Abstract

The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc.

Highlights

  • The regulated polymerization of actin monomers into filaments underlies several cellular processes including the protrusion of cell membranes, endocytosis and the establishment of polarity [Ridley, 2006]

  • We noted that the WAVE2-verprolin central acidic (VCA) domain contains several potential Casein Kinase 2 (CK2) sites (Fig. 1A) and were interested to know if phosphorylation of this region influenced its actin nucleation activity

  • Three residues within the WAVE2-VCA domain fall within the strictest CK2 consensus sequence of ser/thr-X-X-asp/glu [Kuenzel et al, 1987; Meggio and Pinna, 2003], namely ser482, ser484, and ser489, making them candidates for phosphorylation

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Summary

Introduction

The regulated polymerization of actin monomers into filaments underlies several cellular processes including the protrusion of cell membranes, endocytosis and the establishment of polarity [Ridley, 2006]. The formation of new actin filaments is energetically unfavourable under cellular conditions This enables the tight regulation of actin dynamics via a number of catalytic factors which allow for the spatial and temporal control of Factin production [Pollard, 2007]. The first of these factors identified was the Arp2/3 complex, which stimulates the formation of branched actin structures seen in the leading edge lamellipodia of migrating cells [Machesky et al, 1999]. VCA domains bind both globular actin and the Arp2/3 complex and are thought to stimulate a conformational change in the quaternary structure of the Arp2/3 complex [Robinson et al, 2001; Goley et al, 2004; Rodal et al, 2005]. In this state the Arp2/3 complex could potentially mimic an actin dimer, overcoming the initial barrier to Contract grant sponsor: Wellcome Trust and the Medical Research Council, UK

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