Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells.
Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells.
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PC12 细胞中含有 ERK/MEK 和 B-Raf 的独特信号颗粒。
DOI：
10.1042/bj20040272
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Grimes,MarkL
中科院分区：
文献类型：
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作者：
MacCormick,Matt;Moderscheim,Tanja;vanderSalm,LouiseWM;Moore,Anna;Pryor,ShonaClements;McCaffrey,Gretchen;Grimes,MarkL
虽然几个多蛋白复合物含有MAPK（丝裂原活化蛋白激酶）已被确定使用过表达的激酶和支架蛋白，复合物的成分和它们的物理性质在内源性表达水平尚未确定。我们的特点是一个大的蛋白质复合物含有神经生长因子激活的ERK（细胞外信号调节激酶）和MEK（MAPK/ERK激酶）在大鼠嗜铬细胞瘤（PC 12）细胞。这种蛋白质复合物分馏成一个高速小球，并耐非离子洗涤剂处理，溶解膜。通过高盐处理破坏蛋白质-蛋白质相互作用，以促进活性ERK 1的免疫沉淀和MEK 1的共沉淀。微管片段也存在于耐洗涤剂的高速小球中，一些激酶与它们结合，特别是ERK 1b（ERK 1的选择性剪接异构体），它显示出对结合微管的强烈偏好。含有ERK 1和MEK 1的大蛋白质复合物通过微管片段的速度沉降来解决;然而，它不包含已知结合ERK和MEK的其他支架成分。B-Raf也存在于一个独特的耐洗涤剂，微管独立的蛋白复合物略大于含有ERK和MEK。我们的结论是，有两个独立的神经生长因子调节的“信号颗粒”，估计大小为60-75 S，一个包含ERK 1和MEK 1，另一个包含B-Raf。这些信号颗粒可能在细胞内激酶活性的时间和空间调节中发挥作用。
Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein–protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated ‘signalling particles’ with an estimated size of 60–75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.