Bokoch G M, Quilliam L A
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Biochem J. 1990 Apr 15;267(2):407-11. doi: 10.1042/bj2670407.
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.
利用蛋白激酶A磷酸化形式和非磷酸化形式的rap1蛋白，对从人中性粒细胞中纯化出的rap1蛋白的鸟嘌呤核苷酸结合特性进行了研究。发现在存在游离Mg2+的情况下，GTP[S]（鸟苷5'-[γ-硫代]三磷酸）或GDP的结合较慢，但在不存在Mg2+的情况下非常迅速。发现鸟嘌呤核苷酸的结合与rap1蛋白内源性核苷酸的丢失相关，在不存在Mg2+的情况下这种丢失很快。Mg2+的存在调节了GTP和GDP对rap1结合位点的相对亲和力，仅在不存在这种二价阳离子的情况下观察到对GTP有优先亲和力（约15倍）。GDP从rap1上的解离不受G蛋白β/γ亚基复合物的影响。蛋白激酶A在体外对rap1的磷酸化并未改变任何观察到的核苷酸结合参数。此外，胞质rap1 GTP酶激活蛋白刺激中性粒细胞rap1 GTP水解的能力并未因磷酸化而改变。这些数据表明，体内rap的激活可能受内源性GDP释放的调节，但蛋白激酶A的磷酸化并不影响鸟嘌呤核苷酸的结合或水解。
