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). Our results indicate that the rapid phosphorylation of the 80 kDa substrate occurs primarily by protein kinase C whereas the delayed phosphorylation of vimentin occurs primarily by protein kinase A and is enhanced by calcium/calmodulin-dependent protein kinase. The potentiated late phosphorylation of a 19-kDa and 24-kDa substrate by A21387 and PMA suggests mediation by calcium/calmodulin-dependent protein kinase as well as protein kinase C. From these alterations in the protein phosphorylation patterns by different kinase activators, we conclude that these phosphoproteins are regulated by multiple events in the signal transduction pathways which underlie the production of aqueous humor of the eye.
- human ciliary epithelial cells
- phorbol esters
- protein kinases
- protein phosphorylation
- two-dimensional gel electrophoresis
ASJC Scopus subject areas
- Sensory Systems