Roles of the Golgi apparatus in cell growth regulation

GRASP65在S277位点被cdc2/cyclin B磷酸化，可能参与有丝分裂进入的调控。通过两种方法分析GRASP65 S277磷酸化在细胞周期调控中的作用。(1)重组GRASP65 G2A突变体不能定位于高尔基体并在细胞质中积累，将其注射到细胞中，可抑制前期早期组蛋白H3的磷酸化，但对中心体分离无抑制作用。这一结果表明，组蛋白H3磷酸化和中心体分离在有丝分裂开始时是独立调控的，GRASP65的S277磷酸化可能在这些事件的调控中起一定作用。(2)生化分析表明，Plk 1与有丝分裂e磷酸化的GRASP65结合，这依赖于S277的磷酸化。这种结合由Plk1的polo-box结构域介导。这一结果表明GRASP65在S277招募Plk1位点磷酸化，并在有丝分裂开始时作为信号支架发挥作用。

GRASP65 is phosphorylated at S277 by cdc2/cyclin B and may participate in the regulation of mitotic entry. The role of GRASP65 S277 phosphorylation in cell cycle regulation was analyzed by two approaches. (1) Injection of recombinant GRASP65 G2A mutant, which cannot localize to the Golgi apparatus and accumulate in the cytoplasm, into the cells inhibited the phosphorylation of histone H3 in early prophase but not the centrosome separation. This result suggested that phosphorylation of histone H3 and centrosome separation are independently regulated at the onset of mitosis and S277 phosphorylation of GRASP65 may have some role in the regulation of those events. (2) Biochemical analyses revealed that Plk 1 binds to GRASP65 phosphorylated by mitotic e and this was dependent on the phosphorylation of S277. The binding is mediated by polo-box domain of Plk1. This result suggested that GRASP65 phosphorylated at S277 recruit Plk1 and function as a signaling scaffold at the onset of mitosis.