Figure 2. The Arp2/3 complex must be phosphorylated to nucleate actin ﬁlaments. (A) Polymerization assays show that Arp2/3 complex in the absence (red) and presence of 500 nM ScarVCA (blue) nucleates actin faster than actin alone (black). Arp2/3 complex dephosphorylated with the dual-speciﬁcity phosphatase AP (AP-Arp2/3) did not nucleate actin ﬁlaments in the absence (green) or pres- ence of ScarVCA (orange). (B and C) Arp2/3 complex dephosphorylated with the tyrosine- speciﬁc phosphatase YOP (YOP-Arp2/3; B) or the serine/threonine phosphatase PP2C (PP2C -Arp2/3; C) nucleated actin ﬁlaments at rates similar to control Arp2/3 complex in the absence (green) or presence of ScarVCA (orange). (D) Arp2/3 complex dephosphory- lated in series with YOP and PP2C (PP2C / YOP-Arp2/3) did not nucleate actin ﬁlaments either alone (green) or with ScarVCA (orange). (E) Phosphorylation of the Arp2/3 complex is necessary to cap the pointed ends of actin ﬁl- aments. 100 nM actin ﬁlaments capped at the barbed end with gelsolin were capped at the pointed end by mock-treated Arp2/3 complex (blue circles) but not by AP-dephosphorylated Arp2/3 complex (red triangles). (F) Gelsolin- capped actin ﬁlaments were titrated with mock-treated Arp2/3 complex (blue) or AP- dephosphorylated Arp2/3 complex. either tyrosine or serine/threonine phosphorylation being neces- sary for activation. Phosphorylation of the Arp2/3 complex is necessary to cap the pointed ends of actin ﬁlaments Phosphorylation could control nucleation activity by regulating several properties, including the association of subunits, the affin- ity of the Arp2/3 complex for NPFs, the affinity of the complex for the sides of actin filaments, and/or the ability of the complex to undergo the conformational change required for nucleation. Affinity purification using N-WASP VCA bound to agarose beads indicated an intact complex with and without AP treatment (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/ jcb.200802145/DC1). These data also indicated that AP-treated Arp2/3 complex binds to NPFs and we found that phosphory- lation did not affect the affinity of the Arp2/3 complex for binding ScarVCA (Fig. S2 B) or N-WASP VCA (Fig. S2 C). The affinity of untreated and mock-treated Arp2/3 complex for ScarVCA (Kd = 0.88 μM) and for N-WASP VCA (Kd = 0.34 μM) was simi- lar to previous findings (Pan et al., 2004; Kelly et al., 2006) and did not change with AP treatment. Additionally, phosphorylation did not affect affinity of the Arp2/3 complex for actin filaments, as determined by cosedimentation with filamentous actin. The Arp2/3 complex untreated or dephosphorylated with AP (Fig. S2 D), YOP, PP2C , or YOP and PP2C (unpublished data) bound actin filaments with a Kd of 1 μM, which is consistent with published data (Mullins et al., 1998). We used pointed-end binding to assay for the ability of the complex to undergo the conformational change required for nucle- ation. Nucleation of a new actin filament or binding of Arp2/3 complex to the pointed end of preformed actin filaments induces nucleotide hydrolysis on the Arp2 subunit (Dayel and Mullins, 2004). Actin filaments capped at the barbed end with gelsolin (100 nM gelsolin-actin seeds) elongated from the pointed end in the absence of Arp2/3 complex and excess actin monomer (Fig. 2 E). The addition of mock-treated Arp2/3 complex, a pointed end– capping protein (Mullins et al., 1998), slowed pointed-end elongation. ARP2/3 COMPLEX PHOSPHORYLATION • LeClaire et al. 649