Function and Regulation of Protein Scaffolds and Signal Modulators in Signaling

在我们以前的研究中，我们已经采取了蛋白质组学的方法来表征动态信号事件过程中的KSR 1支架的组件。通过这项工作，我们已经发现，KSR 1易位从细胞质到质膜Ras激活后，并协调组装一个大的多蛋白复合物，其功能是调节ERK级联信号的强度和持续时间。更具体地说，我们已经确定了在MEK 1/2的富含脯氨酸的序列中介导与KSR 1支架的组成性结合的疏水基序，并发现KSR 1与B-Raf和MEK形成三元复合物，以响应增强B-Raf介导的MEK活化的生长因子处理。引人注目的是，我们还发现，活性ERK与KSR 1支架的对接允许ERK在反馈位点磷酸化KSR 1和B-Raf。反馈位点的磷酸化通过促进B-RAF/KSR 1/MEK复合物的解离并导致KSR 1从质膜释放来减弱ERK级联信号传导。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，KSR 1与C-Raf竞争与B-Raf的结合，从而减弱这些药物对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支架的功能和调节方面的成功，我们已经扩大了这些技术的使用，以研究哺乳动物CNK支架家族，包括CNK 1，CNK 2和CNK 3蛋白。考虑到CNK家族成员相似的结构域结构，这项分析鉴定了几种常见的CNK相互作用蛋白，这并不奇怪;然而，它也揭示了CNK复合物中的关键差异，表明了重要的功能多样性。特别地，我们发现CNK 1与Arf鸟嘌呤核苷酸交换因子的细胞粘连蛋白家族的成员相互作用，并且CNK 1/细胞粘连蛋白相互作用对于胰岛素和IGF-1受体下游的PI 3 K/AKT级联的激活是至关重要的。胰岛素途径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。这些发现提供了关于这一重要途径的调节的新的机制见解，并定义了CNK 1作为细胞粘连素功能和胰岛素/IGF-1信号传导的调节剂的作用。在本财政年度，我们还表征了CNK 2支架的主要结合伙伴，并发现CNK 2与Rho家族GTP酶的关键调节因子相互作用。此外，我们的研究表明CNK 2在神经元形态发生过程中整合Arf和Rho家族信号转导中起关键作用。与这些发现相一致，CNK 2的功能缺失突变最近在非综合征性X连锁精神发育迟滞的病例中被报道。有趣的是，在某些癌症类型中观察到CNK 2的表达增加，表明CNK 2也可以在肿瘤发生期间调节Arf和Rho家族信号传导。在本预算年度，我们还启动了一项新的研究，其中我们对另一种Ras通路调节剂Sur 8/Shoc 2的结合伴侣进行了表征。以前的研究表明，Sur 8/Shoc 2与已知参与Ras途径信号传导的蛋白质相互作用，如M-Ras，K-Ras，蛋白磷酸酶1催化亚基，并且Sur 8/Shoc 2中的种系突变与某些发育性Ras病相关。通过鉴定Sur 8/Shoc 2的新结合伙伴，我们希望更好地了解其在正常和疾病相关信号传导中的功能。

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. We have also initiated a new study during this budget year in which we have characterized the binding partners of another Ras pathway modulator Sur8/Shoc2. Previous studies have shown that Sur8/Shoc2 interacts with proteins known to be involved in Ras pathway signaling, such as M-Ras, K-Ras, protein phosphatase 1 catalytic subunit, and a germline mutation in Sur8/Shoc2 has been associated with certain developmental Rasopathies. By identifying new binding partners of Sur8/Shoc2, we hope to better understand its function in normal and disease-associated signaling.