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

在我们之前的研究中，我们已经采取了蛋白质组学的方法来表征动态信号事件中KSR1支架的成分。通过这项工作，我们发现KSR1在RAS激活后从胞浆转移到质膜上，并协调一个大的多蛋白复合体的组装，该复合体的功能是调节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Pro的序列中发现了一个疏水基序，它介导了与KSR1支架的结构性结合，并发现KSR1与B-Raf和MEK形成三元复合体，以响应生长因子处理增强B-Raf介导的MEK活性。引人注目的是，我们还发现，活性ERK与KSR1支架的对接允许ERK在反馈位点上磷酸化KSR1和B-Raf。反馈位点的磷酸化通过促进B-RAF/KSR1/MEK复合体的解离并导致KSR1从质膜上释放来减弱ERK级联信号。此外，我们还发现，KSR的表达水平可以改变ATP竞争性Raf抑制剂对致癌RAS/ERK信号的影响。具体地说，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信号的矛盾激活效应。由于蛋白质组学方法在阐明KSR支架的功能和调控方面取得了成功，我们扩大了这些技术的使用范围，以研究哺乳动物CNK支架家族，包括CNK1、CNK2和CNK3蛋白。考虑到CNK家族成员相似的结构域结构，这一分析发现了几种常见的CNK相互作用蛋白，这并不令人惊讶；然而，它也揭示了CNK复合体中的关键差异，这表明了重要的功能多样性。特别是，我们发现CNK1与Arf鸟氨酸核苷酸交换因子的细胞粘素家族成员相互作用，并且CNK1/细胞粘素相互作用对于胰岛素和IGF-1受体下游的PI3K/AKT级联激活至关重要。胰岛素途径对能量代谢和生长至关重要，其失调是导致人类疾病的主要因素。这些发现为这一重要途径的调控提供了新的机制洞察力，并确定了CNK1作为细胞粘附素功能和胰岛素/IGF-1信号调节因子的作用。在本财政年度，我们还表征了CNK2支架的主要结合伙伴，并发现CNK2与Rho家族GTP酶的关键调控因子相互作用。此外，我们的研究还揭示了CNK2在神经元形态发生过程中整合Arf和Rho家族信号的关键作用。与这些发现一致的是，最近有报道称，在非综合征性X连锁精神发育迟滞的病例中，CNK2的功能丧失突变。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在肿瘤发生过程中也可能调节Arf和Rho家族的信号。

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.