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. During the reporting period, we have participated in a study identifying KSR1 as a component of perinuclear signaling complexes (PSC) present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. In addition, our group collaborated on a study characterizing the regulation of ARHGEF2 by MARK3-dependent phosphorylation and 14-3-3 binding, which couples microtubules to the actin cytoskeleton for the establishment of epithelial cell polarity. As well, we have initiated a new study in our laboratory to further characterize the function of the Shoc2 scaffold in Ras pathway signaling. Shoc2 was first discovered in genetic screens conducted 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 M-Ras (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 M-Ras, PP1c and the Raf kinases have all been identified as disease drivers in the developmental RASopathies. Our current work in this project is to investigate whether the Shoc2 scaffold has additional functions in RTK/Ras signaling that may impact tumor formation and/or contribute to the developmental defects associated with the RASopathies. Towards this end, we have identified a new function for the M-Ras/Shoc2 complex in the dynamic regulation of cell-cell adhesion that is required for collective cell migration.
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