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

Jürgen S. Scheele, Renate B. Pilz, Lawrence Quilliam, Gerry R. Boss

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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 increase 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.

Original languageEnglish (US)
Pages (from-to)18599-18606
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number28
StatePublished - Jul 15 1994
Externally publishedYes

Fingerprint

ras Proteins
Leukemia, Erythroblastic, Acute
Cyclic AMP-Dependent Protein Kinases
Phosphotransferases
Substrates
Proteins
Phosphorylation
hexamethylene bisacetamide
8-Bromo Cyclic Adenosine Monophosphate
Phosphopeptides
Electrophoresis, Gel, Two-Dimensional
Guanosine Triphosphate
Electrophoresis
Molecular Weight
Gels
Molecular weight
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Scheele, Jürgen S.; Pilz, Renate B.; Quilliam, Lawrence; Boss, Gerry R.

In: Journal of Biological Chemistry, Vol. 269, No. 28, 15.07.1994, p. 18599-18606.

Research output: Contribution to journalArticle

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