Cancer often arises when the control of normal cell function goes awry due to defects in critical signal transduction pathways. The signaling pathway regulated by the RasGTPase is one such pathway and it functions to modulate vital cellular processes, including proliferation, differentiation, survival, and senescence. Members of the Raf serine/threonine kinase family are key intermediates in the Ras pathway, serving to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. There are three mammalian Raf proteins, A-Raf, B-Raf, and C-Raf (also known as Raf-1). As might be expected for proteins so centrally involved in cell signaling, the Raf kinases can directly contribute to oncogenic transformation and other human disease states. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently results in constitutive signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. In addition, mutations in the Raf proteins themselves can function as disease drivers. Germline-mutations in C-Raf are causative for Noonan and LEOPARD syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) syndromes, with B-Raf mutations occurring in 75% of CFC patients. Moreover, somatic mutations in B-Raf are observed in 70% of malignant melanomas as well as in many colorectal, ovarian, lung and papillary thyroid carcinomas. During this past fiscal year, our research has elucidated several important mechanisms contributing to the regulation of both normal and mutant Raf signaling. Our studies have revealed that the KSR1 scaffold plays a critical role in modulating the intensity and duration of Raf signaling emanating from the plasma membrane in response to growth factor treatment. In addition, we have found that the KSR expression levels can alter the effects of Raf inhibitors on oncogenic Ras to 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. Our studies have also shown that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events (e. g., phosphorylation on inhibitory feedback sites and the phosphorylation of residues that mediate 14-3-3 binding) and protein interactions (e.g., 14-3-3 binding and Raf dimerization). Moreover, we have found that Raf dimerization is critical for upregulated signaling induced by human disease-associated Raf mutants with moderate, low or impaired kinase activity, or in cases where the pathway is induced by activated RTK or Ras proteins. Our work has further revealed that somatic mutations which modulate Raf dimerization have the potential to alter the progression and treatment of human disease states with elevated Ras pathway signaling. Finally, our work has provided the first 'proof of principle'that inhibiting Raf dimerization can suppress Raf signaling under conditions where dimerization is required. Taken together, these findings have important implications for the treatment of human disease states with elevated Ras pathway signaling and identify the Raf dimer interface as a therapeutic target.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010329-13
Application #
8552667
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2012
Total Cost
$643,054
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
Ritt, Daniel A; Abreu-Blanco, MarĂ­a T; Bindu, Lakshman et al. (2016) Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-Induced Phospho-Regulatory Circuit. Mol Cell 64:875-887
Holderfield, Matthew; Morrison, Deborah K (2016) RAS signaling: Divide and conquer. Nat Chem Biol 13:7-8
Kortum, Robert L; Morrison, Deborah K (2015) Path Forward for RAF Therapies: Inhibition of Monomers and Dimers. Cancer Cell 28:279-81
Freeman, Alyson K; Ritt, Daniel A; Morrison, Deborah K (2013) The importance of Raf dimerization in cell signaling. Small GTPases 4:
Freeman, Alyson K; Ritt, Daniel A; Morrison, Deborah K (2013) Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. Mol Cell 49:751-8
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
Morrison, Deborah K (2012) MAP kinase pathways. Cold Spring Harb Perspect Biol 4:
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

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