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

Division Of Basic Sciences - Nci

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Abstract

Many extracellular cues are transduced through the RAS pathway, and qualitative differences in the duration, amplitude, and subcellular localization of signaling events are essential for achieving the appropriate biological response. These signaling parameters are heavily influenced by the actions of scaffold proteins, which modulate the activation, function, and/or localization of core pathway components. In addition, scaffold proteins can facilitate cross-talk between the RAS pathway and other signaling networks, thereby integrating diverse cellular signals. Over the years, our research has defined important roles for multiple scaffolds in RAS pathway regulation. We have shown that 14-3-3 proteins act as key modulators of the conformational switch between active and inactive RAF kinases, and that KSR family members are critical for the spatiotemporal control of ERK cascade signaling. Our earlier studies also identified CNK1 as a positive modulator of Arf GTPase activation and insulin signaling, while CNK2 was shown to regulate Rac GDP/GTP cycling during spine morphogenesis in hippocampal neurons. More recently, our laboratory has focused on further characterizing the role 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. Subsequent studies revealed that SHOC2 functions as a binding partner of protein phosphatase 1 (PP1) and contributes to RAF kinase activation. Specifically, the SHOC2/PP1 complex, upon binding to GTP-bound MRAS (a RAS-related GTPase), dephosphorylates a negative regulatory 14-3-3 binding site on RAF kinases, facilitating RAF dimerization and ERK cascade activation. Notably, germline mutations in SHOC2, MRAS, PP1c, and RAF kinases have been identified as drivers of specific Noonan syndrome subtypes, which are part of a broader group of developmental disorders collectively known as the RASopathies. Our work on SHOC2 identified a novel role for the SHOC2/MRAS/PP1 (SMP) complex in regulating cell-cell adhesion, a process critical for collective cell migration. Specifically, we found that SMP-dependent ERK signaling modulates the p120-catenin/E-cadherin interaction, affecting E-cadherin surface expression and junctional turnover. Specifically, we found that SMP-dependent ERK signaling regulates the interaction between p120-catenin and E-cadherin, which in turn controls E-cadherin surface expression and junctional turnover-key determinants of intercellular adhesion and tissue integrity. Using depletion/reconstitution experiments, we demonstrated upregulated activity for the RASopathy-associated Myr-SHOC2 mutant. Cells expressing this mutant, or either of two RASopathy-associated CRAF mutants (S257L or P261S), exhibited less cohesive migratory behavior, increased junctional turnover, and reduced E-cadherin expression at cell-cell junctions. Importantly, expression of these mutant proteins-unlike their wild-type counterparts-also disrupted convergent extension movements during zebrafish gastrulation, reinforcing the role of SMP and ERK signaling in cell migration and morphogenesis. During the current reporting period, we have continued to investigate the role of the SHOC2/MRAS/PP1 (SMP) complex in RAS-dependent RAF activation. We are also continuing our studies into the molecular mechanisms regulating CNK2 function during spine morphogenesis in hippocampal neurons.