1,25-DIHYDROXYVITAMIN D-3 STIMULATES THE PHOSPHORYLATION OF 2 ACIDIC MEMBRANE-PROTEINS OF 42,000 AND 48,000 DALTONS IN RAT COLONOCYTES - ANEFFECT 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 bre akdown and increased intracellular calcium, as well as activated prote in kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effec ts of 1,25(OH)(2)D-3 were, however, lost in vitamin D-insufficient rat s and restored by the in vivo repletion of 1,25(OH)(2)D-3. In the pres ent studies we have examined the ability of 1,25(OH)(2)D-3 to stimulat e the phosphorylation of colonic membrane proteins in intact D-suffici ent cells. In addition, we investigated the effects of vitamin D statu s on the phosphorylation of these membrane proteins in broken cell pre parations. These studies demonstrated that 1,25(OH)(2)D-3 increased th e phosphorylation of at least two colonic membrane proteins with appar ent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cel ls of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rat s, treatment of colonocytes with 1,25(OH)(2)D-3 or 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly incr eased 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 oka daic acid (OA), a type 1 and 2A protein phosphatase inhibitor, Further augmented their phosphorylation in response to 1,25(OH)(2)D-3. The is oelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, an d both were predominantly phosphorylated on threonine residues. In con trast to our findings in colonocytes from vitamin D-sufficient animals , basal phosphorylation of pp42 and pp48 were increased in membranes p repared from vitamin D-insufficient rats. Moreover, these phosphorylat ions failed to change in response to 1,25(OH)(2)D-3-treatment of colon ocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ab ility to respond to the direct addition of 1,25(OH)(2)D-3 following th e in vivo repletion of vitamin D-insufficient rats with this secostero id. In summary, we have identified two acidic membrane proteins from r at colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)(2)D-3 treatment, an event modulat ed by vitamin D status and mediated, at least in part, by PKC. (C) 199 5 Wiley-Liss, Inc.