Modulation and phosphorylation of calbindin-D28K correlates with protein kinase C activation1.
Calbindin-D28K is a vitamin D3 dependent calcium-binding protein expressed in renal distal tubules. We previously reported that 24 h treatment with 1 alpha, 25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D3, induces calbindin-D28K and activates protein kinase C (PKC) in MDBK (Madin-Darby bovine kidney) cells. In contrast, 24 h treatment with the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (TPA) downregulates calbindin-D28K and PKC activity. In the present studies, we demonstrate that TPA rapidly enhances calbindin-D28K expression in MDBK cells in the absence of 1,25-D3. The enhancement of calbindin-D28K expression is preceded by activation and translocation of PKC alpha. Further, we show that PKC directly phosphorylates calbindin-D28K in a calcium- and phospholipid-dependent manner in vitro. In MDBK cells, the calbindin-D28K antibody immunoprecipitates a 28 kDa protein for which phosphorylation is enhanced after treatment for 1 h with TPA or 24 h with 1,25-D3. Consistent with amino acid sequence analysis of calbindin-D28K indicating two threonine residues that fit the consensus for PKC phosphorylation, TPA-treated MDBK cells exhibit enhanced expression of a phosphothreonine-containing protein that co-migrates with calbindin-D28K. These studies offer the first report that calbindin-D28K is a phosphoprotein and implicate the PKC signal transduction pathway in its regulation.
Gagnon
A
A
Department of Biochemistry, University of Ottawa, ON, Canada.
Welsh
J
J
D000818:Animals; D064026:Calbindins; D002117:Calcitriol / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
D064026:Calbindins; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D011500:Protein Synthesis Inhibitors; D064030:S100 Calcium Binding Protein G; D003513:Cycloheximide; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium