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.
Highlights
The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers
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 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 plasma membrane (PM)
Summary
Mathematical models were used to allow quantification of the SPR sensorgrams and subsequent biological conclusions. The previously described steady-state and dissociation models were used as a basis [11] They were further implemented with free energy and membrane-associated protein fraction calculations to allow representation of the fractional membrane-associated values with respect to the free energy of membrane partitioning. A new formalism is described to extract the average amount of protein adsorbed at the surface of individual liposomes. Free energy and fraction of membrane-associated proteins— The mathematical model described by Figueira et al [11], Equations 1 and 2, was applied to calculate the partition coefficient from the SPR sensorgrams shown in Equation 1: RUS RUL ϭ. Stoichiometry—Conventional SPR formalism at the steady state was used to determine the average amount of protein adsorbed at the surface of individual liposomes as seen in Equation 8:. The above equation was simplified to a DOPC containing liposome only using Equation 10:
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