The Biological and Biochemical Characterization of KRAS4B Q61 Mutants

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  • February 26, 2019
Creator
  • Cooke, Adelaide
    • Affiliation: College of Arts and Sciences, Department of Biology
Abstract
  • Nearly 30% of human cancers have mutations in one of the three RAS genes. Despite over 30 years of dedicated research, no effective therapies against cancers caused by RAS oncogene mutations have reached clinical application. Striking differences in the frequencies of missense mutations seen in RAS-mutant cancers suggest that RAS mutations previously assumed to equally result in activation have distinct consequences on protein structure and function. We hypothesized that different point mutations at the hotspot residue Q61 will have distinct consequences on RAS structure and function, resulting in unique biological activities. We evaluated the effects on biochemical and biological properties of the common mutants KRAS Q61H, KRAS Q61L, and KRAS Q61R and the rare KRAS Q61E and KRAS Q61P. We found that these KRAS Q61 mutants exhibited varying trends of guanine nucleotide exchange as well as binding to RAS effector domains in vitro. KRAS Q61L exhibited increased GEF-stimulated nucleotide exchange, whereas the Q61R and Q61P mutants were poorly responsive to GEFs. Ectopic expression of KRAS Q61 mutants in NIH 3T3 and RIE-1 cells revealed variable mutation-dependent effector signal activation, morphologic transformation and anchorage-independent growth. Unlike the other mutants, KRAS Q61E and KRAS Q61P did not appear to be activated based on morphological traits. Analysis of signaling through RAS effectors indicated these mutants were still activated; however, it still suggested that they are biologically different from each other. Q61 mutants do not equally induce actin stress fiber formation, cell migration and KRAS-dependent macropinocytosis. Our studies suggest that determination of RAS mutation-specific effects will allow for identification of mutation-selective vulnerabilities and therapies for KRAS Q61 mutants.
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  • Funding: NIH NCI, Pancreatic Cancer Action Network, and CDMRP
Advisor
  • Der, Channing
Degree
  • Bachelor of Science
Academic concentration
  • Biology
Honors level
  • Honors
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2018
Language
  • English

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