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 presence 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 PP2Cα (PP2Cα-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 dephosphorylated in series with YOP and PP2Cα (PP2Cα/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 filaments. 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.