Regulation of protein phosphorylation in ocular ciliary epithelial cells by A, C and Ca2+/calmodulin-dependent protein kinases.
We have studied protein phosphorylation events in a cell line (8-SVHCE) derived from the human ocular ciliary epithelium after transformation within Simian Virus-40. We have investigated the time-course and identification of intracellular phosphorylated protein substrates in response to isoproterenol, phorbol-12-myristate-13-acetate (PMA), and ionophore A23187, which are activators of protein kinase A, C and calcium/calmodulin-dependent protein kinase, respectively. Five major endogenous phosphoproteins were readily identified by two-dimensional polyacrylamide gel electrophoresis with the following molecular weights: 80, 57, 24 and 19 kDa. Tryptic peptide analysis and phosphoaminoacid composition were utilized to aid the identification of the phosphoproteins. From these studies we have observed the following: (a) the most prominent phosphorylation of the 80-kDa protein occurs rapidly (1 min) in response to PMA treatment and is potentiated by isoproterenol, (b) the phosphorylation of the 57-kDa substrate (vimentin) occurs preferentially with isoproterenol treatment and increases gradually from 1 to 30 min, (c) late phosphorylation (60 min) of the 80-kDa protein by PMA is potentiated by isoproterenol, and (d) late phosphorylation of 19-kDa and 24-kDa substrates occurs with PMA treatment and is potentiated by A21387. The desensitization of adenylate cyclase activity by PMA or isoproterenol in 8-SVHCE cells results in altered adenylate cyclase activity, which appears to be correlated with similar alterations in the phosphorylation of the 57-kDa substrate (vimentin).(ABSTRACT TRUNCATED AT 250 WORDS)
Lopez-Briones
L G
LG
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06512.
Wax
M B
MB
Coca-Prados
M
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects / Q000201:enzymology*; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate
D005136:Eye Proteins; D000001:Calcimycin; D011494:Protein Kinases; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate