1,25-DIHYDROXYVITAMIN D-3 STIMULATES THE PHOSPHORYLATION OF 2 ACIDIC MEMBRANE-PROTEINS OF 42,000 AND 48,000 DALTONS IN RAT COLONOCYTES - AN EFFECT MODULATED BY VITAMIN-D STATUS

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D-3(1,25(OH)(2)D-3) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)(2)D-3 were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)(2)D-3. In the present studies we have examined the ability of 1,25(OH)(2)D-3 to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)(2)D-3 increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)(2)D-3 or 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)(2)D-3 were both rapid and transient. In addition, PKC19-36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, Further augmented their phosphorylation in response to 1,25(OH)(2)D-3. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)(2)D-3-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)(2)D-3 following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)(2)D-3 treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. (C) 1995 Wiley-Liss, Inc.