Nongenomic action of 1α,25(OH)2-vitamin D3 -: Activation of muscle cell PLCγ through the tyrosine kinase c-Src and PtdIns 3-kinase

We have previously demonstrated that the steroid hormone 1alpha,25(OH)(2)-vitamin D-3 [1alpha,25(OH)(2)D-3] stimulates the production of inositol trisphosphate (InsP(3)), the breakdown product of phosphatidylinositol 4,5-biphosphate (PtdInsP(2)) by phospholipase C (PtdIns-PLC), and activates the cytosolic tyrosine kinase c-Src in skeletal muscle cells. In the present study we examined whether 1alpha,25(OH)(2)D-3 induces the phosphorylation and membrane translocation of PLCgamma and the mechanism involved in this isozyme activation. We found that the steroid hormone triggers a significant phosphorylation on tyrosine residues of PLCgamma and induces a rapid increase in membrane-associated PLCgamma immunoreactivity with a time course that correlates with that of phosphorylation in muscle cells. Genistein, a tyrosine kinase inhibitor, blocked the phosphorylation of PLCgamma. Inhibition of 1alpha,25(OH)(2) D-3 -induced c-Src activity by its specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA, prevented hormone stimulation of PLCgamma tyrosine phosphorylation. The isozyme phosphorylation is also blocked by both wortmannin and LY294002, two structurally different inhibitors of phosphatidyl inositol 3-kinase (PtdIns3K), the enzyme that produces PtdInsP (3) known to activate PLCgamma isozymes specifically by interacting with their SH2 and pleckstrin homology domains. The hormone also increases the physical association of c-Src and PtdIns3K with PLCgamma and induces a c-Src-dependent tyrosine phosphorylation of the p85 regulatory subunit of PtdIns3K. The time course of hormone-dependent PLCgamma phosphorylation closely correlates with the time course of its redistribution to the membrane, suggesting that phosphorylation and redistribution to the membrane of PLCgamma are two interdependent events. 1alpha,25(OH)(2)D-3-induced membrane translocation of PLCgamma was prevented to a great extent by c-Src and PtdIns3K inhibitors, PP1 and LY294002. Taken together, the present data indicates that the cytosolic tyrosine kinase c-Src and PtdIns 3-kinase play indispensable roles in 1alpha,25(OH)(2)D-3 signal transduction cascades leading to PLCgamma activation.