EXAMINATION OF REGULATROY MECHANISMS FOR CELL FUNCTIONS BY THE GOLGI APPARATUS

我们发现GRASP65(S277)的第277个氨基酸残基在间期细胞中被磷酸化，并且包括EGF在内的生长因子处理显著增强了该信号。ERK被EGF诱导的生长因子信号激活，激活的ERK直接磷酸化S277。我们进一步发现S277在M期高度磷酸化，并分析了这种磷酸化上调的分子机制。在哺乳动物CTRASP65同源物中，S277(PGSPG)附近的氨基酸序列非常保守。该序列与CDK1/cyClinB的靶序列吻合较好。S277被M期细胞胞浆提取液强烈磷酸化，并被CDK特异性抑制剂rocovitine完全抑制。S277还被在MEK抑制剂U0126存在下制备的M期细胞ERK活性不活跃的细胞质提取液磷酸化。这些结果有力地表明，S277在M期的磷酸化是由CDK1/cyclinB而不是ERK引起的。令人惊讶的是，没有N-末端肉豆蔻酰化的纯化的GRASP65(Δm-GRASP65)的微量注射强烈地抑制了有丝分裂的进入。微量注射Δm-GRASP65(其中S277被丙氨酸改变)则没有观察到这一现象。这些结果表明，Δm-GRASP65与某些细胞质因子相互作用，抑制有丝分裂进入。我们已经发现Plk1与GRASP65的S277区的磷酸化结合，并且有一些细胞质因子与GRASP65的非磷酸化的S277区结合。

We have shown that a 277th amino acid residue of GRASP65 (S277) is phosphorylated in interphase cells and the phosphorylation signal is markedly enhanced by the growth factor treatment including EGF. ERK is activated by the EGF induced growth factor signal and the activated ERK phosphorylates S277 directly. We further found that S277 is heavily phosphorylated during M phase and analyzed the molecular mechanism for this up regulation of the phosphorylation. The amino acid sequence around S277 (PGSPG) is well conserved among mammalian CTRASP65 homologues. This sequence is well fitted with the target sequence of cdk1/cyclinB. S277 was strongly phosphorylated by a cytoplasmic extract of M phase cells and this was completely inhibited by roscovitine, a cdk specific inhibitor. S277 was also phosphorylated by an ERK inactive cytoplasmic extract of M phase cells that was prepared in the presence of U0126, a MEK inhibitor. These results strongly suggested that cdk1/cyclinB, and not ERK, is responsible for the phosphorylation of S277 in M phase. Surprisingly, the mitotic entry was strongly inhibited by the microinjection of purified GRASP65 without N-terminal myristoylation (Δm-GRASP65). This was not observed by the microinjection of Δm-GRASP65 in which S277 was changed with alanine. These results suggested that Δm-GRASP65 interact with some cytoplasmic factors and inhibits the mitotic entry. We have found that Plk1 specifically binds to phosphorylated S277 region of GRASP65 and there are some cytoplasmic factors that bind to unphosphorylated S277 region of GRASP65.