Role of Protein Scaffolds in RTKRas-dependent Signal Transduction

蛋白质支架在 RTKRas 依赖性信号转导中的作用

• 批准号: 9153776
• 负责人: Deborah Morrison
• 金额: $ 66.94万
• 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/9153776
• 关键词: ABCG2 gene; Animal Model; Attenuated; Binding; Binding Sites; Biological Process; Budgets; CNKSR1 gene; Caenorhabditis elegans; Catalytic Domain; Cell membrane; Cells; Cognitive deficits; Complex; Cytosol; Defect; Dendrites; Dendritic Spines; Dissociation; Docking; Drosophila melanogaster; Energy Metabolism; Enhancers; Ephrin-B1; Equilibrium; Event; Family; Family member; Feedback; GIT1 gene; GTP Binding; Genes; Genetic Screening; Goals; Growth; Growth Factor; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Hereditary Disease; Hippocampus (Brain); Human; Human Genetics; Insulin; Insulin-Like Growth Factor I; KRAS2 gene; Laboratories; MAP2K1 gene; MAPK8 gene; MEKs; Mediating; Monitor; Morphogenesis; Multiprotein Complexes; N-terminal; Neurons; Non-Small-Cell Lung Carcinoma; Oncogenic; PI3K/AKT; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Play; Proline; Protein Analysis; Protein Dephosphorylation; Protein Family; Proteins; Proteomics; Ras/Raf; Regulation; Relative (related person); Reporting; Role; Scaffolding Protein; Shapes; Signal Transduction; Site; Structure; Techniques; Vertebral column; Work; X-Linked Mental Retardation; cancer type; clinically relevant; genetic regulatory protein; human disease; inhibitor/antagonist; insight; member; mutant; overexpression; protein expression; raf Kinases; ras Proteins; receptor; research study; response; scaffold; success; tumor; tumor progression; 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 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 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. In collaborative work with Dr. Ira Daar's laboratory, a role for CNK1 in facilitating JNK pathway activation downstream of Ephrin-B1 was also defined. In this budget year, we have completed a study analyzing the the major binding partners of the neuronally-expressed CNK2 scaffold and find that CNK2 complexes are enriched for components involved in Rac/Cdc42 signaling, including Rac1 itself, alpha/beta-PIX (RacGEFs), GIT1/2 (ArfGAPs that modulate Rac signaling via interactions with alpha/beta-PIX), and PAK3/4 (Rac/Cdc42 effector kinases). Through mutant analysis, protein depletion/rescue experiments and the monitoring of intracellular RacGTP levels, our work identified CNK2 as a spatial modulator of Rac GDP/GTP cycling. This study also had clinical relevance in that it defined a mechanism for how loss of CNK2 function contributes to a human genetic disorder - non-syndromic, X-linked mental retardation (MRX). In 2012, deletions in the human CNK2 gene were reported in patients with MRX, and given that MRX patients display cognitive defects often associated with abnormalities in the number and shape of their dendritic spines, suggested that CNK2 may have a biological function in spine morphogenesis. In our study, we found that CNK2 localizes to the dendrites of hippocampal neurons, and by interacting with regulators of Rac cycling as well as Rac itself, CNK2 functions to maintain RacGTP/GDP levels at a concentration conducive for spine formation. Thus, when CNK2 is not present or when the interaction between CNK2 and a key regulator of Rac cycling, such as Vilse, is disrupted, the localized balance in RacGTP/GDP levels is perturbed, resulting in spine defects. Interestingly, increased protein expression of CNK2 has been observed in certain cancer types, such as non-small cell lung carcinomas, suggesting that CNK2 may also function to regulate Rac/Cdc42 signaling during tumorigenesis. We have also initiated a new study that further investigates the function and regulation of the Sur8/Shoc2 scaffold. Initial studies characterizing the mammalian Sur8 protein found that when overexpressed, Sur8 could enhance Raf activation by promoting the Ras/Raf interaction. Subsequently, Sur8 was reported to function as a regulatory protein for the catalytic subunit of protein phoshatase 1 (PP1) and contribute to Raf activation. More specifically, binding of the Sur8/PP1 complex to GTP-bound M-Ras (a relative of the prototypical H-, N- and K-Ras proteins), was found to promote the dephosphorylation of the inhibitory N-terminal 14-3-3 binding site on the Raf kinases in growth factor-treated cells and thereby facilitate Raf activation. Our goal in this project is to determine whether Sur8 has additional functions in RTK/Ras signaling that may impact tumor formation and/or cancer progression.

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。在与Ira Daar博士实验室的合作中，CNK1在促进Ephrin-B1下游JNK通路激活中的作用也被确定。在本预算年度，我们完成了一项研究，分析了神经元表达的CNK2支架的主要结合伙伴，发现CNK2复合物富含参与Rac/Cdc42信号传导的成分，包括Rac1本身、α / β - pix （RacGEFs）、GIT1/2（通过与α / β - pix相互作用调节Rac信号传导的ArfGAPs）和PAK3/4 （Rac/Cdc42效应激酶）。通过突变体分析、蛋白质消耗/拯救实验和细胞内RacGTP水平监测，我们的工作确定了CNK2是Rac GDP/GTP循环的空间调节剂。该研究还具有临床意义，因为它确定了CNK2功能缺失如何导致人类遗传疾病——非综合征性x连锁智力迟钝（MRX）的机制。2012年，MRX患者报告了人类CNK2基因缺失，并且考虑到MRX患者表现出的认知缺陷通常与树突棘的数量和形状异常有关，这表明CNK2可能在脊柱形态发生中具有生物学功能。在我们的研究中，我们发现CNK2定位于海马神经元的树突，通过与Rac循环的调节因子以及Rac本身相互作用，CNK2的功能是将RacGTP/GDP水平维持在有利于脊柱形成的浓度。因此，当CNK2不存在或当CNK2与Rac循环的关键调节因子（如Vilse）之间的相互作用被破坏时，RacGTP/GDP水平的局部平衡就会受到干扰，从而导致脊柱缺陷。有趣的是，在某些癌症类型（如非小细胞肺癌）中观察到CNK2的蛋白表达增加，这表明CNK2也可能在肿瘤发生过程中调节Rac/Cdc42信号传导。我们还启动了一项新的研究，进一步研究了Sur8/Shoc2支架的功能和调控。对哺乳动物Sur8蛋白特征的初步研究发现，当Sur8过表达时，Sur8可以通过促进Ras/Raf相互作用来增强Raf的激活。随后，Sur8被报道为蛋白phoshatase 1 （PP1）催化亚基的调控蛋白，并参与Raf的激活。更具体地说，Sur8/PP1复合物与gtp结合的M-Ras（原型H-， N-和K-Ras蛋白的亲戚）结合，被发现促进生长因子处理细胞中Raf激酶的抑制N端14-3-3结合位点的去磷酸化，从而促进Raf活化。我们在这个项目中的目标是确定Sur8是否在RTK/Ras信号中具有可能影响肿瘤形成和/或癌症进展的其他功能。