N-WASP as a PIP 2 Density Sensor

Abstract

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a stimulator of actin nucleation through the actin-related protein complex, ARP2/3. N-WASP is activated in a cooperative manner by binding of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] to the polybasic B motif and binding of the guanosine triphosphatase (GTPase) Cdc42 to the GTPase binding domain (GBD). Papayannopoulos et al. explored the mechanism by which PI(4,5)P 2 regulates N-WASP activity. Using in vitro assays with N-WASP peptide fragments, they demonstrated that the B motif bound with highest affinity to PI(4,5)P 2 compared with other phosphoinositide lipids and that the 10-lysine motif was the minimum sequence that bound PI(4,5)P 2 . In vesicle sedimentation assays, PI(4,5)P 2 concentration can be increased by either increasing the number of PI(4,5)P 2 -containing vesicles in the preparation or by increasing the concentration of PI(4,5)P 2 within a given vesicle [the mole percent (mol%) of PI(4,5)P 2 ]. The curve representing binding of the B-GBD fragment of N-WASP to vesicles with increasing mol% of PI(4,5)P 2 showed a sigmoidal shape, indicating cooperative binding. Furthermore, binding of B-GBD to PI(4,5)P 2 -containing vesicles that also included cholesterol to induce the formation of membrane microdomains was higher affinity (with cooperative binding) than binding in the absence of cholesterol. Using two N-WASP constructs (one only lacking the EVH1 domain and one consisting of the B, GBD, and VCA domains--called mN-WASP), the authors demonstrated a sigmoidal, highly cooperative curve (Hill coefficient of ~18) for stimulation of ARP2/3-mediated actin polymerization as a function of mol% PI(4,5)P 2 . The sensitivity of N-WASP to PI(4,5)P 2 -containing vesicles was increased by the presence of prenylated Cdc42 in the vesicles. Increasing or decreasing the number of lysines in the B motif in the context of mN-WASP changed the sensitivity to PI(4,5)P 2 density, with an increase from the native 9 lysines to 14 lysines yielding a Hill coefficient for actin nucleation of ~50. Similar dependency on the number of lysines was observed for the ability of mN-WASP to stimulate endosomal vesicle motility in Xenopus egg extracts. The 14-lysine version of mN-WASP also stimulated a phenomenon called vesicle rocketing in transfected cells, even in the absence of conditions that increased basal PI(4,5)P 2 concentrations. Thus, using multiple in vitro and in vivo tests, the B motif of N-WASP appears to confer switchlike activity to N-WASP, allowing N-WASP to be poised to respond to changes in the density of PI(4,5)P 2 , which may allow cells to link changes in the actin cytoskeleton to signals that alter membrane microdomains and prevent activation of N-WASP in response to "noise" during the quiescent state. V. Papayannopoulos, C. Co, K. E. Prehoda, S. Snapper, J. Taunton, W. A. Lim, A polybasic motif allows N-WASP to act as a sensor of PIP 2 density. Mol. Cell 17 , 181-191 (2005). [PubMed]