controls, Arp2/3 complex was incubated with heat-inactivated phospha- tase (65°C for 30 min) in HipH reaction buffer. For YOP dephosphoryla- tion, Arp2/3 complex was incubated with 1 U YOP (New England Biolabs, Inc.) in 50 mM Tris-HCl, pH 7, 100 mM NaCl, 5 mM DTT, and 0.01% Brij 35 at 30°C for 1 h. For PP2C dephosphorylation, the Arp2/3 complex was incubated with 1 U PP2C (EMD) in 25 mM MES, 50 mM NaCl, 2 mM MnCl2, and 1 mM DTT at 30°C for 1 h. The Arp2/3 complex was afﬁnity puriﬁed after phosphatase treatment by using N-WASP VCA coupled to activated CH–sepharose 4B (GE Healthcare). Actin polymerization Pyrene actin polymerization assays were performed with 4 μM monomeric actin containing 5% pyrene-labeled actin in KMEI (50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole, pH 7), 5 nM Arp2/3 com- plex, and 500 nM ScarVCA/N-WASP VCA domain. Ca-ATP actin was converted to Mg-ATP actin by incubation in ME (50 mM MgCl2 and 0.2 mM EGTA) before adding other assay components. Pyrene actin was excited in an RF-5301PC spectrophotometer (Shimadzu) at 365 nm, and ﬂuorescence was measured at 407 nm at 1-s intervals. Actin ﬁlament barbed ends were calculated as described previously (Higgs, et al., 1999). Pointed elongation from gelsolin-capped actin ﬁlaments was measured as described previ- ously (Mullins et al., 1998). Gelsolin-capped actin ﬁlaments (100 nM) were used for pointed end–binding assays with untreated and dephos- phorylated Arp2/3 complex. Quantiﬁcation of Arp2/3 complex binding constants Binding constants of Arp2/3 complex for NPFs were determined by using GST-ScarVCA and GST-N-WASP VCA covalently coupled to activated CH–sepharose 4B (GE Healthcare). GST-NPF–coupled beads were added to mock-treated or AP-treated Arp2/3 complex and incubated at room temperature for 30 min. NPF-coupled beads were spun at 700 g for 5 min, the supernatant was removed, and beads were resuspended in SDS-PAGE sample buffer. Coomassie-stained gels were scanned and quantiﬁed using a LabWorks imaging system and LabWorks Software (UVI). The data were plotted and ﬁtted using GraphPad Prism software (GraphPad Software, Inc.). Binding constants for Arp2/3 complex for actin ﬁlaments were determined by actin cosedimentation as described previously (Mullins et al., 1997). Mass spectrometry Proteins were separated on 12.5% SDS-PAGE gels, individual bands were excised, and gel slices were destained with 25 mM NH4HCO3/50% aceto- nitrile. After reduction with 10 mM DTT and alkylation with 55 mM iodo- acetamide, proteins were digested with 12.5 ng/μl of recombinant porcine trypsin (Roche) in 25 mM NH4HCO3 at 37°C for 16 h. Peptides were extracted from the gel slices and cleaned using a ZipTipC18 (Millipore) pipette tip. Mass spectrometry was performed with a matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometer (Voyager-DE STR; Applied Biosystems). MTLn3 cell culture and metabolic labeling MTLn3 rat adenocarcinoma cells (provided by J. Condeelis, Albert Einstein College of Medicine, New York, NY) were maintained as described previ- ously (Segall et al., 1996). For metabolic labeling, 3 × 106 cells were plated on 150-mm dishes and grown to 85% conﬂuence in MEM without nucleosides supplemented with 10% FBS at 37°C, 5% CO2. Cells were transferred to phosphate-free MEM without nucleosides supplemented with 0.2 mCi/ml [32P]orthophosphate for 4 h and then stimulated with 10 nM EGF. Cells were lysed at the indicated times with modiﬁed RIPA buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 1% NP-40, 1 mg/ml aprotinin, 1 mM pefabloc, and 1 mg/ml leupeptin) containing phospha- tase inhibitors (1 mM EGTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM -glycerophosphate, and 1 mM sodium orthovanadate), and the Arp2/3 complex was immunoprecipitated with anti-ARPC1 antibody (a gift from M. Welch, University of California, Berkeley) as described previously (Machesky and Insall, 1998). S2 cell culture, Arp2 RNAi, and Arp2 mutants D. melanogaster Schneider S2 cells were cultured as described previ- ously (Rogers et al., 2002). Arp2 siRNA was performed with dsRNA to the 3 untranslated region (UTR) coding sequence of D. melanogaster Arp2 (5 -GUGUGUGUGCGGACCGCAAGAAAUAGGAUAAAAAAGU- GAUAGAUUUCUUUUCUCUAUUUUCUATAGGUUUAAACCUUUCAGAU- UUACGUGAUAUAUCCGUCUAUAUAUGUUUUUUUUUU-3 ). The Arp2 3 UTR was cloned into a TOPO plasmid (Invitrogen) containing a T7 promoter. RNA was ampliﬁed using a T7 Megascript RNA ampliﬁcation kit (Ambion). Plasmids expressing D. melanogaster Arp2 were constructed using Gate- way cloning technology (Invitrogen). D. melanogaster Arp2 was cloned from an S2 cDNA library, sequenced, and inserted into a pENTR-D-TOPO plasmid (Invitrogen). Arp2 was then cloned into an expression vector con- taining a single C-terminal GFP. Mutant Arp2 sequences were constructed using a QuikChange Mutagenesis kit (Stratagene). RNAi was performed over 7 d as described by Rogers et al. (2003). In brief, 5 μg Arp2 double- stranded RNA was added to 24-well plates containing 3 × 105 cells on day one and again on day three. Cells were transformed with plasmids using Cellfectin LTX (Invitrogen) on day ﬁve. Plasmid transfection in S2 cells was 30% efﬁcient. On day seven, S2 cells were prepared for microscopy as described previously (Rogers et al., 2003) and mounted in ﬂuorescent mounting medium (Dako). Images were acquired with a Roper SPOT charge-coupled device camera (Roper Scientiﬁc) on an Axiophot microscope (Carl Zeiss, Inc.) at 40× magniﬁcation at room temperature with Roper SPOT acquisition software. Images were assembled into ﬁgures using Photo- shop (Adobe). Arp2/3 structural model Arp2/3 complex structure was analyzed using PyMol molecular viewing software and PDB 1K8K (Robinson et al., 2001). Images were imported and notated using Illustrator software (Adobe). Online supplemental material Fig. S1 shows data related to AP treatment of Arp2/3 complex with mock-treated samples, N-WASP VCA, and phosphatase inhibitors. Fig. S2 shows the effect of AP treatment on Arp2/3 complex subunit association, NPF-binding, and F-actin binding. Fig. S3 shows mass spectrometry data for phospho-Arp2 and immunoblots of Arp2-GFP from S2 cells. Online sup- plemental material is available at http://www.jcb.org/cgi/content/full/ jcb.200802145/DC1. We thank Jenny Chen, Orkun Akin, Ewa Witkowski, and Steven Hall for tech- nical assistance, the Matthew Welch laboratory for reagents, and the Barber and Mullins laboratories for discussions. This work was supported by National Institutes of Health grants to R.D. Mullings (GM61010) and D.L. Barber (GM58642). L.L. LeClaire was sup- ported by a National Institutes of Health training grant (NIH T32-DE7306-11). Work by L.L. LeClaire and D.L. Barber was conducted in a facility constructed with support from Research Facilities Improvement Program Grant No. C06 RR16490 from the National Center for Research Resources, National Institutes of Health. Submitted: 22 February 2008 Accepted: 24 July 2008 References Dayel, M.J., and R.D. Mullins. 2004. Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2. PLoS Biol. 2:E91. Goley, E.D., and M.D. Welch. 2006. The ARP2/3 complex: an actin nucleator comes of age. Nat. Rev. Mol. Cell Biol. 7:713–726. Goley, E.D., S.E. Rodenbusch, A.C. Martin, and M.D. Welch. 2004. Critical con- formational changes in the Arp2/3 complex are induced by nucleotide and nucleation promoting factor. Mol. Cell. 16:269–279. Higgs, H.N., L. Blanchoin, and T.D. Pollard. 1999. Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. Biochemistry. 38:15212–15222. Iwasa, J.H., and R.D. Mullins. 2007. Spatial and temporal relationships be- tween actin-filament nucleation, capping, and disassembly. Curr. Biol. 17:395–406. Kelly, A.E., H. Kranitz, V. Dotsch, and R.D. Mullins. 2006. Actin binding to the central domain of WASP/Scar proteins plays a critical role in the activa- tion of the Arp2/3 complex. J. Biol. Chem. 281:10589–10597. Machesky, L.M., and R.H. Insall. 1998. Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr. Biol. 8:1347–1356. Martin, A.C., M.D. Welch, and D.G. Drubin. 2006. Arp2/3 ATP hydrolysis- catalysed branch dissociation is critical for endocytic force generation. Nat. Cell Biol. 8:826–833. Mullins, R.D., W.F. Stafford, and T.D. Pollard. 1997. Structure, subunit topology, and actin-binding activity of the Arp2/3 complex from Acanthamoeba. J. Cell Biol. 136:331–343. ARP2/3 COMPLEX PHOSPHORYLATION • LeClaire et al. 653