Abstract
ABSTRACTPodosomes 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- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and 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 (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor.
Highlights
Adhesion and migration of monocyte-derived cells is regulated by actin-rich integrin-containing structures termed podosomes (Calle et al, 2006; Linder and Aepfelbacher, 2003)
PKCh-dependent phosphorylation of S488 of WIP in response to T-cell receptor activation has been demonstrated in Jurkat T-cells (Dong et al, 2007), but increasing evidence indicates that this is not associated with disruption of the WIP–Wiskott–Aldrich syndrome protein (WASP) complex as previously proposed (Sasahara et al, 2002)
In the monocytic THP-1 cell line, we demonstrate that WASP is bound to both unphosphorylatable and phosphomimetic variants of S488 suggesting that phosphorylation of this residue is not associated with the dissociation of the WIP– WASP complex in monocytic cells (Fig. 1B)
Summary
Adhesion and migration of monocyte-derived cells is regulated by actin-rich integrin-containing structures termed podosomes (Calle et al, 2006; Linder and Aepfelbacher, 2003). The rapid formation and turnover of podosomes during migration is dependent upon the actions of the Wiskott–Aldrich syndrome protein (WASP) (Calle et al, 2004b; Calle et al, 2004a), a key regulator of Arp2/3-dependent de novo actin polymerisation (Millard et al, 2004). WASP is associated with the WASP-interacting protein (WIP, known as WIPF1) WASP is indispensable for normal leukocyte function and its importance is highlighted in the congenital disorder Wiskott– Aldrich syndrome in which missense mutations in the WAS gene result in severe immunodeficiency (Derry et al, 1994; Ochs and Thrasher, 2006; Thrasher and Burns, 2010)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
