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 phosphatase YOP did not affect labeling of pThr antibodies but abolished 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.

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.

The putative phosphorylation sites are conserved. (A) Phosphorylated T237, T238, and Y202 of Arp2 were mapped to the Arp2/3 complex crystal structure obtained by Robinson et al. (2001). From the crystal structure, T237 and T238 of Arp2 are adjacent to R105 and R106 of ARPC4, and Y202 of Arp2 is adjacent to R409 of Arp3. (inset) Enlarged view of the boxed region. (B) The proposed phosphorylation sites on the Arp2 subunit and adjacent arginine residues in ARPC4 and Arp3 are conserved in nine organisms.

Phosphorylation of the Arp2 is regulated in cells. (A) Immunoprecipitation of the Arp2/3 complex with antibodies to ARPC1 from MTLn3 epithelial cells metabolically labeled with [³²P]orthophosphate indicated that the Arp2 subunit is phosphorylated in unstimulated cells, and phosphorylation increased after stimulation with EGF. (B) Quantification of time-dependent Arp2 phosphorylation. Data are expressed 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.

Phosphorylation of Arp2 is necessary for lamellipodia formation. (A) Control, luciferase RNAi-treated D. melanogaster S2 cells on concanavalin A–coated coverslips spread symmetrical, round lamellipodia. S2 cells depleted of the Arp2 subunit by siRNA (Arp2 RNAi) exhibited a stellate morphology with membrane protrusions radiating from the cell body. A wild-type morphology of circular lamellipodia was restored in Arp2-depleted S2 cells expressing wild-type Arp2-GFP (Arp2-WT-GFP), or mutated Arp2-T237/238A-GFP or Arp2-Y202A-GFP. Arp2-depleted S2 cells expressing the triple mutant Arp2-T237/238A-Y202A-GFP remained stellate. Bar, 5 μM. (B) The number of cells with wild-type or stellate morphology were scored for the indicated conditions and expressed as the mean ± SD of three separate cell preparations, with >500 cells scored for each condition per cell preparation. (C) Model for activation of the Arp2/3 complex by phosphorylation of the Arp2 subunit. In the “inactive” form of the Arp2/3 complex, the Arp2 and Arp3 subunits are splayed open. Phosphorylated T237/T238 and Y202 residues in Arp2 interact with arginine residues on adjacent subunits to maintain a conformation that stabilizes the complex in the “active” form. This conformation aligns the Arp2 and Arp3 subunits to form a protonucleus for actin nucleation.