GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin
GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin
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DOI：
10.1046/j.1432-1327.1998.2550363.x
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发表时间：
1998-07-15
期刊：
影响因子：
--
通讯作者：
Murofushi, H
中科院分区：
文献类型：
--
作者：
Haga, K;Ogawa, H;Murofushi, H
发现微管蛋白与谷胱甘肽S-转移酶融合蛋白结合，该融合蛋白含有GTP结合蛋白偶联受体激酶2（GRK 2）的羧基末端结构域（残基467-689），已知GRK 2含有普列克底物蛋白同源位点并结合GTP结合蛋白β γ亚基。微管蛋白与融合蛋白的结合不受GTP结合蛋白β γ亚基的影响，表明微管蛋白和β γ亚基独立地结合GRK 2。抗GRK 2 IG蛋白的Western-blotting分析表明GRK 2与微管蛋白通过聚合-解聚过程共纯化。微管蛋白被GRK 2磷酸化，这与GRK 2的已知底物仅限于GTP结合蛋白偶联受体的活化形式以及微管蛋白是大多数激酶的不良底物的事实相反。GRK 2不磷酸化微管相关蛋白（MAP）：在MAP被微管蛋白共纯化的内源性激酶磷酸化的条件下。微管蛋白的Km估计为3 μ M，掺入1.3 mol磷酸盐/微管蛋白二聚体。β-γ亚基和激动剂结合的毒蕈碱受体刺激微管蛋白的磷酸化。磷酸化的微管蛋白可以聚合成微管，聚合的微管蛋白也可以被GRK 2磷酸化。
Tubulin was found to bind to a glutathione S-transferase fusion protein containing the carboxy-terminal domain of GTP-binding-protein-coupled receptor kinase 2 (GRK2) (residues 467-689), which is known to contain a pleckstrin homology site and to bind GTP-binding protein beta gamma subunits. The binding of tubulin to the fusion protein was not affected by GTP-binding protein beta gamma subunits, indicating that tubulin and beta gamma subunits bind GRK2 independently. Western-blotting analysis with anti-GRK2 Ig indicated that GRK2 was copurified with tubulin through the polymerization-depolymerization procedure. Tubulin was phosphorylated by GRK2, in contrast with the facts that the known substrates of GRK2 are restricted to activated forms of GTP-binding-protein-coupled receptors and that tubulin is a poor substrate fur most kinases. GRK2 did not phosphorylate microtubule-associated proteins (MAPs): under conditions where MAPs were well phosphorylated by endogenous kinases copurified with tubulin. The K-m for tubulin was estimated to be 3 mu M, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by beta gamma subunits and agonist-bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2.