Abstract
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.
Highlights
From the Departments of IMedicine and $Chemistry, Universityof California, Sun Diego, California 92093-0652and the Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolin2a7599
Murine erythroleukemia (MEL) cells deficient in differentiation [1].The cells proliferate as proerythroblasts and CAMP-dependent protein kinase (A-kinase)activity are in responseto a number of chemical inducers differentiateover impaired in chemically induced differentiation (Pilz, R. 5-7 days into hemoglobin-producing normoblast-like cells [1]
A-kinase-deficient murine erythroleukemic (MEL) cells, were treated with the membrane-permeableCAMPanalog 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
Summary
Phosphorylation of proteins i n cell extracts byA-kinase.Extracts of parental cells wienrceubawteidth. A-kinase; the extracts were applied 2to-D gels which were exposed tox-ray film. Proteins are labeled as in Fig.1except the number 4 is right of the designated proteirne;ferenpceroteins. Parental MEL cells incubated with [:j2P1orthophosphatefor 3 h were extracted, and the proteins were separated on 2-D gels a s described under “Experimental Procedures.”. Autoradiographs of the gels were scanned by laser densitometry, and the densiotyf the spotcorresponding to p2p1-2 was normalized ttohe densityof three reference proteins. The HMBA-treated cells received 4 mM HMBA for 13 h prior to adding [‘”Plorthophosphate, andthe 8-bromo-CAMP-treatedcells received 1msc. The valuaerse the mean S.D. offour separate gels scannedfor each condition; the densityof the spot corresponding to pp in untreated cells was assigned a value of 1
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