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Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
Published on: January 16, 2020
A Proteomic Approach Identifies Isoform-Specific and Nucleotide-Dependent RAS Interactions
Seth P Miller1, George Maio1, Xiaoyu Zhang1
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Abstract:
Active mutations in the RAS genes are found in ∼30% of human cancers. Although thought to have overlapping functions, RAS isoforms show preferential activation in human tumors, which prompted us to employ a comparative and quantitative proteomics approach to generate isoform-specific and nucleotide-dependent interactomes of the four RAS isoforms, KRAS4A, KRAS4B, HRAS, and NRAS. Many isoform-specific interacting proteins were identified, including HRAS-specific CARM1 and CHK1 and KRAS-specific PIP4K2C and IPO7. Comparing the interactomes of WT and constitutively active G12D mutant of RAS isoforms, we identified several potential previously unknown effector proteins of RAS, one of which was recently reported while this article was in preparation, RADIL. These interacting proteins play important roles as knockdown or pharmacological inhibition leads to potent inhibition of cancer cells. The HRAS-specific interacting protein CARM1 plays a role in HRAS-induced senescence, with CARM1 knockdown or inhibition selectively increasing senescence in HRAS-transformed cells but not in KRAS4B-transformed cells. By revealing new isoform-specific and nucleotide-dependent RAS interactors, the study here provides insights to help understand the overlapping functions of the RAS isoforms.
Insights
This study reveals distinct protein interactions for each RAS protein isoform, uncovering new potential cancer drug targets. These findings help clarify the specific roles of RAS isoforms in cancer development and treatment.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- RAS proteins (KRAS4A, KRAS4B, HRAS, NRAS) are frequently mutated in human cancers.
- Despite known overlapping functions, RAS isoforms exhibit preferential activation in tumors, suggesting isoform-specific roles.
Purpose of the Study:
- To comparatively analyze and quantitatively determine the isoform-specific and nucleotide-dependent interactomes of the four RAS isoforms.
- To identify novel effector proteins and therapeutic targets for RAS-driven cancers.
Main Methods:
- Quantitative proteomics was employed to generate comprehensive interactome maps for each RAS isoform.
- Wild-type and constitutively active mutant RAS isoforms were analyzed to identify differences in protein interactions.
Main Results:
- Numerous isoform-specific interacting proteins were identified, including HRAS-specific CARM1 and CHK1, and KRAS-specific PIP4K2C and IPO7.
- Potential novel RAS effector proteins, such as RADIL, were discovered.
- The HRAS-specific protein CARM1 was found to play a role in HRAS-induced senescence, with its inhibition selectively affecting HRAS-transformed cells.
Conclusions:
- The study identified new isoform-specific and nucleotide-dependent RAS interactors.
- These findings provide insights into the distinct functions of RAS isoforms and offer potential therapeutic strategies for targeting RAS-driven cancers.
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