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.

我们重构了高尔基膜的碎裂和分散 通过孵育透化的正常大鼠肾（NRK）细胞与 有丝分裂细胞质和ATP再生系统在32 C.我们发现 P34CDC2激酶耗尽的有丝分裂细胞溶胶（激酶必需的激酶 进入有丝分裂）导致高尔基体碎片化。 耗尽或 胞质促丝分裂激活蛋白激酶激酶的失活 （MEK1）但是，抑制了高尔基体破裂。 将MEK1添加到MEK1 耗尽的有丝分裂细胞质可恢复高尔基体碎片。 我们发现 下游（以及相关的）胞质Mapkinase，ERK1和ERK2不是 此过程需要。 有趣的是，MAPKINASE，认可 对ERK2具有广泛交叉反应的抗体密切相关 带有高尔基膜。 我们的这一建议的总体目标是 了解MEK1激活并测试我们的机制 高尔基地图酶是直接下游目标的假设 激活的MEK1。 我们的研究应揭示监管机构的身份 称为MEK1激活所需的MEK1-激酶，组件是 参与分散的高尔基膜，最后 高尔基膜相关蛋白，包括MEK1底物 所需的高尔基分裂。 这些研究应提供不足 高尔基膜经历广泛的机制的站立 有丝分裂发作时的破碎。