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

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 (PDE6?), 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.

SUBMITTER: Lakshman B

PROVIDER: S-EPMC6369290 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Lakshman Bindu B Messing Simon S Schmid Eva M EM Clogston Jeffrey D JD Gillette William K WK Esposito Dominic D Kessing Bailey B Fletcher Daniel A DA Nissley Dwight V DV McCormick Frank F Stephen Andrew G AG Jean-Francois Frantz L FL

The Journal of biological chemistry 20181217 6

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 ...[more]

PMID: 30559287

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Project description:KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, Kd, for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers.

Dataset Information

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

Publications

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

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