Figure 1. The Arp2/3 complex is threonine- and tyrosine- phosphorylated in cells. (A) The Arp2/3 complex puriﬁed from A. castellani separated by 2D electrophoresis revealed that Arp2 migrated as five spots and Arp3 as four spots (arrowheads). Pretreatment of the complex with the dual- speciﬁcity AP resulted in Arp2 migrating as two spots and Arp3 migrating as two spots at a more basic pI relative to a ﬁduciary marker (asterisks). (B) The Arp2, Arp3, and ARPC1 subunits of the Arp2/3 complex puriﬁed from A. castellani labeled with antibodies to pThr and pTyr. Dephosphorylation of the Arp2/3 complex with AP abolished labeling of Arp2 with pThr and pTyr antibodies, decreased labeling of ARPC1 by pTyr antibodies to ARPC1, and did not change labeling of Arp3. Dephosphorylation with the tyrosine-speciﬁc phospha- tase YOP did not affect labeling of pThr antibodies but abol- ished labeling of Arp2 with pTyr antibodies and decreased labeling of ARPC1. (C) The Arp2 subunit of puriﬁed bovine Arp2/3 complex labeled with antibodies to both pThr and pTyr. (D) The Arp3 and Arp2 subunits of recombinant human Arp2/3 complex labeled with antibodies to pThr and pTyr. Arp2/3 complex from Acanthamoeba castellani, Arp2 and Arp3 migrated as multiple spots (Fig. 1 A). After dephos- phorylation with Antarctic phosphatase (AP), a dual-speci- ficity alkaline phosphatase, Arp2 and Arp3 migrated as fewer spots at a more basic pI. These data suggest multiple phosphorylated species of Arp2 and at least one of Arp3. The presence of multiple spots for both subunits after AP treatment suggests posttranslational modifications in addi- tion to phosphorylation. We also used immunoblotting to determine phosphory- lation of the Arp2/3 complex purified from A. castellani. Arp2, Arp3, and ARPC1 subunits labeled with antibodies to phosphothreonine (pThr) and phosphotyrosine (pTyr; Fig. 1 B). Labeling of ARPC1 with pThr antibodies is consistent with a previous report that this subunit is phosphorylated on Thr21 (Vadlamudi et al., 2004). When the A. castellani complex was dephosphorylated with AP, Arp2 labeling with pThr and pTyr antibodies was abolished, ARPC1 labeling with pTyr anti- bodies was decreased, and Arp3 labeling was unchanged. Because AP changed the mobility of Arp3 on 2D gels but not Arp3 immunolabeling, we suspect the immunoblot signal may be nonspecific. Arp3 could be phosphorylated on serine residues; however, we were unable to confirm specific label- ing of any Arp2/3 complex subunit with antiphosphoserine antibodies. Tyrosine phosphorylation of Arp2 and ARPC1 subunits was confirmed by dephosphorylating the Arp2/3 complex with the tyrosine-specific phosphatase from Yersinia entercolitica (YOP; Fig. 1 B). Using Arp2/3 complex purified from bovine thymus, the Arp2 but not the Arp3 or ARPC1 subunits labeled with antibodies to pThr and pTyr, and label- ing was abolished after treatment with AP (Fig. 1 C). Immuno- blotting recombinant human Arp2/3 complex purified from insect cells also showed pThr and pTyr labeling of Arp2 be- fore but not after AP treatment (Fig. 1 D). These data suggest that phosphorylation of the Arp2/3 complex, particularly Arp2, is not species specific. Arp2/3 complex phosphorylation is required for actin nucleation We found that a dephosphorylated Arp2/3 complex does not nucleate actin filaments. Mock-treated (heat-inactivated phos- phatase) Arp2/3 complex purified from A. castellani nucleated actin filaments with the C-terminal domain of the nucleation- promoting factor (NPF) Scar (ScarVCA) faster than with actin alone (Fig. 2 A), and at rates similar to an untreated complex (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/ jcb.200802145/DC1). We calculated the number of barbed ends formed by 5 nM of mock-treated Arp2/3 complex, 500 nM ScarVCA, and 4 μM actin to be 3 nM. The number of free barbed ends is comparable to similar studies (Higgs et al., 1999) and in agreement with ScarVCA being a less potent Arp2/3 activator compared with N-WASP-VCA (Zalevsky et al., 2001). The Arp2/3 complex dephosphorylated with AP did not nucleate actin filaments with actin alone or in the presence of ScarVCA (Fig. 2 A) or N-WASP VCA (Fig. S1 B). Arp2/3 nucleating activity decreased with increasing time of incuba- tion with the phosphatase (Fig. S1 C). Activity of the Arp2/3 complex incubated with AP in the presence of phosphatase inhibitors was identical to the activity of mock-treated con- trols (Fig. S1 D). Arp2/3 complex purified from bovine thy- mus also nucleated actin filaments but not after treatment with AP (Fig. S1 E). We next asked whether tyrosine or serine/threonine phos- phorylation is necessary for Arp2/3 nucleation activity. Arp2/3 complex purified from A. castellani and dephosphorylated with YOP or the serine/threonine protein phosphatase 2C (PP2C ) nucleated actin at rates identical to mock-treated controls (Fig. 2, B and C). However, when samples were dephosphorylated in se- ries with YOP and PP2C , the Arp2/3 complex was unable to nucleate actin filaments (Fig. 2 D), which suggests that either ty- rosine phosphorylation or serine/threonine phosphorylation is required. These findings indicate that regulation of actin nucle- ation by the Arp2/3 complex may act as a logical “or gate,” with 648 JCB • VOLUME 182 • NUMBER 4 • 2008