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.
| Identifer | oai:union.ndltd.org:UMIAMI/oai:scholarlyrepository.miami.edu:oa_dissertations-1124 |
| Date | 20 June 2008 |
| Creators | Ragan, Timothy James |
| Publisher | Scholarly Repository |
| Source Sets | University of Miami |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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