From the Department of Medical Science, Graduate School of Medicine, University of Hiroshima, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima 734-8553, Japan.
Departments of Internal Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Biol Chem. 2010 Oct 22;285(43):33018-33027. doi: 10.1074/jbc.M110.137836. Epub 2010 Jul 30.
Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser(136) in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP·CRTC·CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.
Pin1 是一种独特的调节剂，可催化靶蛋白中特定的磷酸化 Ser/Thr-Pro 基序的转化。在此，我们通过在小鼠肝脏中转染 Myc 和 FLAG 标签过表达 Pin1 并随后纯化包含 Pin1 的复合物，鉴定出 CRTC2 是 Pin1 的结合蛋白。Pin1 与 CRTC2 的关联不仅在过表达实验中观察到，而且在小鼠肝脏中也观察到。有趣的是，CRTC2 核定位信号中的 Ser(136) 被证明与 Pin1 相关联。HepG2 细胞中 Pin1 的过表达减弱了 forskolin 诱导的 CRTC2 核定位和 cAMP 反应元件 (CRE) 转录活性，而 siRNA 基因敲低 Pin1 则增强了这两种活性。Pin1 还与 CRTC1 相关联，导致它们在细胞质中的定位，这与 CRTC2 的作用基本相似。此外，还表明与 Pin1 结合的 CRTC2 不与 CREB 结合。综上所述，这些观察结果表明 Pin1 与 CRTC2 的关联减少了核 CBP·CRTC·CREB 复合物。事实上，将 Pin1 的腺病毒基因转移到糖尿病小鼠中，可改善高血糖症，并使受 CRE 转录活性调节的磷酸烯醇丙酮酸羧激酶 mRNA 表达水平正常化。总之，Pin1 通过与 CRTC1 或 CRTC2 结合来调节 CRE 转录活性。
