RAS genes comprise the most commonly mutated class of oncogenes in human cancer (33%). Their protein counterparts function as GDP-GTP regulated switches to modulate signaling networks that control cell growth, differentiation and apoptosis. Mutationally activated Ras proteins are refractory to inactivation by GTPase activating proteins, and are persistently GTP- bound, leading to chronic downstream effectors activation and signaling. Despite three decades of intensive effort, no effective inhibitors that directly target mutant Ras proteins have successfully reached clinical application. Therefore, much of the past and current ongoing efforts have taken indirect approaches, which have met with failure or lack of anti-tumor efficacy. Herein, we investigate a novel mechanism of Ras activation involving posttranslational modification by ubiquitination. Recent findings that covalen modification of Ras by monoubiquitination may be key for its ability to drive human oncogenesis, suggest one possible direction for developing direct Ras inhibitors. To rigorously assess this possibility, two key issues need to be addressed. First, what are the direct consequences of monoubiquitination on intrinsic Ras function? Second, the previous study showed that preventing monoubiquitination impaired the ability of activated K-Ras to promote tumor xenograft growth when ectopically- expressed in NIH3T3 mouse fibroblasts. How important is this modification for endogenous mutant and normal K-Ras in human colorectal tumor cells with validated addiction to mutant K- Ras and normal K-Ras function in cell proliferation? We will address these issues, by performing biochemical and structural studies on monoubiquitinated Ras as well as biological studies with ubiquitination-deficient K-Ras to rigorously assess its role in wild type and mutant K-Ras biological activity, effectors signaling and regulation.

Public Health Relevance

Despite three decades of effort, to date, no effective anti-K-Ras therapies have reached the clinic. The goal of this effort is to investigate a novel mechanism of Ras activation involving posttranslational modification by ubiquitination that may be key for its ability to drive oncogenesis. Our findings may identify novel directions to target K Ras for cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM106227-02
Application #
8669021
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2013-06-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$386,813
Indirect Cost
$128,806
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Hobbs, G Aaron; Gunawardena, Harsha P; Campbell, Sharon L (2014) Biophysical and proteomic characterization strategies for cysteine modifications in Ras GTPases. Methods Mol Biol 1120:75-96