Neuronal Wiskott–Aldrich syndrome protein (N-WASP) activates the Arp2/3 complex, which stimulates actin polymerization. N-WASP activity is repressed by intramolecular interactions whereby the N-terminal polybasic (B) motif and GTPase-binding domain (GBD) inhibit the activity of the C-terminal Arp2/3-stimulating VCA domain. Such autoinhibition is relieved by the binding of phosphatidylinositol 4,5- bisphosphate (PtdIns(4,5)P2) and Cdc42·GTP to the B motif and GBD, respectively. Papayannopoulos et al. have discovered that PtdIns(4,5)P2 binds to the B domain in a multivalent and cooperative manner. This means that, above a certain threshold, small increases in the concentration of PtdIns(4,5)P2 confer a switch-like activation on N-WASP.

A polypeptide that corresponded to the GBD and B motif bound most strongly to PtdIns(4,5)P2 from a panel of lipids. Deletion analysis narrowed the region down to a 15-residue fragment (encompassing the B motif) that contained 10 basic residues (mainly lysines), and Papayannopoulos et al. showed that this high positive-charge density was required for binding.

The authors then showed that the B motif bound PtdIns(4,5)P2 with higher affinity when it was present at higher density, so they measured the influence of this effect on N-WASP-mediated actin polymerization. Activation, even more than binding, of N-WASP depended on PtdIns(4,5)P2 density, and only occurred in vitro above a steep threshold.

The apparent affinity of PtdIns(4,5)P2 for the B motif alone seemed higher than for the full-length form. The authors proposed that the B motif is occluded by autoinhibition in the full-length form of N-WASP, and that, if several adjacent PtdIns(4,5)P2-binding sites are occluded by autoinhibitory interactions, cooperativity of PtdIns(4,5)P2 binding might increase the apparent affinity of subsequent PtdIns(4,5)P2 binding by disrupting autoinhibitory interactions. This proved to be correct. Adding Cdc42 also decreased the N-WASP-activation thresholds, in keeping with Cdc42 relieving the autoinhibitory interactions. Adding both PtdIns(4,5)P2 and prenylated Cdc42 (to mimic its membrane localization) further activated N-WASP.

Next, Papayannopoulos et al. investigated the motility rates of partially purified endosomal vesicles to assess actin-mediated vesicle motility. They depleted endogenous N-WASP, substituted N-WASP variants and artificially induced vesicle motility. Wild-type N-WASP could mediate vesicle motility, but deleting the B motif or some of the basic lysine residues within it couldn't reconstitute motility. But when the number of lysines was increased, motile vesicles with longer actin-containing comet tails were generated, and moved faster than the wild-type-N-WASP-driven vesicles. A significant amount of N-WASP was also activated at resting levels of PtdIns(4,5)P2 in vivo when extra lysines were present in the B motif. Consistent with this, the construct containing extra lysines showed a lower activation threshold and increased threshold steepness, most likely because the multivalency (the number and the affinity) of PtdIns(4,5)P2-binding sites was increased.

The finding that the polybasic region of N-WASP binds to PtdIns(4,5)P2 in a multivalent manner, and is involved in the sharpness of the PtdIns(4,5)P2-mediated transition, provides another example of cooperative activation in biology. This sharp activation threshold probably confers on N-WASP the ability to respond to subtle, signal-induced changes in the local concentrations of PtdIns(4,5)P2.