Characterization of the actin binding properties of the vasodilator‐stimulated phosphoprotein VASP

Hüttelmaier, Stefan; Harbeck, Birgit; Steffens, Nils Ole; Meßerschmidt, Tania; Illenberger, Susanne; Jockusch, Brigitte M.

doi:10.1016/s0014-5793(99)00546-3

Cited by 121 publications

(146 citation statements)

References 22 publications

Supporting

Mentioning

130

Contrasting

Unclassified

Order By: Relevance

“…7C, inset). This result is in agreement with published data showing that VASP nucleates polymerization of Mg 2ϩ /ATP/actin only at low, nonphysiological salt concentrations (20,40). Under these low salt conditions (2 mM MgCl 2 and 15 mM KCl), dephospho-VASP had strong nucleating activity, whereas phospho-VASP did not nucleate actin polymerization (Fig.…”

Section: Fig 1 Endogenous Evl Redistributes To Sites Of Actin Polymsupporting

confidence: 93%

cAMP-dependent Protein Kinase Phosphorylation of EVL, a Mena/VASP Relative, Regulates Its Interaction with Actin and SH3 Domains

Lambrechts¹,

Kwiatkowski²,

Lanier³

et al. 2000

Journal of Biological Chemistry

168

205

View full text Add to dashboard Cite

Proteins of the Ena/VASP family are implicated in processes that require dynamic actin remodeling such as axon guidance and platelet activation. In this work, we explored some of the pathways that likely regulate actin dynamics in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, were highly expressed in hematopoietic cells of thymus and spleen. In CD3-activated T-cells, EVL was found in F-actin-rich patches and at the distal tips of the microspikes that formed on the activated side of the T-cells. Like the other family members, EVL localized to focal adhesions and the leading edge of lamellipodia when expressed in fibroblasts. EVL was a substrate for the cAMP-dependent protein kinase, and this phosphorylation regulated several of the interactions between EVL and its ligands. Unlike VASP, EVL nucleated actin polymerization under physiological conditions, whereas phosphorylation of both EVL and VASP decreased their nucleating activity. EVL bound directly to the Abl, Lyn, and nSrc SH3 domains; the FE65 WW domain; and profilin, likely via its proline-rich core. Binding of Abl and nSrc SH3 domains, but not profilin or other SH3 domains, was abolished by cAMP-dependent protein kinase phosphorylation of EVL. We show strong cooperative binding of two profilin dimers on the polyproline sequence of EVL. Additionally, profilin competed with the SH3 domains for binding to partially overlapping binding sites. These data suggest that the function of EVL could be modulated in a complex manner by its interactions with multiple ligands and through phosphorylation by cyclic nucleotide dependent kinases.To respond properly to environmental cues, cells possess multiple complex signal transduction networks. Many pathways lead to dynamic changes of the actin cytoskeleton that form the basis for cell movement in a wide variety of biological phenomena. In recent years, many proteins participating in one or more signal transduction pathways have been identified. One group of multifunctional proteins, involved in actin-based motility, is the Ena/VASP family of proteins that include Drosophila Ena (Enabled), Mena (mammalian Ena), VASP (vasodilator-stimulated phosphoprotein), and EVL (Ena/VASP-like protein) (1). Ena was identified through genetic interactions with the Drosophila Abl homologue (2, 3), whereas VASP was identified as a prominent target for cAMP (PKA) 1 -and cGMPdependent protein kinases in platelets (4). Mena and EVL were identified by similarity to Ena (1). Ena, Mena, and VASP are important in processes that require highly dynamic actin reorganization, including axon guidance (5), platelet aggregation (6, 7), and fibroblast motility (8). The proteins are concentrated in regions of the cell associated with movement and adhesion, including the leading edge of lamellipodia, focal adhesions, and adherens junctions (1, 9, 10).The Ena/VASP proteins share a common domain structure that consists of an amino-terminal Ena/VASP homology (EVH) 1 domain, a carboxyl-terminal EVH2 domain, and a central proline-rich domain...

show abstract

Section: Fig 1 Endogenous Evl Redistributes To Sites Of Actin Polymsupporting

confidence: 93%

cAMP-dependent Protein Kinase Phosphorylation of EVL, a Mena/VASP Relative, Regulates Its Interaction with Actin and SH3 Domains

Lambrechts¹,

Kwiatkowski²,

Lanier³

et al. 2000

Journal of Biological Chemistry

168

205

View full text Add to dashboard Cite

show abstract

“…The EVH2 domain of Ena/VASP proteins has been implicated in the interaction of this protein family with F-actin in vitro (Bachmann et al, 1999;Hü ttelmaier et al, 1999). Kuo and McGrath (2000) have recently demonstrated that Listeria are tightly linked to their own actin tails raising the possibility that this tight interaction limits bacterial motility.…”

Section: Discussionmentioning

confidence: 99%

“…The EVH2 domain of Ena/VASP proteins has also been implicated in the interaction of this protein family with F-actin in vitro (Bachmann et al, 1999;Hü ttelmaier et al, 1999;Harbeck et al, 2000). Moreover, Ena/VASP proteins stimulate actin polymerization by shortening the lag phase of actin filament formation, an effect that can be reversed by adding a peptide (corresponding to amino acids 261-283 of EVL) that has been shown to interact with F-actin in sedimentation assays (Laurent et al, 1999;Harbeck et al, 2000;Lambrechts et al, 2000).…”

Section: Deletion Of F-actin-binding Site Of Ena/vasp Proteins Enhancmentioning

confidence: 99%

Contribution of Ena/VASP Proteins to Intracellular Motility ofListeriaRequires Phosphorylation and Proline-rich Core but Not F-Actin Binding or Multimerization

Geese

Loureiro

Bear

et al. 2002

MBoC

101

View full text Add to dashboard Cite

The Listeria model system has been essential for the identification and characterization of key regulators of the actin cytoskeleton such as the Arp2/3 complex and Ena/vasodilator-stimulated phosphoprotein (VASP) proteins. Although the role of Ena/VASP proteins in Listeria motility has been extensively studied, little is known about the contributions of their domains and phosphorylation state to bacterial motility. To address these issues, we have generated a panel of Ena/ VASP mutants and, upon expression in Ena/VASP-deficient cells, evaluated their contribution to Ena/VASP function in Listeria motility. The proline-rich region, the putative G-actin binding site, and the Ser/Thr phosphorylation of Ena/VASP proteins are all required for efficient Listeria motility. Surprisingly, the interaction of Ena/VASP proteins with F-actin and their potential ability to form multimers are both dispensable for their involvement in this process. Our data suggest that Ena/VASP proteins contribute to Listeria motility by regulating both the nucleation and elongation of actin filaments at the bacterial surface.

show abstract

“…In experiments with untransfected human umbilical vein endothelial cells (HUVEC), confluent cells were placed in serum-free Medium 199 for 5 min before challenge with control vehicle or 10 ng/ml PMA for 30 min. Cells were then washed twice with PBS before cross-linking essentially as described previously (34,35). Briefly, cells were incubated at room temperature for 30 min in PBS containing 0.5 mM dithiobis(succinimidyl propionate) before quenching by the addition of 0.1 ml of 1 M Tris, pH 7.5, followed by two washes with ice-cold PBS.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Heterotypic Interaction between the Receptor Tyrosine Kinases TIE-1 and TIE-2

Marron

Hughes

Edge

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membranelocalized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

show abstract

Characterization of the actin binding properties of the vasodilator‐stimulated phosphoprotein VASP

Cited by 121 publications

References 22 publications

cAMP-dependent Protein Kinase Phosphorylation of EVL, a Mena/VASP Relative, Regulates Its Interaction with Actin and SH3 Domains

cAMP-dependent Protein Kinase Phosphorylation of EVL, a Mena/VASP Relative, Regulates Its Interaction with Actin and SH3 Domains

Contribution of Ena/VASP Proteins to Intracellular Motility ofListeriaRequires Phosphorylation and Proline-rich Core but Not F-Actin Binding or Multimerization

Evidence for Heterotypic Interaction between the Receptor Tyrosine Kinases TIE-1 and TIE-2

Contact Info

Product

Resources

About