Lipid binding and the scaffolding function of the Kinase Suppressor of Ras
The signal transduction field has recently seen a surge of interest in cascade scaffolding proteins. One of these, the Kinase Suppressor of Ras (KSR), has received a great deal of attention as a scaffold for the Ras/ERK signaling pathway. KSR interacts with both MEK and ERK, and possibly binds to Raf-1 as well. Very little is known about the regulation of KSR; however, it has been determined that membrane association is essential for its function in signal augmentation. KSR shares a high degree of sequence homology to Raf-1, including an almost identical phosphatidic acid binding region (PABR). Previous work in the Romero lab has determined the direct interaction of Raf-1 with phosphatidic acid is critical for its membrane recruitment. The PABR is a 35 amino acid sequence consisting of a poly-basic motif (PBM) flanked by two hydrophobic regions. Neutralization of the two arginine residues in the PBM abrogates the binding of Raf-1 to phosphatidic acid (PA), and consequently disrupts its membrane association. This thesis examines lipid-binding properties of the PABR and their potential role in the traffic and function of KSR. Using peptides corresponding to the PABR and tryptophan fluorescence spectroscopy, the data presented in the first section demonstrate that PA induces a blue-shift in the tryptophan emission spectra of WT KSR PABR, and this shift is specific for PA. The second section explores the cellular consequence of KSR PABR mutation. A KSR protein lacking the arginine residues in the PBM expressed in HIRcB fibroblasts retains its membrane-binding ability, but inhibits MEK and ERK phosphorylation in a dominant negative fashion. The data presented here support the conclusion that, although an intact PABR may not be essential for the membrane localization of KSR, it is essential for proper coupling of the pathway.
Advisor:Guillermo Romero, Ph.D.; Daniel L. Altschuler, Ph.D.; Edwin S. Levitan, Ph.D.; Judith Klein-Seetharaman, Ph.D.; Thomas E. Smithgall, Ph.D.; Jes Klarlund, Ph.D.
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:12/09/2005