Sahin Bogachan, Shu Hongjun, Fernandez Joseph, El-Armouche Ali, Molkentin Jeffery D, Nairn Angus C, Bibb James A
Department of Psychiatry, Protein Chemistry Laboratory, Alliance for Cellular Signaling, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
J Biol Chem. 2006 Aug 25;281(34):24322-35. doi: 10.1074/jbc.M603282200. Epub 2006 Jun 13.
Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.
抑制剂 -1在苏氨酸（35）被环磷酸腺苷依赖性蛋白激酶磷酸化后，成为蛋白磷酸酶1的有效抑制剂。此外，抑制剂 -1的丝氨酸（67）是大脑中细胞周期蛋白依赖性激酶5的底物。在此，我们报告去磷酸化的抑制剂 -1而非磷酸化丝氨酸（67）的抑制剂 -1在体外能被蛋白激酶C在丝氨酸（65）位点有效磷酸化。相反，细胞周期蛋白依赖性激酶5介导的丝氨酸（67）磷酸化不受磷酸化丝氨酸（65）的影响。纹状体组织中蛋白激酶C的激活导致抑制剂 -1在丝氨酸（65）和丝氨酸（67）位点同时发生磷酸化，但并非仅在丝氨酸（65）位点。对蛋白磷酸酶活性的选择性药理学抑制表明，磷酸化丝氨酸（65）的抑制剂 -1在纹状体中被蛋白磷酸酶1去磷酸化。体外研究证实了这些发现，并表明磷酸化丝氨酸（67）可保护磷酸化丝氨酸（65）的抑制剂 -1在体内不被蛋白磷酸酶1去磷酸化。I组代谢型谷氨酸受体的激活导致该组织中二磷酸化丝氨酸（65）/丝氨酸（67）的抑制剂 -1上调。相反，N - 甲基 - D - 天冬氨酸型离子型谷氨酸受体的激活则抑制纹状体中二磷酸化丝氨酸（65）/丝氨酸（67）的抑制剂 -1水平升高。丝氨酸（65）和/或丝氨酸（67）的磷酸模拟突变并未使抑制剂 -1转变为蛋白磷酸酶1抑制剂。另一方面，体外和体内研究表明，二磷酸化丝氨酸（65）/丝氨酸（67）的抑制剂 -1是环磷酸腺苷依赖性蛋白激酶的不良底物。这些观察结果扩展了关于磷酸化丝氨酸（67）功能的早期研究，并强调了抑制剂 -1这一区域被多种蛋白激酶磷酸化可能作为一种整合信号机制来控制抑制剂 -1对环磷酸腺苷依赖性蛋白激酶激活反应性的可能性。
