JCB: REPORT Phosphor ylation of the Arp2/3 complex is necessar y to nucleate actin ﬁlaments Lawrence L. LeClaire III,1 Martin Baumgartner,1 Janet H. Iwasa,2 R. Dyche Mullins,2 and Diane L. Barber1 1Department of Cell and Tissue Biology and 2Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143 Y G O L O I B L L E C F O L A N R U O J E H T T the primary nucleator of new actin ﬁlaments in most he actin-related protein 2/3 (Arp2/3) complex is crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 com- plex must be phosphorylated on either threonine or tyro- sine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin ﬁlaments but is critical for binding the pointed end of actin ﬁlaments and nucleating actin ﬁlaments. Introduction Eukaryotic cells change shape and move by assembling ordered networks of actin filaments at their leading edges (Pollard et al., 2001). The heart of this actin-based motility engine is the actin- related protein 2/3 (Arp2/3) complex, a seven-subunit protein complex that builds cross-linked filament arrays by nucleating new filaments from the sides of preexisting filaments (Goley and Welch, 2006). Actin-nucleating and cross-linking activities of the Arp2/3 complex are required to build many cellular structures, including lamellipodia and phagocytic cups, and to move endosomes and intracellular pathogens through the cytoplasm (Pollard and Borisy, 2003). The Arp2/3 complex, which is composed of two actin- related proteins (Arp2 and Arp3) and five accessory proteins, nu- cleates a new actin filament by first binding to the side of a preexisting filament. Next, the Arp2 and Arp3 subunits form a protonucleus that mimics a new fast-growing barbed end of an ac- tin filament (Robinson et al., 2001; Goley et al., 2004; Nolen et al., 2004). Actin monomers polymerize at this barbed end, elongating rapidly toward the plasma membrane, whereas the Arp2/3 complex Correspondence to Diane L. Barber: diane.barber@ucsf.edu M. Baumgartner’s present address is Vetsuisse Faculty, Department of Clinical Research and Veterinary Public Health, Section of Molecular Pathobiology, University of Bern, Bern CH-3012, Switzerland. Abbreviations used in this paper: AP, Antarctic phosphatase; Arp, actin- related protein; NPF, nucleation-promoting factor; PP2C , protein phosphatase 2C ; pThr, phosphothreonine; pTyr, phosphotyrosine; WASP, Wiskott-Aldrich syndrome protein. The online version of this article contains supplemental material. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 182 No. 4 647–654 www.jcb.org/cgi/doi/10.1083/jcb.200802145 Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved resi- dues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine sub- stitutions of Arp2 T237 and T238 or Y202 inhibits mem- brane protrusion. These ﬁndings reveal an additional level of regulation of actin ﬁlament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical “or gate” capable integrating diverse upstream signals. remains bound to the slow-growing pointed end, thereby cross- linking the new filament to the mother filament (Mullins et al., 1998). Arp2 and Arp3 subunits of the complex bind ATP. Although ATP hydrolysis is not necessary for actin nucleation, it is thought to be necessary for debranching of Arp2/3-branched actin filaments (Martin et al., 2006). Wiskott-Aldrich syndrome protein (WASP) family proteins are the predominant activators of the Arp2/3 com- plex. However, in addition to regulation by WASP proteins, recent evidence indicates the Arp2/3 complex is phosphorylated by serine/threonine kinases. The p21-activated kinase (PAK) phos- phorylates Thr21 in the ARPC1 subunit (Vadlamudi et al., 2004), and the MAPK-activated protein kinase 2 phosphorylates Ser77 in the ARPC5 subunit (Singh et al., 2003). Although phosphorylation of the Arp2/3 complex is suggested to increase cell motility, whether phosphorylation directly regulates actin nu- cleating activity of the Arp2/3 complex has not been reported. Results and discussion Puriﬁed Arp2/3 complex is phosphorylated We found that several subunits of the Arp2/3 complex are phosphorylated in cells. Using 2D electrophoresis of purified © 2008 LeClaire et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the ﬁrst six months after the publica- tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 647