The KSR1 scaffold 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. In the past fiscal year, we have identified a hydrophobic motif in the proline-rich sequence of MEK1 and MEK2 that is required for 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 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 include the the mammalian CNK scaffold family, comprised of the CNK1, CNK2A, CNK2B 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, our studies revealed that the major binding partners of the CNK1 scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream of insulin and IGF-1 receptors. We have identified an 83 amino acid domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins and find that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and protein add-back experiments, we find that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the PIP5Ks to generate a PIP2-rich microenvironment that is critical for both the membrane recruitment of IRS1 and signal transmission to the PI3K/AKT cascade. The insulin pathway is vital for energy metabolism and growth, and its dysregulation is a major contributor to human disease. These findings provide important new mechanistic insight regarding insulin pathway regulation and define a role for CNK1 as a regulator of cytohesin function and a positive modulator of insulin signaling.

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National Cancer Institute (NCI)
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Basu, Sandip K; Lee, Sook; Salotti, Jacqueline et al. (2018) Oncogenic RAS-Induced Perinuclear Signaling Complexes Requiring KSR1 Regulate Signal Transmission to Downstream Targets. Cancer Res 78:891-908
Sandí, María-José; Marshall, Christopher B; Balan, Marc et al. (2017) MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity. Sci Signal 10:
Zhou, Bingying; Ritt, Daniel A; Morrison, Deborah K et al. (2016) Protein Kinase CK2? Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2? Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma. J Biol Chem 291:17804-15
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Koveal, Dorothy; Schuh-Nuhfer, Natasha; Ritt, Daniel et al. (2012) A CC-SAM, for coiled coil-sterile ? motif, domain targets the scaffold KSR-1 to specific sites in the plasma membrane. Sci Signal 5:ra94
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McKay, Melissa M; Freeman, Alyson K; Morrison, Deborah K (2011) Complexity in KSR function revealed by Raf inhibitor and KSR structure studies. Small GTPases 2:276-281

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