Hata Y, Kaibuchi K, Kawamura S, Hiroyoshi M, Shirataki H, Takai Y
Department of Biochemistry, Kobe University School of Medicine, Japan.
J Biol Chem. 1991 Apr 5;266(10):6571-7.
We have previously shown that cyclic AMP-dependent protein kinase (protein kinase A) phosphorylates smg p21A and -B, ras p21-like small GTP-binding proteins. In the present study, we investigated the function(s) of this phosphorylation by use of the smg p21B purified from human platelets. smg p21B bound to plasma membranes and the protein kinase A-catalyzed phosphorylation of smg p21B reduced this binding. Moreover, the phosphorylation of smg p21B enhanced the two actions of its specific GDP/GTP exchange protein, named GDP dissociation stimulator, when tested in a cell-free system: one is the action to stimulate the GDP/GTP exchange reaction of smg p21B, and the other is the action to inhibit the binding of smg p21B to membranes. Consistently, smg p21B was translocated from the membranes to the cytoplasm when it was phosphorylated by protein kinase A in intact platelets in response to prostaglandin E1 or dibutyryl cyclic AMP. The protein kinase A-catalyzed phosphorylation of smg p21B affected neither its basal GDP/GTP exchange reaction, basal GTPase activity, nor the GTPase activity stimulated by its specific GTPase activating protein. On the other hand, we have recently clarified that the structure of the C-terminal region of the post-translationally processed human platelet smg p21B is Lys-Lys-Ser-Ser-all-trans-geranylgeranyl Cys181 methyl ester, and that this modification of the C-terminal region is essential for smg p21B to bind to membranes. We furthermore determined here that protein kinase A phosphorylated Ser179 in this C-terminal region of smg p21B. These results indicate that protein kinase A-catalyzed phosphorylation of smg p21B makes smg p21B sensitive to the actions of smg p21 GDP dissociation stimulator.
我们之前已经表明，环磷酸腺苷依赖性蛋白激酶（蛋白激酶A）可使smg p21A和-B磷酸化，smg p21A和-B是与ras p21类似的小GTP结合蛋白。在本研究中，我们利用从人血小板中纯化的smg p21B来研究这种磷酸化作用的功能。smg p21B与质膜结合，蛋白激酶A催化的smg p21B磷酸化降低了这种结合。此外，当在无细胞系统中进行测试时，smg p21B的磷酸化增强了其特异性GDP/GTP交换蛋白（称为GDP解离刺激因子）的两种作用：一种是刺激smg p21B的GDP/GTP交换反应的作用，另一种是抑制smg p21B与膜结合的作用。一致的是，当完整血小板中的smg p21B在前列腺素E1或二丁酰环磷酸腺苷的作用下被蛋白激酶A磷酸化时，它会从膜转移到细胞质中。蛋白激酶A催化的smg p21B磷酸化既不影响其基础GDP/GTP交换反应、基础GTP酶活性，也不影响其特异性GTP酶激活蛋白刺激的GTP酶活性。另一方面，我们最近阐明，翻译后加工的人血小板smg p21B的C末端区域的结构是赖氨酸-赖氨酸-丝氨酸-丝氨酸-全反式香叶基香叶基半胱氨酸181甲酯，并且C末端区域的这种修饰对于smg p21B与膜结合至关重要。我们在此进一步确定，蛋白激酶A使smg p21B的这个C末端区域中的丝氨酸179磷酸化。这些结果表明，蛋白激酶A催化的smg p21B磷酸化使smg p21B对smg p21 GDP解离刺激因子的作用敏感。
