Editors' ChoiceProtein Domains

N-WASP as a PIP2 Density Sensor

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Science's STKE  25 Jan 2005:
Vol. 2005, Issue 268, pp. tw36
DOI: 10.1126/stke.2682005tw36

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)P2] 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)P2 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)P2 compared with other phosphoinositide lipids and that the 10-lysine motif was the minimum sequence that bound PI(4,5)P2. In vesicle sedimentation assays, PI(4,5)P2 concentration can be increased by either increasing the number of PI(4,5)P2-containing vesicles in the preparation or by increasing the concentration of PI(4,5)P2 within a given vesicle [the mole percent (mol%) of PI(4,5)P2]. The curve representing binding of the B-GBD fragment of N-WASP to vesicles with increasing mol% of PI(4,5)P2 showed a sigmoidal shape, indicating cooperative binding. Furthermore, binding of B-GBD to PI(4,5)P2-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)P2. The sensitivity of N-WASP to PI(4,5)P2-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)P2 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)P2 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)P2, 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 PIP2 density. Mol. Cell 17, 181-191 (2005). [PubMed]

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