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J Biol Chem. 2019 Feb 8;294(6):2193-2207. doi: 10.1074/jbc.RA118.005669. Epub 2018 Dec 17.

Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane.

Author information

1
From the NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland 21702.
2
Department of Bioengineering, University of California Berkeley, Berkeley, California 94720.
3
Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 21702.
4
Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, California 94720.
5
Chan Zuckerberg Biohub, San Francisco, California 94158.
6
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California 94158.
7
From the NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland 21702, Frantz.jean-francois@nih.gov.

Abstract

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.

KEYWORDS:

GTPase; MAPK pathway; Raf kinase; Ras protein; lipid raft; liposome; mitogen-activated protein kinase (MAPK); protein-lipid interaction; surface plasmon resonance (SPR)

PMID:
30559287
PMCID:
PMC6369290
[Available on 2020-02-08]
DOI:
10.1074/jbc.RA118.005669

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