Guanine nucleotide binding properties of rap1 purified from human neutrophils.
The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.
Bokoch
G M
GM
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Quilliam
L A
LA
D001667:Binding, Competitive; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000097:blood* / Q000302:isolation & purification; D006150:Guanine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D013873:Thionucleotides / Q000378:metabolism*; D020668:rap GTP-Binding Proteins
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D012265:Ribonucleotides; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D008274:Magnesium