Effects of phosphorylation on function of the Rad GTPase.
Effects of phosphorylation on function of the Rad GTPase.
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磷酸化对 Rad GTPase 功能的影响。
DOI：
10.1042/bj3330609
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Kahn,CR
中科院分区：
文献类型：
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作者：
Moyers,JS;Zhu,J;Kahn,CR
Rad、Gem和Kir与其他Ras样GTP酶相比具有独特的结构特征，包括C-末端31个残基的延伸，缺乏典型的异戊烯化基序。我们最近表明，拉德和宝石结合钙调素在钙离子依赖性的方式通过这种C-末端的延伸，涉及残基278-297在人类拉德。该结构域还包含几个丝氨酸磷酸化的共有位点，并且Rad与C2 C12细胞中的钙调蛋白依赖性蛋白激酶II（CaMKII）复合。在此，我们发现Rad作为底物被CaMK Ⅱ、cAMP依赖性蛋白激酶（PKA）、蛋白激酶C（PKC）和酪蛋白激酶Ⅱ（CKII）磷酸化，其化学计量比为0.2-1.3 mol磷酸盐/mol Rad。通过缺失和点突变分析，我们发现CaMK II和PKA的磷酸化发生在273位的单个丝氨酸残基上，而PKC和CKII磷酸化多个C-末端丝氨酸残基，包括Ser 214，Ser 257，Ser 273，Ser 290和Ser 299。将Rad与PKA孵育可降低60- 70%的GTP结合，但这种效应似乎与磷酸化无关，因为在PKA磷酸化位点含有突变的Rad的Ser 273 → Ala突变体中观察到了这种效应。其余的丝氨酸激酶对Rad GTP结合、内在GTP水解或由推定的肿瘤转移抑制因子nm 23刺激的GTP水解没有影响。然而，磷酸化的Rad的PKC和CKII废除的Rad与钙调蛋白的相互作用。这些研究结果表明，结合的RAD钙调蛋白，以及其能力，结合GTP，可能是由几个丝氨酸激酶的激活调节。
Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278–297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometriesin vitroof 0.2–1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290and Ser299. Incubation of Rad with PKA decreases GTP binding by 60–70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273→ Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.