Phosphorylation of MP26, a lens junction protein, is enhanced by activators of protein kinase C.
MP26, a protein thought to form gap junctional channels in the lens, and other lens proteins were phosphorylated under conditions that activate protein kinase C. Phosphorylation was detected both in lens fiber cell fragments in an "in vivo" labeling procedure with 32P-phosphate and in cell homogenates with 32P-ATP. In these experiments, both calcium and 12-O-tetradecanoylphorbol 13-acetate (TPA) were necessary for maximal phosphorylation of MP26. Calcium stimulated the phosphorylation of MP26 approximately fourfold and TPA with calcium led to a sevenfold increase. If TPA was present, 1 microM calcium was sufficient for maximal labeling. Phosphoamino acid analysis demonstrated approximately 85% phosphoserine, 15% phosphothreonine, and no phosphotyrosine when MP26 was phosphorylated in lens homogenates in the presence of TPA and calcium and then electrophoretically purified. Phosphorylation occurred near the cytoplasmic, C-terminal of MP26. The possible involvement of other kinases was also examined. The Walsh inhibitor, which affects cAMP-dependent protein kinases, had no influence on the TPA-mediated increase in phosphorylation. In studies with isolated membranes and added kinases, MP26 was also found to not be a substrate for calcium/calmodulin-dependent protein kinase II. Thus, protein kinase C may have phosphorylated MP26 in a direct manner.
Lampe
P D
PD
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Johnson
R G
RG
D000818:Animals; D020346:Aquaporins; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005136:Eye Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007365:Intercellular Junctions / Q000378:metabolism; D008562:Membrane Glycoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; C030328:aquaporin 0; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium