Serine 113 is the site of receptor-mediated phosphorylation of the Dictyostelium G protein alpha-subunit G alpha 2.
The G protein alpha-subunit G alpha 2 is essential to the developmental program of Dictyostelium. G alpha 2 is transiently phosphorylated on a serine residue(s) following stimulation with extracellular cAMP (Gundersen, R. E., and Devreotes, P.N. (1990) Science 248, 591-593). To aid in defining the function of alpha-subunit phosphorylation, we identified the site of G alpha 2 phosphorylation. Comparison of the isoelectric points (pI) of the phosphorylated and nonphosphorylated forms indicated that a single mole of phosphate is added to G alpha 2. Cleavage at tryptophan residues and immunoprecipitation with a specific peptide antibody localized the phosphorylated serine in the N-terminal 119 residues. Analysis of a series of G alpha 1 and G alpha 2 chimeras further confined the site between amino acids 33 and 215. Site-directed mutagenesis of serines between amino acids 33 and 119 produced two mutants that were not phosphorylated, S45A and S113A. Ser113 was identified as the site by sequential Edman degradation of 32P-radiolabeled G alpha 2 digested with endoproteinase Glu-C. We have expressed the G alpha 2 mutants S113A, S113I, S113T, and S113D in a G alpha 2 null cell line to examine the function of phosphorylation.
Chen
M Y
MY
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Devreotes
P N
PN
Gundersen
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RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004023:Dictyostelium / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D017931:DNA Primers; D012694:Serine; D019204:GTP-Binding Proteins