Profilin Interacts with the Gly-Pro-Pro-Pro-Pro-Pro Sequences of Vasodilator-Stimulated Phosphoprotein (VASP):  Implications for Actin-Based <i>Listeria</i> Motility

Kang, Fan; Laine, Roney O.; Bubb, Michael R.; Southwick, Frederick S.; Purich, Daniel L.

doi:10.1021/bi970065n

Cited by 112 publications

(106 citation statements)

References 29 publications

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“…Therefore, it is possible that nucleation of actin polymerization by EVL could be enhanced in the presence of profilin. Consistent with this are the observations that addition of the VASP-derived prolinerich peptide arrests or decelerates Listeria motility in PtK2 cells (48) and that profilin is recruited only to motile Listeria in an Ena/VASP-dependent manner (49). It is clear, however, that Ena/VASP proteins can promote Listeria motility independently of profilin (20,50).…”

Section: Discussionmentioning

confidence: 56%

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

216

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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...

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Section: Discussionmentioning

confidence: 56%

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

216

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“…Recombinant human profilin I, rat profilin I, and rat thymosin ␤ 4 were purified as previously described (25,26). The rat profilin was mutated to have a cysteine at position 41 (C41S) (27) and the thymosin ␤ 4 contained an added C-terminal cysteine.…”

Section: Methodsmentioning

confidence: 99%

Formation and Implications of a Ternary Complex of Profilin, Thymosin β4, and Actin

Yarmola

Parikh

Bubb³

2001

Journal of Biological Chemistry

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Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin ␤ 4 , and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin ␤ 4 (residues 6 -22) confirm the presence of an extensive binding surface between actin and thymosin ␤ 4 , and explain why thymosin ␤ 4 and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin ␤ 4 peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin ␤ 4 . Intracellular concentrations of thymosin ␤ 4 and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin ␤ 4 -actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin ␤ 4 can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.The amount of unpolymerized actin in many cells is large. Several actin-monomer sequestering proteins have been identified that are responsible for maintaining this pool, and attempts have been made to account for the quantity of unpolymerized actin by calculation of the sum of sequestered actin in cells (1-3). These calculations depend not only on the concentration of each sequestering protein and its equilibrium dissociation constant for actin, but also on several other parameters, including an estimate of the critical concentration of actin (i.e. the amount of unpolymerized, unsequestered actin), the stoichiometry with which the sequestering proteins bind to actin, the potential qualitative and quantitative effects should different sequestering proteins interact simultaneously with a single actin subunit, and on assumptions regarding the effects of cytoplasmic molecular crowding on equilibrium association constants. Not surprisingly, with so many parameters to evaluate, even small quantitative errors in measurement or qualitative errors in mechanism result in a wide range of plausible estimates of total sequestered actin. In this report we investigate assumptions that have very significant effects on predictions related to the amount of sequestered actin, finding significant discrepancies with previously reported results, and discuss the expected consequences of these observations. Based on assays measuring steady-state F-actin levels and the failure to obtain a covalently cross-linked ternary complex, thymosin ␤ 4 and profilin have been reported to bind ...

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“…Profilin is the major actin monomer carrier in the cell (1). By delivering actin monomers to growing actin filaments, profilin stimulates actin filament elongation by VASP (28) and WASP͞ WAVE (29)(30)(31). The role of profilin in elongation appears to be common among cytoskeletal proteins.…”

Section: Control Of Actin Nucleotide Exchange By Wh2 and T␤mentioning

confidence: 99%

Actin-bound structures of Wiskott–Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly

Chéreau

Kerff

Graceffa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

236

375

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Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) is a small and widespread actin-binding motif. In the WASP family, WH2 plays a role in filament nucleation by Arp2͞3 complex. Here we describe the crystal structures of complexes of actin with the WH2 domains of WASP, WASP-family verprolin homologous protein, and WASP-interacting protein. Despite low sequence identity, WH2 shares structural similarity with the N-terminal portion of the actin monomer-sequestering thymosin ␤ domain (T␤). We show that both domains inhibit nucleotide exchange by targeting the cleft between actin subdomains 1 and 3, a common binding site for many unrelated actin-binding proteins. Importantly, WH2 is significantly shorter than T␤ but binds actin with Ϸ10-fold higher affinity. WH2 lacks a C-terminal extension that in T␤4 becomes involved in monomer sequestration by interfering with intersubunit contacts in F-actin. Owing to their shorter length, WH2 domains connected in tandem by short linkers can coexist with intersubunit contacts in F-actin and are proposed to function in filament nucleation by lining up actin subunits along a filament strand. The WH2-central region of WASP-family proteins is proposed to function in an analogous way by forming a special class of tandem repeats whose function is to line up actin and Arp2 during Arp2͞3 nucleation. The structures also suggest a mechanism for how profilin-binding Pro-rich sequences positioned N-terminal to WH2 could feed actin monomers directly to WH2, thereby playing a role in filament elongation.x-ray crystallography ͉ isothermal titration calorimetry ͉ nucleotide exchange T he actin cytoskeleton plays an essential role in many cellular functions, including intracellular transport and the control of cell shape and polarity (1). In the cell, a vast number of actin-binding proteins (ABPs) direct the location, rate, and timing for actin assembly into different structures, such as filopodia, lamellipodia, stress fibers, and focal adhesions. ABPs are commonly multidomain proteins, containing signaling domains and structurally conserved actin-binding motifs. One of the most abundant actin-binding motifs is Wiskott-Aldrich syndrome protein (WASP)-homology domain 2 (WH2) (2). The hematopoietic-specific protein, WASP, and its ubiquitously expressed ortholog N-WASP form part of a family that also includes the three WASP-family verprolin homologous protein (WAVE͞SCAR) isoforms: WAVE1, WAVE2, and WAVE3 (1, 3). Members of this family activate Arp2͞3-dependent actin nucleation and branching in response to signals mediated by Rho-family GTPases. Although the domain structure of these proteins varies, reflecting different modes of regulation, they all share a common C-terminal WH2 central-acidic region (CA region) (Fig. 1A), which constitutes the smallest fragment necessary for Arp2͞3 activation (4). WH2 is also present in members of the WASP-interacting protein (WIP) family, which form complexes with WASP͞N-WASP and modulate their functions in vivo (5, 6). Members of this family include ...

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Profilin Interacts with the Gly-Pro-Pro-Pro-Pro-Pro Sequences of Vasodilator-Stimulated Phosphoprotein (VASP): Implications for Actin-Based Listeria Motility

Cited by 112 publications

References 29 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

Formation and Implications of a Ternary Complex of Profilin, Thymosin β4, and Actin

Actin-bound structures of Wiskott–Aldrich syndrome protein (WASP)-homology domain 2 and the implications for filament assembly

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