1,25-DIHYDROXYVITAMIN-D-3 STIMULATES PHOSPHOLIPASE C-GAMMA IN RAT COLONOCYTES - ROLE OF C-SRC IN PLC-GAMMA ACTIVATION

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D-3 (1,25[OH](2)D-3) rapidly stimulated polyphosphoinositide (PI) hydr olysis, raised intracellular Ca2+, and activated two Ca2+-dependent pr otein kinase C (PKC) isoforms, PKC-alpha and -beta(II) in the rat larg e intestine. We also showed that the direct addition of 1,25(OH)(2)D-3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the i nvolvement of a soluble factor. Furthermore, this PI hydrolysis was re stricted to the basal lateral plasma membrane of these cells. In the p resent studies, therefore, we examined whether polyphosphoinositide-ph ospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1, 25(OH)(2)D-3. This isoform has been shown to be activated and membrane -associated by tyrosine phosphorylation. We found that 1,25(OH)(2)D-3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifica lly in the basal lateral membranes. This secosteroid also induced a tw ofold increase in the activity of Src, a proximate activator of PLC-ga mma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)(2)D-3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isola ted from colonocytes treated with 1,25(OH)(2)D-3, demonstrated an incr eased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibitio n of 1,25(OH)(2)D-3-induced Src activation by PP1, a specific Src fami ly protein tyrosine kinase inhibitor, blocked the ability of this seco steroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was last in D deficiency, and was reversibly restored with the in vivo repletio n of 1,25(OH)(2)D-3 These studies demonstrate for the first time that 1,25(OH)(2)D-3 stimulates PLC-gamma as well as c-Src in rat colonocyte s, and indicate that PLC-gamma is a direct substrate of secosteroid-ac tivated c-Src in these cells.