Biochemical characterization of the interaction between KRAS and Argonaute 2

Jessica J. Waninger; Tyler S. Beyett; Varun V. Gadkari; Ronald F. Siebenaler; Carson Kenum; Sunita Shankar; Brandon T. Ruotolo; Arul M. Chinnaiyan; John J.G. Tesmer

Biochemistry and Biophysics Reports (Mar 2022)

Biochemical characterization of the interaction between KRAS and Argonaute 2

Jessica J. Waninger,
Tyler S. Beyett,
Varun V. Gadkari,
Ronald F. Siebenaler,
Carson Kenum,
Sunita Shankar,
Brandon T. Ruotolo,
Arul M. Chinnaiyan,
John J.G. Tesmer

Affiliations

Jessica J. Waninger: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Medical Education, University of Michigan, Ann Arbor, MI, USA; Department of Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA
Tyler S. Beyett: Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Varun V. Gadkari: Department of Chemistry, University of Michigan, Ann Arbor, MI, USA
Ronald F. Siebenaler: Department of Medical Education, University of Michigan, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA
Carson Kenum: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, 48109, USA
Sunita Shankar: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Pathology, University of Michigan, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA
Brandon T. Ruotolo: Department of Chemistry, University of Michigan, Ann Arbor, MI, USA
Arul M. Chinnaiyan: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Pathology, University of Michigan, Ann Arbor, MI, USA; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA; Department of Urology, University of Michigan, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA
John J.G. Tesmer: Departments of Biological Sciences and Medicinal Chemistry & Molecular Pharmacology, Purdue University, Indiana, USA; Corresponding author.

Journal volume & issue: Vol. 29
p. 101191

Abstract

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Oncogenic mutations in KRAS result in a constitutively active, GTP-bound form that in turn activates many proliferative pathways. However, because of its compact and simple architecture, directly targeting KRAS with small molecule drugs has been challenging. Another approach is to identify targetable proteins that interact with KRAS. Argonaute 2 (AGO2) was recently identified as a protein that facilitates RAS-driven oncogenesis. Whereas previous studies described the in vivo effect of AGO2 on cancer progression in cells harboring mutated KRAS, here we sought to examine their direct interaction using purified proteins. We show that full length AGO2 co-immunoprecipitates with KRAS using purified components, however, a complex between FL AGO2 and KRAS could not be isolated. We also generated a smaller N-terminal fragment of AGO2 (NtAGO2) which is believed to represent the primary binding site of KRAS. A complex with NtAGO2 could be detected via ion-mobility mass spectrometry and size exclusion chromatography. However, the data suggest that the interaction of KRAS with purified AGO2 (NtAGO2 or FL AGO2) is weak and likely requires additional cellular components or proteo-forms of AGO2 that are not readily available in our purified assay systems. Future studies are needed to determine what conformation or modifications of AGO2 are necessary to enrich KRAS association and regulate its activities.

Published in Biochemistry and Biophysics Reports

ISSN: 2405-5808 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Science: Biology (General); Science: Chemistry: Organic chemistry: Biochemistry
Website: http://www.journals.elsevier.com/biochemistry-and-biophysics-reports/

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