Figure 5. Phosphorylation of Arp2 is necessary for lamellipodia formation. (A) Control, luciferase RNAi-treated D. melanogaster S2 cells on con- canavalin A–coated coverslips spread symmetrical, round lamellipodia. S2 cells depleted of the Arp2 subunit by siRNA (Arp2 RNAi) exhibited a stellate morphology with membrane protrusions radiating from the cell body. A wild-type morphology of circular lamellipodia was restored in Arp2- depleted S2 cells expressing wild-type Arp2-GFP (Arp2-WT-GFP), or mu- tated Arp2-T237/238A-GFP or Arp2-Y202A-GFP. Arp2-depleted S2 cells expressing the triple mutant Arp2-T237/238A-Y202A-GFP remained stel- late. Bar, 5 μM. (B) The number of cells with wild-type or stellate morphol- ogy were scored for the indicated conditions and expressed as the mean ± SD of three separate cell preparations, with >500 cells scored for each condition per cell preparation. (C) Model for activation of the Arp2/3 complex by phosphorylation of the Arp2 subunit. In the “inactive” form of the Arp2/3 complex, the Arp2 and Arp3 subunits are splayed open. Phos- phorylated T237/T238 and Y202 residues in Arp2 interact with arginine residues on adjacent subunits to maintain a conformation that stabilizes the complex in the “active” form. This conformation aligns the Arp2 and Arp3 subunits to form a protonucleus for actin nucleation. 652 JCB • VOLUME 182 • NUMBER 4 • 2008 We also confirmed that recombinant wild-type but not mutant Arp2 in S2 cells was phosphorylated on threonine and tyrosine residues (Fig. S3, B and C). Complexes immunoprecipitated with antibodies to GFP and immunoblotted with antibodies to pThr and pTyr showed labeling of Arp2-WT but not Arp2-T237/238- Y202A, which suggests that Y202 is necessary for tyrosine phosphorylation of Arp2 in cells. Whether this reflects a direct requirement for Y202 phosphorylation or a necessary role of Y202 for phosphorylation of a different Tyr residue in Arp2 re- mains to be determined. Our data show that the Arp2/3 complex must be phosphor- ylated on threonine or tyrosine residues for actin-nucleating activity. Phosphorylation is necessary but not sufficient for nu- cleating activity of the complex, which must be stimulated by NPFs. However, NPFs are not sufficient in the absence of Arp2/3 complex phosphorylation. In cells, lamellipodia formation is inhibited by alanine substitutions in Arp2 T237/238-Y202. Whether phosphorylation of these residues is necessary for nu- cleating activity of the complex or whether they might function as docking sites for a kinase remains to be determined. How- ever, based on their potential charge interactions with other sub- units of the complex, we predict that phosphorylation of Arp2 T237/238 or Y202 stabilizes the Arp2/3 complex in an active conformation (Fig. 5 C). Although current models of Arp2/3 complex regulation do not incorporate a role for phosphory- lation of subunits (Goley and Welch, 2006; Stradal and Scita, 2006), a requirement for either threonine or tyrosine phosphory- lation is an effective mechanism for integrating diverse upstream signals regulating actin filament assembly. Materials and methods Protein puriﬁcation Arp2/3 complex and actin from A. castellani were puriﬁed as described previously (Zalevsky et al., 2001). Bovine Arp2/3 complex was a gift from T. Pollard (Yale University, New Haven, CT), and recombinant human Arp2/3 complex was a gift from M. Welch (University of California, Berke- ley, Berkeley, CA). N-WASP VCA and ScarVCA were expressed in E. coli and puriﬁed as described previously (Zalevsky et al., 2001). 2D electrophoresis The Arp2/3 complex was precipitated by chloroform/methanol extraction and resuspended in ﬁrst-dimension rehydration buffer (8 M urea, 2% CHAPS, 0.5% pharmalyte, pH 3–10, 0.002% bromophenol blue, and 18 mM DTT). Protein samples were separated in the ﬁrst dimension with an Ettan IGphor 3 IEF system (GE Healthcare) on Immobiline Dry Strips (pH 3–10NL, 7 cm; GE Healthcare) at 1,500 volt hours for 10 h. The focused proteins were then separated in the second dimension on Nu-PAGE 12% Bis-Tris precast gels (Invitrogen). SDS-PAGE and immunoblotting Arp2/3 complex was separated on 12.5% SDS-PAGE gels and transferred to Immobilon-P (Millipore) by standard methods. Immunoblots were incu- bated in 3% BSA in TBS-T (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) for 2 h, and monoclonal anti-pTyr (Cell Signaling Technology) or polyclonal anti-pThr (Invitrogen) were added at 200 ng/ml. Primary anti- bodies were detected with horseradish peroxidase–conjugated anti–mouse and anti–rabbit antibodies (BioRad Laboratories), respectively, and visual- ized with an ECL chemiluminescence kit (Pierce). Arp2/3 complex dephosphorylation Arp2/3 complex was dephosphorylated with AP (New England Biolabs, Inc.). Arp2/3 complex was diluted 1:1 with 2 mM Tris, pH 8, and combined with HipH Buffer (50 mM Tris, pH 8, 1 mM Mg2Cl2, and 0.1 mM ZnCl2) containing 1 U AP and incubated at 30°C for 1.5 h. For mock-treated