Mehanisms in Cancer Signaling: The Role of the Membrane in the Recruitment of the Oncogene KRas4b

Abstract

Ras proteins are a key element of signal transduction where extracellular growth factors control nuclear transcription events involved in cell division, proliferation, and apoptosis. Ras activity is controlled, and signaling mediated by, critical protein-protein interactions. GTPase activating proteins (GAPs), such as p120, bind to activated Ras, dramatically increasing the rate of GTP hydrolysis thus returning the system to the inactive GDP bound state. Guanine exchange factors (GEFs), such as Son of Sevenless (SOS), bind and effect the exchange of GDP for GTP, thus turning “on” KRas4b. Most importantly, these multi-protein complexes all operate on a membrane surface, which is a critical partner in signaling. Despite this critical role of the membrane, there is incomplete knowledge as to the role of the bilayer composition in anchoring the protein to the membrane and the importance of specific lipid type in dictating the final orientation of KRas4b on the surface. Using a fully post-translationally modified KRAS4b provided by the Frederick National Laboratory, we investigated the role of specific lipids in the recruitment of KRas4b to a Nanodisc membrane surface of defined composition. Application of a single frequency fluorescence anisotropy decay experiment to this system revealed that KRas4b has a significant binding preference for Nanodisc bilayers containing PIP2. We conducted molecular dynamics simulations to look for an origin of this specificity. In the case of membranes containing PIP2 the protein formed long-lived salt bridges with PIP2 head groups but not the monovalent DMPS, explaining the experimentally observed lipid specificity. Additionally, we report that PIP2 forms key contacts with Helix-4 on the catalytic domain of KRas4b that orient the protein in a manner expected to facilitate association with upstream and downstream signaling partners.