Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

11-POS Board 3 REGULATION OF RAF KINASE THROUGH MEMBRANE RECRUITMENT AND MULTIVALENT PROTEIN-LIPID INTERACTIONS Andres Jimenez Salinas 1 ; Youngkwang Lee 1 ; 1 San Diego State University, Chemistry & Biochemistry, San Diego, CA, USA Multivalent membrane-protein interactions dictate and regulate signaling pathways. The MAPK pathway controls various cellular outcomes including differentiation, proliferation and apoptosis. An important node of the MAPK pathway consists of Raf Kinase and its regulatory domains of Raf, the Ras binding domain (RBD) and the cysteine rich domain (CRD), drives membrane recruitment as an initial step for its activation. Its activation mechanism is not fully understood due to its complexity of molecular events and multivalent interactions and its challenge of investigating molecular processes on a relevant membrane environment. To better understand the activation mechanism of Raf, we quantitively characterized membrane binding of Raf on supported lipid bilayers. Total internal reflectance fluorescent microscopy measurements were preformed to obtain kinetic and thermodynamic parameters. Our data demonstrate the functional coupling between RBD and CRD domain in which binding of RBD to active Ras promotes lipid engagement of CRD. Detailed kinetic analysis revealed that Raf integrates protein and membrane interactions through kinetically distinct binding modes. The RBD and CRD determine membrane association and dissociation rates, respectively. The functional coupling between RBD and CRD is critical to achieve specific and efficient membrane translocation in response to Ras activation. Our studies provide a novel insight into mechanisms of Raf activation, which requires the extraction of CRD from the autoinhibited complex upon binding of RBD to Ras.


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