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1.
Life Sci Alliance ; 2(4)2019 08.
Article in English | MEDLINE | ID: mdl-31296567

ABSTRACT

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras G12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras G12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras G12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.


Subject(s)
Cell Membrane/enzymology , Cell Membrane/ultrastructure , Proto-Oncogene Proteins p21(ras)/metabolism , Cell Line, Tumor , Cell Membrane/chemistry , Epithelial Cells/metabolism , Humans , Liposomes/metabolism , Phosphatidylserines/metabolism , Protein Isoforms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Signal Transduction/genetics , Spatial Analysis , Spatio-Temporal Analysis
2.
J Biol Chem ; 294(6): 2193-2207, 2019 02 08.
Article in English | MEDLINE | ID: mdl-30559287

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.


Subject(s)
Cell Membrane/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Calmodulin/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Humans , MAP Kinase Signaling System , Membrane Proteins/metabolism , Membranes, Artificial , Protein Domains , Proto-Oncogene Mas , Proto-Oncogene Proteins c-raf , Signal Transduction , Static Electricity
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