Tyrosine phosphorylation of villin regulates the organization of the actincytoskeleton

We have previously shown that tyrosine phosphorylation of the actin-regulat ory protein villin is accompanied by the redistribution of phosphorylated v illin and a concomitant decrease in the F-actin content of intestinal epith elial cells. The temporal and spatial correlation of these two events sugge sted that tyrosine phosphorylation of villin may be involved in the rearran gement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetic s of actin polymerization by reconstituting in vitro the tyrosine phosphory lation of villin and its association with actin. Full-length recombinant hu man villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding prope rties of villin were examined using a co-sedimentation assay. Phosphorylati on of villin did not change the stoichiometry (1:2) but decreased the bindi ng affinity (4.4 mum for unphosphorylated versus 0.6 mum for phosphorylated ) of villin for actin. Using a pyrene-actin-based fluorescence assay, we de monstrated that tyrosine phosphorylation had a negative effect on actin nuc leation by villin. In contrast, tyrosine phosphorylation enhanced actin sev ering by villin. Electron microscopic analysis showed complementary morphol ogical changes. Phosphorylation inhibited the actin bundling and enhanced t he actin severing functions of villin. Taken together our data show that ty rosine phosphorylation of villin decreases the amount of villin bound to ac tin filaments, inhibits the actin-polymerizing properties of villin, and pr omotes the actin-depolymerizing functions instead. These observations sugge st a role for tyrosine phosphorylation in modulating the microvillar cytosk eleton in vivo by villin in response to specific physiological stimuli.