Role of Protein Scaffolds in RTK-Ras-dependent Signal Transduction

不同的细胞外信号通过RAS途径传递，信号事件在持续时间、幅度和亚细胞定位方面的质的差异对于实现适当的生物反应至关重要。RAS信号的这些方面受到支架蛋白活性的影响，支架蛋白可以调节核心途径组件的激活、功能和/或定位。此外，这些支架可能会促进与其他信号通路的串扰。多年来，我们对信号支架的研究已经明确了14-3-3蛋白作为RAF家族激酶“活性与非活性”构象的关键调节器的作用，并证明了KSR家族成员在ERK级联信号的时空调控中的重要性。此外，我们以前在蛋白质支架方面的工作发现，CNK1是Arf GTPase激活和胰岛素途径信号的正向调节器，并确定了CNK2支架在海马神经元脊髓形态发生过程中对Rac GDP/GTP循环的空间调节功能。最近，我们的实验室进行了一项研究，以进一步表征SHOC2支架在RAS信号通路中的功能。SHOC2首先在秀丽线虫的遗传筛查中被发现，在那里它被鉴定为RTK和RAS介导的信号的正向调节器。随后，SHOC2被报道作为蛋白磷酸酶1催化亚单位(PP1)的调节蛋白，并在RAF激酶激活中发挥作用。更具体地说，SHOC2/PP1复合体与GTP结合的MRAs(典型的H-、N-和K-RAS蛋白的近亲)被发现去磷酸化RAF激酶上的负调节结合部位14-3-3，从而促进RAF与规范的RAS蛋白结合并促进ERK级联激活。值得注意的是，SHOC2以及MRAs、PP1c和RAF激酶的胚系突变都被确定为Noonan综合征特定亚型的疾病驱动因素，Noonan综合征是一组被称为Rasopathies的相关发育障碍之一。我们在这个项目中的工作是确定SHOC2支架是否在RTK/RAS信号中具有额外的作用，这些信号可能会影响肿瘤的形成和/或导致与Rasopathies相关的发育缺陷。为此，我们确定了MRAS/SHOC2复合体在细胞-细胞黏附的动态调节中的一个新功能，这是集体细胞迁移所必需的。更具体地说，我们发现MRAS/SHOC2/ERK级联信号在调节p120-catenin/E-cadherin相互作用中发挥作用，而p120-catenin/E-cadherin反过来控制细胞表面E-cadherin的表达和连接周转，E-cadherin是控制细胞间黏附的细胞连接的关键成分。通过耗尽/重建实验，这些研究还揭示了与Rasathy相关的MyR-SHOC2突变体的功能获得活性。特别是，我们发现表达MyR-SHOC2突变体或两个Rasathy相关CRAF突变体(S257L或P261S)的细胞表现出较不粘合的迁移行为，这与连接周转增加和E-钙粘素连接表达减少相关。MyR-SHOC2或CRAF突变体的表达，而不是野生型蛋白的表达，也诱导了斑马鱼原肠形成过程中协调的汇聚/伸展细胞运动的缺陷，进一步支持了MRAS/SHOC2/ERK级联信号轴在正常发育所需的细胞迁移事件中的调节作用。鉴于集体细胞迁移在人类胚胎发育中的重要作用，我们对MRAS/SHOC2在这一高度调控过程中的功能的阐明可能有助于深入了解Noonan综合征和其他Rasopathies相关缺陷的细胞学基础。在本报告所述期间，我们继续调查SHOC2支架的调节工作，并启动了新的研究，调查突变的14-3-3蛋白在Rasopathies中作为疾病驱动因素的作用。最后，我们正在继续研究在海马神经元中调节CNK2支架在脊髓形态发生过程中的分子机制。

Diverse extracellular cues are transduced through the RAS pathway, and qualitative differences in the duration, amplitude, and subcellular localization of signaling events are critical for achieving the appropriate biological response. These aspects of RAS signaling are influenced by the activities of scaffold proteins that can modulate the activation, function, and/or localization of the core pathway components. In addition, these scaffolds may facilitate cross-talk with other signaling pathways. Over the years, our research on signaling scaffolds has defined a role for the 14-3-3 proteins as key modulators of the "active versus inactive" conformation of the RAF family kinases and has demonstrated the importance of the KSR family members in the spatio/temporal control of ERK cascade signaling. In addition, our previous work on protein scaffolds identified CNK1 as a positive modulator of Arf GTPase activation and insulin pathway signaling and defined a function for the CNK2 scaffold in the spatial regulation of Rac GDP/GTP cycling during spine morphogenesis in hippocampal neurons. More recently, our laboratory conducted a study to further characterize the function of the SHOC2 scaffold in RAS pathway signaling. SHOC2 was first discovered in genetic screens performed in Caenorhabditis elegans, where it was identified as a positive modulator of RTK- and RAS-mediated signaling. Subsequently, SHOC2 was reported to function as a regulatory protein for the catalytic subunit of protein phosphatase 1 (PP1) and to play a role in RAF kinase activation. More specifically, binding of the SHOC2/PP1 complex to GTP-bound MRAS (a relative of the prototypical H-, N- and K-RAS proteins) was found to dephosphorylate a negative regulatory 14-3-3 binding site on the RAF kinases, which promotes RAF binding to the canonical RAS proteins and facilitates ERK cascade activation. Of note, germline mutations in SHOC2 as well as MRAS, PP1c and the RAF kinases have all been identified as disease drivers in specific subtypes of Noonan syndrome, one of a group of related developmental disorders known as the RASopathies. Our work in this project has been to determine whether the SHOC2 scaffold has additional roles in RTK/RAS signaling that may impact tumor formation and/or contribute to the developmental defects associated with the RASopathies. Towards this end, we identified a new function for the MRAS/SHOC2 complex in the dynamic regulation of cell-cell adhesion that is required for collective cell migration. More specifically, we found that MRAS/SHOC2/ERK cascade signaling plays a role in regulating the p120-catenin/E-cadherin interaction, which in turn controls the cell surface expression and junctional turnover of E-cadherin, a critical component of cell junctions that controls intercellular adhesiveness. Through depletion/reconstitution experiments, these studies also revealed a gain-of-function activity for the RASopathy-associated Myr-SHOC2 mutant. In particular, we found that cells expressing the Myr-SHOC2 mutant or either of two RASopathy-associated CRAF mutants (S257L or P261S) displayed a less cohesive migratory behavior, which correlated with increased junction turnover and reduced junctional expression of E-cadherin. Expression of Myr-SHOC2 or CRAF mutants, but not the wild-type proteins, also induced defects in coordinated convergent/extension cell movements during zebrafish gastrulation, further supporting a regulatory role for the MRAS/SHOC2/ERK cascade signaling axis in cell migratory events required for normal development. Given the essential role of collective cell migration in human embryogenesis, our elucidation of MRAS/SHOC2 function in this highly regulated process may provide insight regarding the cellular basis of defects associated with Noonan syndrome and other RASopathies. During the reporting period, we have continued our work investigating the regulation of the SHOC2 scaffold and we have initiated new studies investigating the role of mutant 14-3-3 proteins as disease drivers in the RASopathies. Finally, we are continuing to investigate the molecular mechanism that regulat the CNK2 scaffold during spine morphogenesis in hippocampal neurons.