As direct activators of the Arp2/3 complex, Wiskott–Aldrich syndrome proteins (WASPs) link Cdc42 activation to the formation of actin filaments, and WASP phosphorylation on tyrosine 291 enhances WASP activity toward the Arp2/3 complex. In Molecular Cell, Torres and Rosen further explore Y291 phosphorylation, while Cory et al. identify two new WASP phosphorylation sites.

Autoinhibition of WASP — by the carboxy-terminal VCA (verprolin homology, central hydrophobic, and acidic) domain binding to the GTPase-binding domain (GBD) — is relieved by active Cdc42. Torres and Rosen investigated cooperation between Cdc42 and tyrosine kinases in WASP regulation. Y291 is in the GBD, within the fold of the autoinhibited domain, and so is only expected to be phosphorylated when autoinhibition is relieved. Indeed, adding the VCA domain inhibited Y291 phosphorylation; this was relieved by adding a mimic of GTP-bound Cdc42. Similarly, the autoinhibited fold protected phosphoY291 (pY291)-WASP against tyrosine phosphatases. So, WASP Y291 phosphorylation seems to be altered only when activated Cdc42 is present, which, in the context of positive signalling, would allow WASP to remain phosphorylated long after the initial stimulus had subsided.

It has previously been proposed that Y291 phosphorylation might destabilize autoinhibition and constitutively activate WASP. Indeed, the pY291-GBD still bound VCA, but with a lower affinity — its basal activity towards the Arp2/3 complex, though, increased. Another consequence was that Src, through its Src-homology-2 (SH2) domain, could displace pY291-GBD from the VCA, which, again, increases Arp2/3 activity. Src kinase activity might also potentiate WASP activation by phosphorylating Y291.

Cory et al. identified two phosphorylation sites — serine 483 and serine 484 — in the VCA domain and showed that these sites in WASP and its relative N-WASP are phosphorylated in various cell types. S483 is within a consensus casein kinase 2 (CK2) phosphorylation site, and CK2 inhibitors decrease endogenous levels of pS483/pS484-WASP. Cory et al. also showed that S483 and S484 are substrates for CK2 in vitro. S483/S484 phosphorylation enhanced VCA–Arp2/3 binding, and phosphorylation of S483/S484 enhanced actin polymerization that was induced by activated Cdc42 or by a Y291 mutation that renders WASP constitutively active. S483/S484 phosphorylation, therefore, seems to be important for optimal functioning of activated WASP.

So, as Cory et al. conclude, “...post-translational modification of WASP is important in its regulation...and it will be of great interest to determine the interplay between phosphorylation and other cellular regulators of WASP function”.