Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.
rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.
Rubinfeld
B
B
Department of Molecular Biology, Onyx Pharmaceuticals, Emeryville, California.
Crosier
W J
WJ
Albert
I
I
Conroy
L
L
Clark
R
R
McCormick
F
F
Polakis
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D004247:DNA; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D020690:GTPase-Activating Proteins; D011506:Proteins; D012694:Serine; D004247:DNA