A reciprocal interdependence between Nck and PI(4,5)P(2) promotes localized N-WASp-mediated actin polymerization in living cells.
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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.