Cell Type-Specific Importance of Ras-c-Raf Complex Association Rate Constants for MAPK Signaling
Cell Type-Specific Importance of Ras-c-Raf Complex Association Rate Constants for MAPK Signaling
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DOI：
10.1126/scisignal.2000397
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发表时间：
2009-07-28
影响因子：
7.3
通讯作者：
Serrano, Luis
中科院分区：
文献类型：
--
作者：
Kiel, Christina;Serrano, Luis
我们产生了17个c-Raf（RAF原癌基因丝氨酸-苏氨酸蛋白激酶）突变体与改变的Ras-Raf协会和解离速率，研究静电驱动的Ras-Raf协会的作用表皮生长因子（EGF）激活的丝裂原活化蛋白激酶（MAPK）信号转导。这些突变体中的一些具有补偿性的缔合和解离速率的变化，使得缔合速率的变化的影响与亲和力的变化的影响区分开来。在兔肾（RK 13）细胞中，这些突变体影响下游信号传导，Ras-c-Raf结合率的变化对MAPK信号传导的影响大于解离率的类似变化。突变体与补偿减少协会和解离速率刺激细胞外信号调节激酶（ERK）依赖的报告活性比野生型c-Raf，而匡威的是真实的突变体增加协会和解离速率。与此形成鲜明对比的是，突变体对人胚肾（HEK）293细胞中的信号传导几乎没有影响或没有影响。这两种细胞系还显示出不同的EGF依赖性ERK磷酸化和信号传导模式：ERK激活和信号传导在HEK 293细胞中是短暂的，在RK 13细胞中是持续的，差异是由于后者缺乏ERK对Sos（Son of Sevenless）的负反馈。计算机模拟显示，在负反馈的存在下，Ras-c-Raf结合率的变化对ERK激活的影响很小。因此，EGF-MAPK激活动力学和反馈调节是细胞类型特异性的，并取决于网络拓扑结构。
We generated 17 c-Raf (RAF proto-oncogene serine-threonine protein kinase) mutants with altered Ras-Raf association and dissociation rates to investigate the role of electrostatically driven Ras-Raf association rates on epidermal growth factor (EGF)-activated mitogen-activated protein kinase (MAPK) signal transduction. Some of these mutants had compensating changes in association and dissociation rates, enabling the effects of changes in association rate to be distinguished from those of changes in affinity. In rabbit kidney (RK13) cells, these mutants affected downstream signaling, with changes in Ras-c-Raf association rates having a greater effect on MAPK signaling than did similar changes in dissociation rates. Mutants with compensating decreases in both association and dissociation rates stimulated less extracellular signal regulated kinase (ERK)-dependent reporter activity than did wild-type c-Raf, whereas the converse was true for mutants with increased association and dissociation rates. In marked contrast, the mutants had little or no effect on signaling in human embryonic kidney (HEK) 293 cells. These two cell lines also showed distinct patterns of EGF-dependent ERKphosphorylation and signaling: ERK activation and signaling were transient in HEK293 cells and sustained in RK13 cells, with the difference resulting from the lack of negative feedback from ERK to Sos (Son of Sevenless) in the latter. Computer simulation revealed that, in the presence of negative feedback, changes in the rate of Ras-c-Raf binding have little effect on ERK activation. Thus, EGF-MAPK activation kinetics and feedback regulation is cell type specific and depends on the network topology.