Identification of a ras-related protein in murine erythroleukemia cells that is a cAMP-dependent protein kinase substrate and is phosphorylated during chemically induced differentiation

• Published in: The Journal of biological chemistry Vol. 269; no. 28; pp. 18599 - 18606
• Main Authors: SCHEELE, J. S, PILZ, R. B, QUILLIAM, L. A, BOSS, G. R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.07.1994
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Analytical, structural and metabolic biochemistry; Animals; Autoradiography; Biological and medical sciences; Blotting, Western; Cell Differentiation - drug effects; Colforsin - pharmacology; Cyclic AMP-Dependent Protein Kinases - metabolism; Cysteine - metabolism; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - isolation & purification; GTP-Binding Proteins - metabolism; Guanosine Triphosphate - metabolism; Leukemia, Erythroblastic, Acute - metabolism; Methionine - metabolism; Mice; Miscellaneous; Peptide Mapping; Phosphopeptides - chemistry; Phosphopeptides - isolation & purification; Phosphorylation; Proteins; Proto-Oncogene Proteins - metabolism; rap GTP-Binding Proteins; Recombinant Proteins - metabolism; Substrate Specificity; Sulfur Radioisotopes; Transfection; Tumor Cells, Cultured; Erythroleukemia; Phosphorylation; Enzyme; Transferases; Rodentia; Cell differentiation; Vertebrata; Immunoprecipitation reaction; Mammalia; Mouse; Phosphoproteins; Protein kinase; Immunoblotting assay; Two dimensional electrophoresis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)32352-9

Murine erythroleukemia (MEL) cells deficient in cAMP-dependent protein kinase (A-kinase) activity are impaired in chemically induced differentiation (Pilz, R. B., Eigenthaler, M., and Boss, G. R. (1992) J. Biol. Chem. 267, 16161-16167). We identified by two-dimensional polyacrylamide gel electrophoresis two low molecular weight proteins (referred to as pp 21-1 and 21-2) that were phosphorylated when parental MEL cells, but not A-kinase-deficient MEL cells, were treated with the membrane-permeable cAMP analog 8-bromo-cAMP. We showed that pp 21-1 and 21-2: (a) were direct A-kinase substrates; (b) bound GTP; and (c) belonged to the ras superfamily of proteins. The only ras-related proteins that are clearly A-kinase substrates both in vitro and in vivo are Rap 1A and 1B while H- and K-Ras can be A-kinase substrates in vitro; we showed by immunological methods, phosphopeptide mapping, and migration on two-dimensional gels that pp 21-1 and 21-2 were not identical to one of these four proteins. We found a 3-fold increase of 32PO4 incorporation into pp 21-2 in hexamethylene bisacetamide-treated parental MEL cells which was not secondary to an increase in pp 21-2 protein but appeared secondary to increased phosphorylation of pp 21-2 by A-kinase. Thus, pp 21-1 and 21-2 are either new ras-related proteins or are previously identified ras-related proteins not known to be A-kinase substrates, and increased phosphorylation of pp 21-2 occurs during differentiation of MEL cells.