Regulation of translation by protein phosphorylation.

为了阐明蛋白磷酸化对翻译的调控作用，我们检测了细胞中两种蛋白磷酸酶PP1和MKP-7的功能。最近我们发现tautomycetin/TC是PP1的抑制剂。为了检测TC对培养细胞中PP1的抑制作用，我们先用TC处理细胞，然后分析细胞提取物中PP1和PP2A的活性。TC完全抑制PP1而不影响细胞中的PP2A。当细胞用TC处理时，MEK/ERK的激活被抑制。通过与野生型或失活突变型PP1催化亚基共同表达raf，我们得出PP1活性是激活raf /MEK/ERK通路所必需的。PP1可以结合并负向调节极光激酶。GSK3β在培养细胞中与PP1/1-2异二聚体结合，磷酸化1-2的Thr72。提示GSK3β通过磷酸化使1-2的抑制活性失活，从而作为PPIC的分子开关。通过两次杂交筛选，确定PP1催化亚基为衣壳素结合蛋白。这表明PP1可以通过scapinin靶向非染色质结构，在那里它可能作为基因表达的调节剂。我们最近发现了一个新的MAPK结合基序，通常在MAPK磷酸酶中保守。MKP-7是一种JNK特异性磷酸酶，在几种刺激下被ERK磷酸化。

In order to clarify regulation of translation by protein phosphorylation, we examined function of two protein phosphatases, PP1 and MKP-7 in cells.1.Recently we found that tautomycetin/TC is an inhibitor of PP1. To examine whether TC inhibits PP1 in cultured cells, cells are treated with TC and then PP1 and PP2A activities in the cell extract were analyzed. TC was shown to inhibit PP1 completely without affecting PP2A in the cells.2.When cells are treated with TC, MEK/ERK activation was inhibited. By co-expression of raf together with either wild type or inactive mutant type of PP1 catalytic subunit, we concluded that PP1 activity is necessary for activation of Raf/MEK/ERK pathway.3. PP1 was shown to bind and negatively regulate Aurora kinases.4.GSK3β was shown to associate with PP1/1-2 heterodimer and phosphorylate Thr72 of 1-2 in the cultured cells. It was suggested that GSK3β functions as molecular switch for PPIC by inactivating the inhibitory activity of 1-2 by phosphorylation5.By two hybrid screening, PP1 catalytic subunit was identified as scapinin binding protein. It was suggested that PP1 is targeted to non-chromatin structure by scapinin, where it may function as a regulator of gene expression.6.We newly identified a novel MAPK binding motif commonly conserved in MAPK phosphatases. MKP-7, a JNK specific phosphatase, was shown to be phosphorylated by ERK upon several stimuli.