The Arp2/3 complex has a fundamental role in the regulation of actin assembly at endocytic sites. In yeast, Pan1 is a key regulator of Arp2/3 that is known to be essential for endocytosis and actin organization, but the intricate details of the molecular mechanisms of Arp2/3 regulation at endocytic vesicles have eluded researchers — until now. Toshima et al. report in Nature Cell Biology that the kinase Prk1 shuts off Arp2/3-mediated actin polymerization during endocytosis by directly phosphorylating and inactivating Pan1.

Using site-directed mutagenesis, the authors showed that a mutant Pan1 protein that is resistant to Prk1-targeted phosphorylation produced large actin clumps and endocytic defects that were similar to those previously seen in Prk1- and Ark1-mutant cells (Ark1 is a Prk1-related kinase). Further analysis revealed that constitutive dephosphorylation of Pan1 caused filamentous (F)-actin structures to associate with endocytic components.

Loss-of-function studies had previously identified Pan1 as a downstream target of Prk1. Toshima et al. showed that Prk1 directly phosphorylates Pan1 at its N terminus to suppress its function as an Arp2/3 activator. Phosphorylation of Pan1 probably precludes its association with F-actin, thereby preventing it from activating Arp2/3, as a Pan1 truncation mutant that was defective in F-actin binding also lost its ability to activate Arp2/3 both in vitro and in vivo. Structure-function analysis revealed that Pan1 contains a Wiskott-Aldrich syndrome protein (WASP) homology-2 (WH2)-like motif that is necessary for Arp2/3 activation. However, in contrast to previously studied WH2 domains, the Pan1 WH2-like motif is unique in that it does not bind globular (G)-actin, but instead binds to F-actin.

So are the actin clumps that were observed in cells expressing phosphorylation-resistant Pan1 a result of uncontrolled actin polymerization? Consistent with unphosphorylated Pan1 being hyperactive, the authors demonstrated that mutating the F-actin-binding sites in constitutively unphosphorylated Pan1 was sufficient to suppress the formation of actin clumps and the associated endocytic defects. This also confirmed the absolute requirement for F-actin binding to Pan1 prior to Arp2/3 activation.

Toshima et al. propose an elegant model whereby, at the onset of endocytosis, Pan1 associates with endocytic proteins at the plasma membrane to activate the Arp2/3 complex and promote actin polymerization. Phosphorylation of Pan1 by Prk1 results in the release of Pan1 from the endocytic machinery as it can no longer bind to F-actin, and so this inhibits its Arp2/3-activator function. Presumably, Pan1 then joins the same machinery as other endocytic components in preparation for starting a new cycle.

Pan1, although an important component of receptor-mediated endocytosis, is not the only activator of the Arp2/3 complex. The authors have already shown that there is considerable overlap between the Pan1 phenotype and phenotypes that are caused by mutations in the WASP-related protein Las17 — a known Arp2/3 activator. It would certainly be interesting to see whether Las17 is also regulated by Prk1-mediated phosphorylation. However, the extent to which the findings reported by Toshima et al. using yeast will translate to more complex organisms remains to be seen, and is certainly something to keep in mind.