Regulation of S6K1 Protein Kinae Activation by its C-Terminal Autoinhibitory Domain
Signal transduction kinases lie at the heart of the cells 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.
Advisor:Vincent J. Hilser; Charles W. Luetje; Arun Malhotra; Terace M. Fletcher; Thomas K. Harris
School:University of Miami
School Location:USA - Florida
Source Type:Master's Thesis
Keywords:biochemistry and molecular biology medicine
Date of Publication:06/20/2008