Jcb_201404095 161..170

The seven subunit actin-related protein (Arp) 2/3 complex nucleates actin filaments that drive diverse cell processes such as vesicle trafficking, plasma membrane protrusion during cell migration, and motility of some pathogens in host cells (Goley and Welch, 2006). Hence, resolving how activity of the Arp2/3 complex is regulated is important for a basic understanding of diverse cell processes and for targeting aberrant actindependent cell behaviors in diseases. Arp2/3 complex binding to nucleation promoting factors (NPFs) such as N-WASP was previously thought to be sufficient to increase nucleating activity (Welch and Mullins, 2002). However, we recently showed that the Arp2 subunit must be phosphorylated for the complex to be activated by NPFs (LeClaire et al., 2008; Narayanan et al., 2011; Choi et al., 2013). The necessary role for Arp2 phosphorylation for nucleation activity suggests that the Arp2/3 complex is a coincidence detector requiring both binding to NPFs and phosphorylation of Arp2 for increased activity. Coincidence detection is emerging as a common regulatory mechanism for many actin-binding proteins, including NPFs (Rohatgi et al., 2001; Rivera et al., 2009) and cofilin (Frantz et al., 2008; Magalhaes et al., 2011). In support of coincidence regulation of the Arp2/3 complex, we show that expression of a phosphorylationdefective Arp2 mutant complexes with endogenous Arp2/3 complex subunits and dominantly suppresses increased actin filament assembly and membrane protrusion in response to EGF. Despite the confirmed importance of phosphorylated Arp2 in the nucleation activity of the Arp2/3 complex, kinases phosphorylating Arp2 have not been identified. We report that the Nck-interacting kinase (NIK), a Ste20/MAP4K4 serine/threonine kinase, phosphorylates Arp2 and primes the Arp2/3 complex for activation by NPFs. NIK and its orthologues have a conserved role in regulating actin cytoskeleton-dependent cell processes. NIK activity is necessary for mesoderm migration (Xue et al., 2001) and for epithelial cell membrane protrusion (Baumgartner et al., 2006) and invasion (Wright et al., 2003). The NIK orthologue misshapen in Drosophila melanogaster functions in determining epithelial polarity, neuronal targeting, and cell invasion (Su et al., 1997; Cobreros-Reguera et al., 2010), and the orthologue MIG-15 in Caenorhabditis elegans controls axonal navigation (Poinat et al., 2002) and neuroblast The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics. The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit