A Reciprocal Interdependence between Nck and PI(4,5)P2 Promotes Localized N-WASp-Mediated Actin Polymerization in Living Cells

Summary Modulation of actin dynamics through the N-WASp/Arp2/3 pathway is important in cell locomotion, membrane trafficking, and pathogen infection. Here, we demonstrate that Nck is essential for actin remodeling stimulated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P 2 ) and, conversely, that PI(4,5)P 2 is necessary for localized actin polymerization induced by Nck in vivo. Nck knockdown or knockout suppressed actin comets induced by phosphatidylinositol 5-kinase (PIP5K), and PIP5K stimulated tyrosine phosphorylation of an Nck SH2 domain binding partner, suggesting that Nck couples phosphotyrosine- and phosphoinositide-dependent signals. We show that PI(4,5)P 2 and PIP5K are both enriched at actin comets induced by Nck aggregates and that formation of actin comets was strongly inhibited by coclustering with an inositol 5-phosphatase domain to decrease local PI(4,5)P 2 levels. The extent of Nck-induced actin polymerization was also modulated by PI(4,5)P 2 -sensitive N-WASp mutants. This study uncovers a strong reciprocal interdependence between Nck and PI(4,5)P 2 in promoting localized N-WASp-mediated actin polymerization in cells.