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.
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.
Scheele
J S
JS
Department of Chemistry, University of California, San Diego 92093-0652.
Pilz
R B
RB
Quilliam
L A
LA
Boss
G R
GR
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013462:Sulfur Radioisotopes; D014162:Transfection; D014407:Tumor Cells, Cultured; D020668:rap GTP-Binding Proteins
D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D006160:Guanosine Triphosphate; D008715:Methionine; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D003545:Cysteine