MODULATION AND PHOSPHORYLATION OF CALBINDIN-D-28K CORRELATES WITH PROTEIN-KINASE-C ACTIVATION

Calbindin-D-28K is a vitamin D-3 dependent calcium-binding protein exp ressed in renal distal tubules. We previously reported that 24 h treat ment with 1 alpha,25-dihydroxyvitamin D-3 (1,25-D-3), the active form of vitamin D-3, induces calbindin-D-28K and activates protein kinase C (PKC) in MDBK (Madin-Darby bovine kidney) cells. In contrast, 24 h tr eatment with the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (TP A) downregulates calbindin-D-28K and PKC activity. In the present stud ies, we demonstrate that TPA rapidly enhances calbindin-D-28K expressi on in MDBK cells in the absence of 1,25-D-3. The enhancement of calbin din-D-28K expression is preceded by activation and translocation of PK C alpha. Further, we show that PKC directly phosphorylates calbindin-D -28K in a calcium- and phospholipid-dependent manner in vitro. In MDBK cells, the calbindin-(28K) antibody immunoprecipitates a 28 kDa prote in for which phosphorylation is enhanced after treatment for 1 h with TPA or 24 h with 1,25-D-3. Consistent with amino acid sequence analysi s of calbindin-D-28K indicating two threonine residues that fit the co nsensus for PKC phosphorylation, TPA-treated MDBK cells exhibit enhanc ed expression of a phosphothreonine-containing protein that co-migrate s with calbindin-D-28K. These studies offer the first report that calb indin-D-28K is a phosphoprotein and implicate the PKC signal transduct ion pathway in its regulation.