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Agonist-induced phosphorylation of an immunologically ras-related protein in human erythroleukemia cells
E R Lazarowski1, J C Lacal, E G Lapetina
1Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, N.C. 27709.
Abstract:
Monoclonal antibody M90 recognizes a specific epitope of the ras-encoded p21 protein. This region comprises amino acids 107-130 containing the residues 116-119, which are related to GTP binding. This antibody strongly reacts on Western Blots with a 22kDa protein from human erythroleukemia (HEL) cells. Treatment of HEL cells with iloprost, an agonist that increases cellular cyclic AMP levels, produces the appearance of a protein with an apparent molecular mass of 24kDa. This protein is also recognized by antiserum M90 on Western Blots; its appearance parallels a decrease of the 22kDa protein, and it can be labeled with 32P. This effect is also observed with dibutyryl cyclic AMP, which indicates phosphorylation of the 22kDa protein by cyclic AMP-dependent protein kinase. This phosphorylation produces an electrophoretic mobility change of the 22kDa protein to a 24kDa region on gels. The change of mobility of the 22kDa protein induced by iloprost in HEL cells is also observed when the protein is labeled with [35S]methionine and immunoprecipitated with antiserum M90. This information indicates a coupling mechanism involving phosphorylation of an oncogene product in HEL cells.
Insights
Monoclonal antibody M90 identifies a ras-encoded p21 protein in human erythroleukemia cells. Iloprost treatment induces phosphorylation, creating a larger 24kDa protein, indicating oncogene product modification.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Oncogene Research
Background:
- The ras-encoded p21 protein plays a crucial role in cellular signaling pathways.
- Monoclonal antibody M90 specifically targets an epitope on the ras p21 protein, including GTP-binding residues.
- Human erythroleukemia (HEL) cells provide a model system to study ras p21 protein modifications.
Purpose of the Study:
- To investigate the effect of iloprost, a cyclic AMP agonist, on the ras p21 protein in HEL cells.
- To characterize the molecular changes in the ras p21 protein upon stimulation with iloprost.
- To elucidate the mechanism of oncogene product modification in response to cellular signaling.
Main Methods:
- Western Blot analysis using monoclonal antibody M90 to detect ras p21 protein.
- Treatment of HEL cells with iloprost and dibutyryl cyclic AMP.
- Radiolabeling of proteins with 32P and 35S-methionine.
- Immunoprecipitation assays.
Main Results:
- Iloprost treatment of HEL cells resulted in the appearance of a 24kDa protein, recognized by M90, alongside a decrease in the 22kDa protein.
- The 24kDa protein could be labeled with 32P, indicating phosphorylation.
- Cyclic AMP-dependent protein kinase mediated the phosphorylation, causing an electrophoretic mobility shift from 22kDa to 24kDa.
Conclusions:
- Iloprost induces phosphorylation of the ras-encoded p21 protein in HEL cells via a cyclic AMP-dependent pathway.
- This phosphorylation event alters the protein's electrophoretic mobility, suggesting a regulatory mechanism.
- The findings reveal a coupling mechanism involving the phosphorylation of an oncogene product in response to cellular signaling.