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

蛋白质支架和信号调制器的功能和调控分析

• 批准号: 8552983
• 负责人: Deborah Morrison
• 金额: $ 64.31万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552983
• 关键词: Attenuated; Binding; CNKSR1 gene; Cell membrane; Cells; Complex; Cytosol; Dissociation; Docking; Energy Metabolism; Enhancers; Event; Family; Family member; Feedback; Goals; Growth; Growth Factor; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Insulin; Insulin-Like Growth Factor I; Laboratories; MAP2K1 gene; MEKs; Mediating; Morphogenesis; Multiprotein Complexes; Neurons; Oncogenic; PI3K/AKT; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Play; Proline; Protein Family; Proteins; Proteomics; Regulation; Reporting; Resistance; Role; Scaffolding Protein; Signal Transduction; Site; Structure; Techniques; Work; X-Linked Mental Retardation; anticancer research; cancer type; human disease; inhibitor/antagonist; insight; loss of function mutation; member; raf Kinases; receptor; response; rho; scaffold; success; tumorigenesis

In our previous studies, we have taken a proteomic approach to characterize the components of the KSR1 scaffold during dynamic signaling events. Through this work, we have found that KSR1 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. More specifically, we have identified a hydrophobic motif in the proline-rich sequence of MEK1/2 that mediates 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 ATP-competitive 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 investigate the mammalian CNK scaffold family, comprised of the CNK1, CNK2 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, we found that CNK1 interacts with members of the cytohesin family of Arf guanine nucleotide exchange factors and that the CNK1/cytohesin interaction is critical for the activation of the PI3K/AKT cascade downstream of insulin and IGF-1 receptors. The insulin pathway is vital for energy metabolism and growth, and its dysregulation is a major contributor to human disease. These findings provide new mechanistic insight regarding the regulation of this important pathway and define a role for CNK1 as a regulator of both cytohesin function and insulin/IGF-1 signaling. During this fiscal year, we have also characterized the major binding partners of the CNK2 scaffold and find that CNK2 interacts with critical regulators of the Rho family GTPases. Moreover, our studies have revealed that CNK2 play a key role in integrating Arf and Rho family signaling during neuronal morphogenesis. Consistent with these findings, loss-of-function mutations in CNK2 have recently been reported in cases of non-syndromic X-linked mental retardation. Interestingly, increased expression of CNK2 has been observed in certain cancer types, indicating that CNK2 may also function to regulate Arf and Rho family signaling during tumorigenesis.

在我们之前的研究中，我们采用了蛋白质组学方法来表征动态信号事件中KSR1支架的成分。通过这项工作，我们发现KSR1在Ras激活后从细胞质溶胶转运到质膜，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。更具体地说，我们已经在MEK1/2富含脯氨酸的序列中发现了一个疏水基序列，该序列介导与KSR1支架的组成性结合，并发现KSR1在生长因子处理下与B-Raf和MEK形成三元复合物，从而增强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atp竞争性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，我们已经扩展了这些技术的使用，以研究由CNK1， CNK2和CNK3蛋白组成的哺乳动物CNK支架家族。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，我们发现CNK1与Arf鸟嘌呤核苷酸交换因子的细胞hesin家族成员相互作用，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。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。这些发现为这一重要通路的调控提供了新的机制见解，并定义了CNK1作为细胞聚丝素功能和胰岛素/IGF-1信号传导的调节因子的作用。在本财政年度，我们还描述了CNK2支架的主要结合伙伴，并发现CNK2与Rho家族GTPases的关键调节因子相互作用。此外，我们的研究表明，在神经元形态发生过程中，CNK2在整合Arf和Rho家族信号方面发挥了关键作用。与这些发现一致的是，最近在非综合征性x连锁智力迟钝的病例中也报道了CNK2的功能缺失突变。有趣的是，在某些类型的癌症中观察到CNK2的表达增加，这表明CNK2也可能在肿瘤发生过程中调节Arf和Rho家族信号。