Figure 4. Phosphorylation of the Arp2 is regulated in cells. (A) Immunoprecipitation of the Arp2/3 complex with antibodies to ARPC1 from MTLn3 epithelial cells metabolically la- beled with [32P]orthophosphate indicated that the Arp2 subunit is phosphorylated in unstimu- lated cells, and phosphorylation increased after stimulation with EGF. (B) Quantiﬁcation of time- dependent Arp2 phosphorylation. Data are ex- pressed as the fold increase of phosphorylation in the absence of EGF and represent means ± SD of ﬁve separate cell preparations. (C) The Arp2/3 complex was immunoprecipitated from quiescent and EGF-stimulated MTLn3 cells. Labeling of Arp2 with antibodies to pThr and pTyr increased 1.3- and 1.21-fold after EGF stimulation, respectively. bridges with R105 and R106 of ARPC4 to perhaps stabilize the Arp2/3 complex in an active conformation. Molecular dynamics simulations suggest that Arp2 pT237 interacts exclusively with R105 of ARPC4 (unpublished data). Because Arp2 pTyr residues were suggested by immunoblotting but were not identified by mass spectrometry, we searched the Arp2 structure for potential tyrosine phosphorylation sites that might interact with basic resi- dues of adjacent subunits. Of particular interest was Y202 in sub- domain IV, which is adjacent to R409 in the Arp3 subunit. Sequence alignments of Arp2/3 subunits of nine organisms re- vealed that Arp2 T237, T238, and Y202; Arp3 R409; and ARPC4 R105 and R106 are evolutionary conserved from yeasts and slime molds to mammals (Fig. 3 B). To determine whether phosphorylation of Arp2/3 complex subunits is regulated in cells, MTLn3 epithelial carcinoma cells were metabolically labeled with [32P]orthophosphate and stimulated with 10 nM EGF, and the Arp2/3 complex was immunoprecipitated with antibodies to the ARPC1 subunit. Phosphorylation of only the Arp2 subunit was detected in un- stimulated and EGF-treated cells (Fig. 4 A). Phosphorylation of other unidentified proteins was detected in the immune com- plex; however, none of these proteins migrated with Arp2/3 complex subunits. EGF induced a time-dependent but transient increase in Arp2 phosphorylation (Fig. 4, A and B). Phosphory- lation rapidly increased at 90 s, was maximal at 180 s, with a twofold increase, and was not significantly different compared with quiescence at 360 s. In response to EGF, labeling by anti- pThr increased 1.3-fold and labeling by anti-pTyr increased 1.21-fold at 180s, as determined by immunoprecipitating Arp2/3 complex from unlabeled cells and immunoblotting with anti- bodies to pThr and pTyr (Fig. 4 C). Phosphorylation of Arp2 is necessary for lamellipodia formation We asked whether the identified pThr residues and the speculated pTyr residue in Arp2 were functionally significant by analyzing lamellipodia formation by Drosophila melanogaster Schneider S2 cells. When plated on coverslips coated with concanavalin A, S2 cells extended a circular, flat lamellipodia (Fig. 5 A), which is similar to previous findings (Rogers et al., 2003). When the Arp2/3 complex is inactivated by depletion of the ARPC3 or ARPC4 subunits by RNAi, S2 cells exhibit a stellate morphology with formations that extend radially around the cell body (Rogers et al., 2002; Iwasa and Mullins, 2007). We observed a similar stellate phenotype when S2 cells were depleted of the Arp2 sub- unit by Arp2 siRNA (Fig. 5 A). Transient expression of wild-type Arp2 fused to GFP (Arp2-WT-GFP) in Arp2-depleted cells re- stored a wild-type phenotype with flat circular lamellipodia (Fig. 5, A and B). Expression of Arp2 containing alanine substitutions for T237 and T238 (Arp2-T237/238A-GFP) or for Y202 (Arp2- Y202A-GFP) also restored lamellipodia formation (Fig. 5, A and B). In contrast, expression of Arp2-T237/238A-Y202A-GFP did not restore lamellipodia formation, and cells retained a stellate phenotype. These findings are consistent with our in vitro actin polymerization data suggesting that either threonine or tyrosine phosphorylation of the complex is necessary for nucleating activity. ARP2/3 COMPLEX PHOSPHORYLATION • LeClaire et al. 651