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 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 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. In collaborative work with Dr. Ira Daar's laboratory, a role for CNK1 in facilitating JNK pathway activation downstream of Ephrin-B1 was also defined. In this budget year, we have completed a study analyzing the the major binding partners of the neuronally-expressed CNK2 scaffold and find that CNK2 complexes are enriched for components involved in Rac/Cdc42 signaling, including Rac1 itself, alpha/beta-PIX (RacGEFs), GIT1/2 (ArfGAPs that modulate Rac signaling via interactions with alpha/beta-PIX), and PAK3/4 (Rac/Cdc42 effector kinases). Through mutant analysis, protein depletion/rescue experiments and the monitoring of intracellular RacGTP levels, our work identified CNK2 as a spatial modulator of Rac GDP/GTP cycling. This study also had clinical relevance in that it defined a mechanism for how loss of CNK2 function contributes to a human genetic disorder - non-syndromic, X-linked mental retardation (MRX). In 2012, deletions in the human CNK2 gene were reported in patients with MRX, and given that MRX patients display cognitive defects often associated with abnormalities in the number and shape of their dendritic spines, suggested that CNK2 may have a biological function in spine morphogenesis. In our study, we found that CNK2 localizes to the dendrites of hippocampal neurons, and by interacting with regulators of Rac cycling as well as Rac itself, CNK2 functions to maintain RacGTP/GDP levels at a concentration conducive for spine formation. Thus, when CNK2 is not present or when the interaction between CNK2 and a key regulator of Rac cycling, such as Vilse, is disrupted, the localized balance in RacGTP/GDP levels is perturbed, resulting in spine defects. Interestingly, increased protein expression of CNK2 has been observed in certain cancer types, such as non-small cell lung carcinomas, suggesting that CNK2 may also function to regulate Rac/Cdc42 signaling during tumorigenesis. We have also initiated a new study that further investigates the function and regulation of the Sur8/Shoc2 scaffold. Initial studies characterizing the mammalian Sur8 protein found that when overexpressed, Sur8 could enhance Raf activation by promoting the Ras/Raf interaction. Subsequently, Sur8 was reported to function as a regulatory protein for the catalytic subunit of protein phoshatase 1 (PP1) and contribute to Raf activation. More specifically, binding of the Sur8/PP1 complex to GTP-bound M-Ras (a relative of the prototypical H-, N- and K-Ras proteins), was found to promote the dephosphorylation of the inhibitory N-terminal 14-3-3 binding site on the Raf kinases in growth factor-treated cells and thereby facilitate Raf activation. Our goal in this project is to determine whether Sur8 has additional functions in RTK/Ras signaling that may impact tumor formation and/or cancer progression.

National Institute of Health (NIH)
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
Lim, Junghwa; Ritt, Daniel A; Zhou, Ming et al. (2014) The CNK2 scaffold interacts with vilse and modulates Rac cycling during spine morphogenesis in hippocampal neurons. Curr Biol 24:786-92
Cho, Hee Jun; Hwang, Yoo-Seok; Mood, Kathleen et al. (2014) EphrinB1 interacts with CNK1 and promotes cell migration through c-Jun N-terminal kinase (JNK) activation. J Biol Chem 289:18556-68
Morrison, Deborah K (2012) MAP kinase pathways. Cold Spring Harb Perspect Biol 4:
Logue, Jeremy S; Morrison, Deborah K (2012) Complexity in the signaling network: insights from the use of targeted inhibitors in cancer therapy. Genes Dev 26:641-50
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
Rouquette-Jazdanian, Alexandre K; Sommers, Connie L; Kortum, Robert L et al. (2012) LAT-independent Erk activation via Bam32-PLC-?1-Pak1 complexes: GTPase-independent Pak1 activation. Mol Cell 48:298-312
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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