Ando S, Tanabe K, Gonda Y, Sato C, Inagaki M
Biophysics Unit, Aichi Cancer Center Research Institute, Nagoya, Japan.
Biochemistry. 1989 Apr 4;28(7):2974-9. doi: 10.1021/bi00433a035.
We reported that stoichiometric phosphorylation by either cAMP-dependent protein kinase or protein kinase C induces disassembly of vimentin filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato, C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263, 5970-5978]. In the present work, we attempted to identify the sites of vimentin phosphorylated by each protein kinase. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were specifically phosphorylated by protein kinase C, whereas Ser-46 was phosphorylated preferentially by cAMP-dependent protein kinase. Both kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific phosphorylation sites for protein kinase C are mostly located close to the amino-terminal side of arginine while those for cAMP-dependent protein kinase are located close to the carboxyl-terminal side of arginine. The phosphorylation sites exclusively occur in the amino-terminal non-alpha-helical head domain, particularly at the beta-turn region. These results provide clues to the molecular mechanisms of phosphorylation-dependent disassembly of vimentin filaments.
我们曾报道，由环磷酸腺苷（cAMP）依赖性蛋白激酶或蛋白激酶C进行的化学计量磷酸化可诱导波形蛋白丝的解聚[稻垣，M.，西，Y.，西泽，K.，松山，M.，& 佐藤，C.（1987年）《自然》328，649 - 652；稻垣，M.， 近田，Y.，松山，M.，西泽，K.，西，Y.，& 佐藤，C.（1988年）《生物化学杂志》263，5970 - 5978]。在本研究中，我们试图确定每种蛋白激酶磷酸化波形蛋白的位点。对纯化的磷酸肽进行序列分析，并结合已知的一级序列，结果显示丝氨酸 - 8、丝氨酸 - 9、丝氨酸 - 20、丝氨酸 - 25、丝氨酸 - 33和丝氨酸 - 41被蛋白激酶C特异性磷酸化，而丝氨酸 - 46优先被cAMP依赖性蛋白激酶磷酸化。两种激酶都能作用于丝氨酸 - 6、丝氨酸 - 24、丝氨酸 - 38、丝氨酸 - 50和丝氨酸 - 65。蛋白激酶C的特异性磷酸化位点大多位于精氨酸的氨基末端一侧附近，而cAMP依赖性蛋白激酶的磷酸化位点则位于精氨酸的羧基末端一侧附近。这些磷酸化位点仅出现在氨基末端非α螺旋头部结构域，特别是在β转角区域。这些结果为波形蛋白丝磷酸化依赖性解聚的分子机制提供了线索。
