Analysis of the Function and Regulation of Protein Scaffolds and Signal Modulato

Ras激活后，KSR1支架从细胞质转移到质膜，并协调一个大的多蛋白复合物的组装，该复合物的功能是调节ERK级联信号的强度和持续时间。在过去的财政年度，我们已经在MEK1和MEK2富含脯氨酸的序列中发现了一个疏水基序列，这是与KSR1支架组成性结合所必需的，并发现KSR1与B-Raf和MEK形成三元复合物，以响应生长因子处理，增强B-Raf介导的MEK激活。引人注目的是，我们还发现，活性ERK与KSR1支架的对接允许ERK在反馈位点磷酸化KSR1和B-Raf。反馈位点的磷酸化通过促进B-RAF/KSR1/MEK复合物的解离和导致KSR1从质膜释放来减弱ERK级联信号。此外，我们发现KSR表达水平可以改变Raf抑制剂对致癌Ras/ERK信号传导的影响。具体而言，KSR1与C-Raf竞争抑制剂诱导的与B-Raf的结合，从而减弱了这些药物对ERK信号传导的矛盾激活作用。由于蛋白质组学方法在阐明KSR支架的功能和调控方面的成功，我们已经扩大了这些技术的使用范围，包括哺乳动物CNK支架家族，包括CNK1， CNK2A， CNK2B和CNK3蛋白。考虑到CNK家族成员相似的结构域结构，该分析确定了几种常见的CNK相互作用蛋白，这并不奇怪；然而，它也揭示了CNK复合物的关键差异，表明重要的功能多样性。特别是，我们的研究揭示了CNK1支架的主要结合伙伴是Arf鸟嘌呤核苷酸交换因子的细胞hesin家族成员，并且CNK1/细胞hesin相互作用对于胰岛素和IGF-1受体下游PI3K/AKT级联的激活至关重要。我们已经确定了位于CNK1 c端区域的83个氨基酸结构域，该结构域与细胞分裂素的卷曲结构域组成性地相互作用，并发现CNK1促进胰岛素刺激后细胞分裂素-2的膜募集。此外，通过蛋白耗尽和蛋白添加回实验，我们发现CNK1/细胞分裂素相互作用促进从质膜结合的Arf GTPases到PIP5Ks的信号传导，从而产生pip2丰富的微环境，这对于IRS1的膜募集和PI3K/AKT级联的信号传递都是至关重要的。胰岛素通路对能量代谢和生长至关重要，其失调是人类疾病的主要原因。这些发现为胰岛素通路调控提供了重要的新的机制见解，并确定了CNK1作为细胞聚丝素功能的调节剂和胰岛素信号的积极调节剂的作用。

The KSR1 scaffold translocates from the cytosol to the plasma membrane upon Ras activation and coordinates the assembly of a large multiprotein complex that functions to regulate the intensity and duration of ERK cascade signaling. In the past fiscal year, we have identified a hydrophobic motif in the proline-rich sequence of MEK1 and MEK2 that is required for constitutive binding to the KSR1 scaffold and find that KSR1 forms a ternary complex with B-Raf and MEK in response to growth factor treatment that enhances B-Raf-mediated MEK activation. Strikingly, we have also found that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback sites. Phosphorylation of the feedback sites attenuates ERK cascade signaling by promoting the dissociation of the B-RAF/KSR1/MEK complex and causing the release of KSR1 from the plasma membrane. In addition, we have found that KSR expression levels can alter the effects of Raf inhibitors on oncogenic Ras/ERK signaling. Specifically, KSR1 competes with C-Raf for inhibitor-induced binding to B-Raf and in doing so attenuates the paradoxical activating effect of these drugs on ERK signaling. Due to success of the proteomic approach in elucidating the function and regulation of the KSR scaffolds, we have expanded our use of these techniques to include the the mammalian CNK scaffold family, comprised of the CNK1, CNK2A, CNK2B and CNK3 proteins. Not surprising given the similar domain structure of the CNK family members, this analysis identified several common CNK-interacting proteins; however, it also revealed key differences in the CNK complexes that suggest important functional diversity. In particular, our studies revealed that the major binding partners of the CNK1 scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream of insulin and IGF-1 receptors. We have identified an 83 amino acid domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins and find that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and protein add-back experiments, we find that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the PIP5Ks to generate a PIP2-rich microenvironment that is critical for both the membrane recruitment of IRS1 and signal transmission to the PI3K/AKT cascade. The insulin pathway is vital for energy metabolism and growth, and its dysregulation is a major contributor to human disease. These findings provide important new mechanistic insight regarding insulin pathway regulation and define a role for CNK1 as a regulator of cytohesin function and a positive modulator of insulin signaling.