Regulation of phospholipase C-γ₁ by the actin-regulatory protein villin

Abstract

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-gamma(1) (PLC-gamma(1)) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-gamma(1) activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-gamma(1). Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-gamma(1). The catalytic activity of PLC-gamma(1) was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 microM. Villin inhibited PLC-gamma(1) activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)), since increasing concentrations of PIP(2) reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-gamma(1) activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP(2). In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-gamma(1) and activates PLC-gamma(1) catalytic activity. Villin regulates PLC-gamma(1) activity by modifying its own ability to bind PIP(2). This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-gamma(1) by villin.

Association of Villin with Phosphatidylinositol 4,5-Bisphosphate Regulates the Actin Cytoskeleton

“…The correlation of these two events suggests that interaction of villin with PIP 2 can regulate epithelial cell structure and function in response to various stimuli. We have shown previously (33) that villin interacts directly with PIP 2 in vitro. The aim of the present study was to investigate the villin-PIP 2 interaction further to include identification of the PIP 2 -binding sites in villin and to investigate the relationship of villin regulation of actin dynamics by phosphoinositides.…”

“…Lipid Vesicles-The lipid binding assay was used to determine the association of PIP 2 with villin as described before (33). Briefly, fusion proteins (wild type or deletion mutants, 2 g) were incubated with PIP 2 containing unilamellar vesicles (20% PIP 2 , 80% PC; final concentration 1.7 mg/ml), and after incubation at room temperature for 10 min and on ice for 5 min, the tubes were centrifuged at 100,000 ϫ g for 30 min at 4°C.…”

“…Phosphoinositides-Association of villin with the lipid bilayer was studied using recombinant villin, which we have shown previously (33) to behave like the native villin protein. Direct interaction between lipid vesicles containing pure phospholipids and fulllength recombinant villin protein (VIL/WT) was examined by solid phase binding assay using different concentrations of PC, PS, PE, or PIP 2 and related phosphoinositides PIP and PI.…”

“…Villin inhibits the catalytic activity of phospholipase C-␥ 1 (PLC-␥ 1 ) by sequestrating its substrate PIP 2 (33). This points to a role for villin in modifying lipid-signaling events.…”

A Novel Role for Villin in Intestinal Epithelial Cell Survival and Homeostasis

“…Cell Culture and Treatment-Full-length human villin was cloned in pTRE-HA as described previously (17,30). MDCK or HeLa Tet-Off cells were stably transfected with HA-tagged wild-type villin protein as described previously (16).…”