Regulation of translation by protein phosphorylation.

为了阐明蛋白磷酸化对翻译的调控，我们检测了两种蛋白磷酸酶PP1和MKP-7在细胞中的功能。1.最近我们发现，交链霉菌素/TC是PP1的抑制剂。为了检测TC是否抑制培养细胞中的PP1，用TC处理细胞，然后分析细胞提取液中PP1和PP2A的活性。TC可完全抑制细胞内的PP1，而不影响细胞内的PP2A。2.TC可抑制MEK/ERK的激活。通过RAF与野生型或失活突变型PP1催化亚基的共表达，我们得出结论：PP1活性是激活Raf/MEK/ERK通路所必需的。4.在培养的细胞中，GSK3β与Pp1/1-2异源二聚体结合，并磷酸化1-2的Thr72。通过两次杂交筛选，初步确定β催化亚基为花椒素结合蛋白。结果表明，PP1是以非染色质结构为靶点的，它可能是基因表达的调节因子。6.我们新发现了一个新的MAPK结合基序，该基序普遍存在于MAPK磷酸酶中。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.