Tyrosine phosphorylation of WIP releases bound WASP and impairs podosome assembly in macrophages

Abstract

Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of podosomes is based on a WASP-Arp2/3 mediated mechanism. WASP also associates with a second protein, WIP, where they co-localise in podosome cores. Here we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP-WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP-WASP binding, cellular WASP is rapidly degraded, leading to disruption of podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP-WASP complex remains intact and podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a critical regulator of WASP stability and function as an actin nucleation promoting factor.