Focal Adhesion Kinase Regulation of N-WASP Subcellular Localization and Function

Abstract

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr 256 . We found that phosphorylation of Tyr 256 affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr 256 of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr 256 plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.

Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions

“…In this regard, Cdc42 and phosphatidylinositol (3,4,5)-triphosphate binding with N-WASP as well as the tyrosine phosphorylation of N-WASP has been shown to induce N-WASP activity (23,24). We previously showed that both Cdc42 and FAK maintain AJs and restore basal endothelial permeability following chal- lenge with edemagenic agents (25,26).…”

“…Thrombin Induces N-WASP Phosphorylation in a FAK-dependent Manner That Restores Barrier Function-Phosphorylation of N-WASP by tyrosine kinases such as FAK has been shown to induce its activation (24). We therefore addressed whether thrombin induces the phosphorylation of N-WASP in a FAK-dependent manner.…”

“…Several mechanisms have been implicated in facilitating the unfolding of the VCA domain from GBD domain, including WASP phosphorylation by tyrosine kinases and interaction with phosphatidylinositol (3,4,5)-triphosphate or Cdc42 GTPases (21,23). Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, was shown to phosphorylate N-WASP at Tyr-256, leading to its activation (24). FAK and Cdc42 are known to maintain AJ function in part by interacting with p120-catenin (25)(26)(27).…”

Focal adhesion kinase-Tyr 407 and -Tyr 397 exhibit antagonistic effects on blood–testis barrier dynamics in the rat

“…2). It is noted that the association between FAK and N-WASP was also observed in NIH 3T3 cells (12). Given the coexistence of FAK and N-WASP within the same macromolecular complex in the seminiferous epithelium, the possible influence of FAK on N-WASP-mediated activation of the Arp2/3 complex was next examined.…”

“…FAK has recently been shown to influence actin-related protein (Arp) 2/3 complex-mediated actin nucleation, modulating actin cytoskeleton, through at least two mechanisms. These include blockade of neuronal Wiskott-Aldrich syndrome protein (N-WASP), an Arp2/3 activator (11), from entering the nucleus following FAK-mediated phosphorylation (12), as well as the direct interaction of FAK 4.1/ezrin/radixin/moesin domain with Arp3, which enhances Arp2/3 activity (13). In short, FAK harnesses multiple levels of control over actin polymerization.…”