MECHANISMS OF GOLGI VESICULATION DURING MITOSIS

有丝分裂过程中高尔基体形成的机制

• 批准号: 6151027
• 负责人: VIVEK MALHOTRA
• 金额: $ 26.97万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6151027
• 关键词: Golgi apparatus; cell cycle; enzyme activity; laboratory rabbit; membrane activity; membrane proteins; mitogen activated protein kinase; phosphorylation; protein sequence

We have reconstituted the fragmentation and dispersal of Golgi membranes by incubation of permeabilized Normal Rat Kidney (NRK) cells with mitotic cytosol and an ATP regenerating system at 32 C. We find that mitotic cytosol depleted of p34cdc2 kinase (the kinase necessary for entry into mitosis) causes Golgi fragmentation. Depletion or inactivation of the cytosolic Mitogen activated protein kinase kinase (MEK1), however, inhibits Golgi fragmentation. Adding back MEK1 to MEK1 depleted mitotic cytosol restores Golgi fragmentation. We find that the downstream (and the relevant) cytosolic MAPkinase, ERK1 and ERK2 are not required for this process. Interestingly, a MAPkinase, recognized by an antibody with broad cross-reactivity to ERK2 is tightly associated with Golgi membranes. Our overall goals for this proposal are to understand the mechanism by which MEK1 is activated and test our hypothesis that Golgi MAPkinase is the immediate downstream target of activated MEK1. Our studies should reveal the identity of a regulator termed MEK1-kinase necessary for MEK1 activation, components that are involved in dispersal of the fragmented Golgi membranes and finally the Golgi membrane associated proteins, including the MEK1 substrate required Golgi fragmentation. These studies should provide an under- standing of the mechanism by which Golgi membranes undergo extensive fragmentation at the onset of mitosis.

我们重建了高尔基膜的破碎和分散 通过将透化的正常大鼠肾 (NRK) 细胞与 有丝分裂细胞质和 32 C 下的 ATP 再生系统。我们发现 有丝分裂细胞质中 p34cdc2 激酶（该激酶是细胞分裂所必需的）耗尽 进入有丝分裂）导致高尔基体分裂。 耗尽或 胞浆丝裂原激活蛋白激酶激酶失活 然而，(MEK1) 会抑制高尔基体断裂。 将 MEK1 添加回 MEK1 耗尽的有丝分裂细胞质恢复高尔基体碎片。 我们发现 下游（和相关）胞质 MAPkinase、ERK1 和 ERK2 不是 此过程所需的。 有趣的是，MAPkinase 被公认 与 ERK2 具有广泛交叉反应性的抗体紧密相关 与高尔基膜。 我们此提案的总体目标是 了解 MEK1 的激活机制并测试我们的 假设高尔基体 MAP 激酶是 激活 MEK1。 我们的研究应该揭示监管者的身份 MEK1 激活所必需的称为 MEK1 激酶，其成分是 参与破碎的高尔基体膜的分散，最后 高尔基体膜相关蛋白，包括 MEK1 底物 需要高尔基体分裂。 这些研究应该提供一个不充分的 高尔基体膜经历广泛的机制的立场 有丝分裂开始时发生碎片。