Usage of tautomycetin, a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo
Usage of tautomycetin, a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo
复制标题
DOI：
10.1074/jbc.m208888200
复制
发表时间：
2003-01-03
影响因子：
4.8
通讯作者：
Kikuchi, K
中科院分区：
文献类型：
--
作者：
Mitsuhashi, S;Shima, H;Kikuchi, K
蛋白磷酸酶1(PP1)与蛋白磷酸酶2A(PP2A)一起，是真核生物中主要的丝氨酸/苏氨酸蛋白磷酸酶，参与多种细胞功能的调节。虽然PP2A的作用已被广泛使用PP2A的抑制剂冈田酸进行了广泛的研究，但由于缺乏特异性的抑制剂，对PP1的生物学分析仍然受到限制。最近，我们报道了交托霉素(TC)是一种高度特异的PP1抑制剂。为了阐明TC的生物学效应，我们在初步实验中证明，用5 MU的TC处理COS-7细胞5h，内源性PP1的抑制率超过90%，而不影响PP2A的活性。因此，以TC作为PP1的特异性抑制剂，研究了PP1对MAPK信号转导的生物学效应。首先，我们发现TC抑制COS-7细胞中的PP1特异性地抑制ERK的激活，这三种MAPK激酶(ERE、JNK和p38)。TC介导的PP1抑制也抑制了Raf-1的激活，导致MEK-ERK途径失活。为了研究PP1在Raf-1调控中的作用，我们在COS-7细胞中过表达了PP1催化亚单位(PP1C)，发现PP1C促进了Raf-1活性的激活，而磷酸酶死亡的PP1C抑制了Raf-1的激活。此外，还观察到PP1C和Raf-1之间的物理相互作用。这些数据有力地表明，PP1在体内对Raf-1具有正向调节作用。
Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 mum TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERE, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.