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Publication Date

2008-06-20

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2009-06-05

First Committee Member

Terace M. Fletcher

Second Committee Member

Charles W. Luetje

Third Committee Member

Arun Malhotra

Fourth Committee Member

Thomas K. Harris

Fifth Committee Member

Vincent J. Hilser

Abstract

Signal transduction kinases lie at the heart of the cell's ability to respond to environmental cues. These kinases are typically controlled by post-translational modification, most commonly by phosphorylation. S6K1alphaII is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (T389) regions of its catalytic kinase domain. In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID, residues 399-502), which inhibits T-loop and hydrophobic motif phosphorylation. Autoinhibition is relieved upon multi-site Ser-Thr phosphorylation of the AID by MAP kinase(s). We developed an optimized PCR-based gene synthesis method, which I utilized to build expression constructs for the AID alone as well as the kinase domain and full length S6K1alphaII. A fully activated form of S6K1alphaII was purified from Sf9 cells by co-expression with PDK1, and was used for in vitro analysis of the signaling pathway. AID was successfully purified in a soluble form from E. coli despite the fact that PONDR analysis predicted a highly disordered structure. Aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC spectra and far UV CD data showing a lack of secondary structure, confirmed that purified recombinant AID is largely unfolded. Despite this, addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1 and inhibition was decreased when the tetraphospho-mimic mutant AID(D2ED) was used. These studies, along with the reagents produced by them, will allow for further exploration of the emerging field of disordered regulatory domains.

Keywords

CD; NMR

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