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Regulation of protein phosphorylation in ocular ciliary epithelial cells by A, C and Ca2+/calmodulin-dependent
L G Lopez-Briones1, M B Wax, M Coca-Prados
1Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06512.
Abstract:
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)
Insights
This study investigated protein phosphorylation in human ocular ciliary epithelium cells, identifying key phosphoproteins and their responses to various kinase activators. Results reveal specific phosphorylation patterns linked to cell signaling pathways.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Biology
Background:
- The human ocular ciliary epithelium (8-SVHCE) cell line, derived from Simian Virus-40 transformed cells, serves as a model for studying intracellular signaling.
- Protein phosphorylation is a critical post-translational modification regulating cellular functions.
Purpose of the Study:
- To investigate the time-course and identify intracellular phosphorylated protein substrates in 8-SVHCE cells.
- To analyze the effects of specific kinase activators on protein phosphorylation patterns.
Main Methods:
- Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for phosphoprotein identification.
- Tryptic peptide analysis and phosphoamino acid composition for phosphoprotein characterization.
- Stimulation of cells with isoproterenol, phorbol-12-myristate-13-acetate (PMA), and ionophore A23187.
Main Results:
- Five major endogenous phosphoproteins (80, 57, 24, and 19 kDa) were identified.
- The 80-kDa protein showed rapid phosphorylation with PMA, potentiated by isoproterenol.
- Vimentin (57-kDa) phosphorylation was preferential with isoproterenol, and late phosphorylation of 19/24-kDa proteins occurred with PMA and A23187.
- Altered adenylate cyclase activity correlated with changes in vimentin phosphorylation.
Conclusions:
- Specific intracellular phosphoproteins in 8-SVHCE cells exhibit distinct phosphorylation dynamics in response to various signaling stimuli.
- PMA, isoproterenol, and A23187 differentially modulate protein phosphorylation, impacting cellular signaling pathways.
- Vimentin phosphorylation is closely linked to adenylate cyclase activity regulation in these cells.