Mutational analysis of the cAMP-dependent protein kinase-mediated phosphorylation site of Rap1b.
Rap1b, a member of the Ras superfamily of low molecular weight GTP-binding proteins, can be phosphorylated by cAMP-dependent protein kinase (protein kinase A). The experiments presented here were undertaken to determine the precise site of this phosphorylation. Because the Rap1 proteins are highly homologous, there are no specific antibodies able to discriminate between them. To overcome this problem, we used a transient expression system of a fused protein containing in the NH2 terminus an epitope for a known antibody. Using this system, the transfected protein was expressed at a high level and was localized in a perinuclear structure, as previously reported for the endogenous Rap1 proteins. The mutational analysis of Rap1b revealed Ser179 as the residue involved in the protein kinase A-mediated phosphorylation. The presence of a Lys179 instead of the wild-type Ser179 (resembling the Rap1a sequence) rendered Ser180 a better substrate for phosphorylation caused by protein kinase A. The mobility shift of Rap1b in SDS gels, observed in cells that were stimulated with agonists that increase cAMP, was caused, at least in part, by the phosphorylation of Rap1b.
Altschuler
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Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Lapetina
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D014162:Transfection; D020668:rap GTP-Binding Proteins
D004247:DNA; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins