al.  Cytoskeleton assembly at endothelial cell–cell contacts is regulated by αII-spectrin–VASP complexes , 2008, The Journal of cell biology.

[9]  C. Gachet,et al.  Testing antiplatelet therapy , 2008 .

[10]  U. Walter,et al.  The role of VASP in regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization. , 2008, American journal of physiology. Cell physiology.

[11]  Laura A. Sullivan,et al.  Global Survey of Phosphotyrosine Signaling Identifies Oncogenic Kinases in Lung Cancer , 2007, Cell.

[12]  R. Bronson,et al.  Ena/VASP is required for endothelial barrier function in vivo , 2007, The Journal of cell biology.

[13]  R. Dominguez,et al.  Structural basis for the recruitment of profilin–actin complexes during filament elongation by Ena/VASP , 2007, The EMBO journal.

[14]  T. Renné,et al.  Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239 , 2007, Journal of Cell Science.

[15]  S. Colgan,et al.  The FASEB Journal • Research Communication Identification of vasodilator-stimulated phosphoprotein , 2022 .

[16]  Sharon Y. R. Dent,et al.  HDAC6 modulates cell motility by altering the acetylation level of cortactin. , 2007, Molecular cell.

[17]  Frank B Gertler,et al.  Ena/VASP proteins have an anti-capping independent function in filopodia formation. , 2007, Molecular biology of the cell.

[18]  T. Renné,et al.  AMP-activated Protein Kinase Impairs Endothelial Actin Cytoskeleton Assembly by Phosphorylating Vasodilator-stimulated Phosphoprotein* , 2007, Journal of Biological Chemistry.

[19]  K. Nakayama,et al.  PKCdelta regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation. , 2006, Blood.

[20]  T. Shishido,et al.  A rapid protein expression and purification system using Chinese hamster ovary cells expressing retrovirus receptor. , 2006, Journal of biotechnology.

[21]  J. Shabanowitz,et al.  Cortactin phosphorylation sites mapped by mass spectrometry , 2006, Journal of Cell Science.

[22]  T. Bretschneider,et al.  The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Poole,et al.  Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets. , 2006, The Biochemical journal.

[24]  J. Shabanowitz,et al.  FAK phosphorylation sites mapped by mass spectrometry , 2005, Journal of Cell Science.

[25]  J. Niu,et al.  A Novel Mechanism of G Protein-dependent Phosphorylation of Vasodilator-stimulated Phosphoprotein* , 2005, Journal of Biological Chemistry.

[26]  T. Renné,et al.  Local Bradykinin Formation Is Controlled by Glycosaminoglycans1 , 2005, The Journal of Immunology.

[27]  D. Hanein,et al.  Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins*[boxs] , 2005, Journal of Biological Chemistry.

[28]  Ilme Schlichting,et al.  The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[29]  L. Holzman,et al.  Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization , 2004, The EMBO journal.

[30]  J. Wehland,et al.  ENA/VASP proteins: multifunctional regulators of actin cytoskeleton dynamics. , 2004, Frontiers in bioscience : a journal and virtual library.

[31]  U. Walter,et al.  Vasodilator-Stimulated Phosphoprotein Regulates Proliferation and Growth Inhibition by Nitric Oxide in Vascular Smooth Muscle Cells , 2004, Arteriosclerosis, thrombosis, and vascular biology.

[32]  U. Walter,et al.  Vasodilator-stimulated Phosphoprotein Activation of Serum-response Element-dependent Transcription Occurs Downstream of RhoA and Is Inhibited by cGMP-dependent Protein Kinase Phosphorylation* , 2004, Journal of Biological Chemistry.

[33]  U. Walter,et al.  Physiology and pathophysiology of vascular signaling controlled by guanosine 3',5'-cyclic monophosphate-dependent protein kinase. , 2004, Acta biochimica Polonica.

[34]  J. Loureiro,et al.  Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. , 2003, Annual review of cell and developmental biology.

[35]  F. Hofmann,et al.  Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3′,5′-Cyclic Monophosphate–Dependent Protein Kinase , 2003 .

[36]  J. Loureiro,et al.  Function and regulation of Ena/VASP proteins. , 2003, Trends in cell biology.

[37]  R. Treisman,et al.  A role for VASP in RhoA‐Diaphanous signalling to actin dynamics and SRF activity , 2003, The EMBO journal.

[38]  R. Firtel,et al.  Requirement of a Vasodilator-stimulated Phosphoprotein Family Member for Cell Adhesion, the Formation of Filopodia, and Chemotaxis in Dictyostelium* 210 , 2002, The Journal of Biological Chemistry.

[39]  R. Juliano,et al.  Regulation of Vasodilator-stimulated Phosphoprotein Phosphorylation and Interaction with Abl by Protein Kinase A and Cell Adhesion* , 2002, The Journal of Biological Chemistry.

[40]  J. Loureiro,et al.  Critical roles of phosphorylation and actin binding motifs, but not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migration. , 2002, Molecular biology of the cell.

[41]  J. Wehland,et al.  Contribution of Ena/VASP proteins to intracellular motility of listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization. , 2002, Molecular biology of the cell.

[42]  R. Treisman,et al.  LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics , 2002, The Journal of cell biology.

[43]  Gary G. Borisy,et al.  Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility , 2002, Cell.

[44]  B. Levi,et al.  Role of vasodilator‐stimulated phosphoprotein in protein kinase A‐induced changes in endothelial junctional permeability , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[45]  D. Portnoy,et al.  Pivotal role of VASP in Arp2/3 complex–mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility , 2001, The Journal of cell biology.

[46]  K. Pryzwansky,et al.  The Vasodilator-Stimulated Phosphoprotein Is Regulated by Cyclic GMP-Dependent Protein Kinase During Neutrophil Spreading1 , 2001, The Journal of Immunology.

[47]  J. Vandekerckhove,et al.  cAMP-dependent Protein Kinase Phosphorylation of EVL, a Mena/VASP Relative, Regulates Its Interaction with Actin and SH3 Domains* , 2000, The Journal of Biological Chemistry.

[48]  S. Illenberger,et al.  Phosphorylation of the Vasodilator-stimulated Phosphoprotein Regulates Its Interaction with Actin* , 2000, The Journal of Biological Chemistry.

[49]  P. Schmieder,et al.  Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity , 2000, The EMBO journal.

[50]  D. Carling,et al.  Characterization of the Role of AMP-Activated Protein Kinase in the Regulation of Glucose-Activated Gene Expression Using Constitutively Active and Dominant Negative Forms of the Kinase , 2000, Molecular and Cellular Biology.

[51]  N. Brindle,et al.  Vasodilator-Stimulated Phosphoprotein Is Involved in Stress-Fiber and Membrane Ruffle Formation in Endothelial Cells , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[52]  U. Walter,et al.  Regulation of Human Endothelial Cell Focal Adhesion Sites and Migration by cGMP-dependent Protein Kinase I* , 2000, The Journal of Biological Chemistry.

[53]  James E Bear,et al.  Negative Regulation of Fibroblast Motility by Ena/VASP Proteins , 2000, Cell.

[54]  Elaine Fuchs,et al.  Directed Actin Polymerization Is the Driving Force for Epithelial Cell–Cell Adhesion , 2000, Cell.

[55]  T. Renné,et al.  Mapping of the Discontinuous H-kininogen Binding Site of Plasma Prekallikrein , 1999, The Journal of Biological Chemistry.

[56]  U. Walter,et al.  The EVH2 Domain of the Vasodilator-stimulated Phosphoprotein Mediates Tetramerization, F-actin Binding, and Actin Bundle Formation* , 1999, The Journal of Biological Chemistry.

[57]  S. Narumiya,et al.  Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. , 1999, Science.

[58]  Richard Treisman,et al.  Signal-Regulated Activation of Serum Response Factor Is Mediated by Changes in Actin Dynamics , 1999, Cell.

[59]  Marie-France Carlier,et al.  Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes , 1999, The Journal of cell biology.

[60]  M. Hall,et al.  Signaling to the actin cytoskeleton. , 1998, Annual review of cell and developmental biology.

[61]  J. Wehland,et al.  Mena, a Relative of VASP and Drosophila Enabled, Is Implicated in the Control of Microfilament Dynamics , 1996, Cell.

[62]  T. Mitchison,et al.  Actin-Based Cell Motility and Cell Locomotion , 1996, Cell.

[63]  U. Walter,et al.  Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets , 1995, FEBS letters.

[64]  U. Walter,et al.  The proline‐rich focal adhesion and microfilament protein VASP is a ligand for profilins. , 1995, The EMBO journal.

[65]  M. Krieger,et al.  cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. , 1994, The Journal of biological chemistry.

[66]  U. Walter,et al.  Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators. , 1990, The Journal of biological chemistry.

[67]  D. Kalderon,et al.  Molecular cloning and predicted full‐length amino acid sequence of the type Iβ isozyme of cGMP‐dependent protein kinase from human placenta , 1989 .

[68]  D. Kalderon,et al.  Molecular cloning and predicted full-length amino acid sequence of the type I beta isozyme of cGMP-dependent protein kinase from human placenta. Tissue distribution and developmental changes in rat. , 1989, FEBS letters.

[69]  T. Kouyama,et al.  Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. , 2005, European journal of biochemistry.