Figure 3. 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. Mock-treated Arp2/3 complex bound pointed ends with an appar- ent Kd of 42 nM, which is similar to previously described values (Mullins et al., 1998). The Arp2/3 complex dephosphorylated with AP did not block elongation of barbed end–capped actin filaments. The rate of pointed-end elongation decreased when titrated with untreated Arp2/3 complex but remained nearly constant when titrated with AP-dephosphorylated Arp2/3 complex (Fig. 2 F). Because capping of the actin filament pointed end is similar to the nucleation step, these data indicate that the complex must be phos- phorylated to undergo the conformation change required for actin nucleation. Phosphorylation of the Arp2 subunit is on conserved residues and is regulated in cells We used nanoelectrospray LC-MS/MS to identify phosphory- lated residues in Arp2/3 complex purified from A. castellani cells. We identified two threonine phosphorylation sites on the Arp2 subunit, T237 and T238 (Fig. S3 A, available at http://www.jcb .org/cgi/content/full/jcb.200802145/DC1), but did not detect pThr residues in Arp3 or pSer residues in Arp2 or Arp3. We also did not detect pTyr residues on Arp2 or Arp3, despite immunoblotting data indicating that Arp2 labels with anti-pTyr antibodies before but not after treatment with AP or YOP. Because the amino acid sequence of A. castellani ARPC1 is not known, we were unable to identify phosphorylated sites by mass spectrometry. Based on the atomic structure of the Arp2/3 complex deter- mined by Robinson et al. (2001), T237 and T238 of the Arp2 sub- unit are in subdomain IV near the interface of the Arp3, ARPC2, and ARPC4 subunits (Fig. 3 A). The side chains of Arp2 T237 and T238 are directly adjacent to the basic side chains of R105 and R106, respectively, in the ARPC4 subunit (Fig. 3 A). Phos- phorylated T237 and T238 of the Arp2 complex could form salt 650 JCB • VOLUME 182 • NUMBER 4 • 2008