+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)



Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)



Protein Expression and Purification 57(2): 271-279



S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 053159838

Download citation: RISBibTeXText

PMID: 17980619

DOI: 10.1016/j.pep.2007.09.014


Related references

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of. 2008

Baculovirus-mediated expression, purification, and characterization of a fully activated catalytic kinase domain construct of the 70 kDa 40S ribosomal protein S6 kinase-1 alphaII isoform (S6K1alphaII). Protein Expression and Purification 58(1): 32-41, 2008

Molecular cloning, over-expression, purification, and functional characterization of the meningococcal HrpA protein C-terminal domain. Journal of Biotechnology 185: S98-S99, 2014

Characterization of the p90 ribosomal S6 kinase 2 carboxyl-terminal domain as a protein kinase. Journal of Biological Chemistry 277(31): 27733-27741, 2002

Purification and characterization of seven chloroplast ribosomal proteins: evidence that organelle ribosomal protein genes are functional and that NH2-terminal processing occurs via multiple pathways in chloroplasts. Plant Molecular Biology 20(3): 459-465, 1992

Purification and characterization of a protein kinase that phosphorylates the carboxyl terminal domain of rna polymerase ii. Journal of Cellular Biochemistry Suppl. (15 Part G): 252, 1991

Expression and purification of the N-terminal regulatory domain of Protein Kinase C for biophysical studies. Protein Expression and Purification 110: 14-21, 2015

Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor. Journal of Bacteriology 176(22): 6907-6914, 1994

Expression, purification, and functional characterization of the carboxyl-terminal domain fragment on bacteriophage P22 repressor. Journal of Biomolecular Structure & Dynamics 12(6): A044, 1995

Purification and Functional Characterization of the C-Terminal Domain of the β-Actin-Binding Protein AIM1 In Vitro. Molecules 23(12):, 2018

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein. Protein Expression & Purification 27(2): 313-318, 2003

Characterization and bacterial expression of the Dictyostelium myosin light chain kinase cDNA. Identification of an autoinhibitory domain. Journal of Biological Chemistry 266(24): 16044-9, 1991

Characterization and bacterial expression of the dictyostelium myosin light chain kinase complementary dna identification of an autoinhibitory domain. Journal of Biological Chemistry 266(24): 16044-16049, 1991

Characterization of an autoinhibitory domain in human mitogen-activated protein kinase-activated protein kinase 2. Journal of Biological Chemistry 270(1): 202-206, 1995

Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues. Journal of Biological Chemistry 267(3): 1761-1768, 1992