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Lipid binding and the scaffolding function of the Kinase Suppressor of Ras

Kraft, Catherine Ann (2005) Lipid binding and the scaffolding function of the Kinase Suppressor of Ras. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kraft, Catherine
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairLevitan, Edwin Slevitan@server.pharm.pitt.eduELEVITAN
Committee MemberAltschuler, Daniel
Committee MemberRomero, Guillermoggr@pitt.eduGGR
Committee MemberKlarlund,
Committee MemberKlein-Seetharaman, Judithjks33@pitt.eduJKS33
Committee MemberSmithgall, Thomas Etsmithga@pitt.eduTSMITHGA
Date: 9 December 2005
Date Type: Completion
Defense Date: 27 September 2005
Approval Date: 9 December 2005
Submission Date: 29 November 2005
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Pharmacology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: KSR; MAPK signaling; phosphatidic acid; Raf; scaffold proteins; endosome; tryptophan fluorescence
Other ID:, etd-11292005-095950
Date Deposited: 10 Nov 2011 20:06
Last Modified: 15 Nov 2016 13:52


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