title: Characterization of the p90 ribosomal S6 kinase 2 carboxyl-terminal domain as a protein kinase.
abstract: The carboxyl-terminal domain (CTD) of the p90 ribosomal S6 kinases (RSKs) is an important regulatory domain in RSK and a model for kinase regulation of FXXFXF(Y) motifs in AGC kinases. Its properties had not been studied. We reconstituted activation of the CTD in Escherichia coli by co-expression with active ERK2 mitogen-activated protein kinase (MAPK). GST-RSK2-(aa373-740) was phosphorylated in the P-loop (Thr(577)) by MAPK, accompanied by increased phosphorylation on the hydrophobic motif site, Ser(386). Activated GST-RSK2-(aa373-740) phosphorylates synthetic peptides based on Ser(386). The peptide RRQLFRGFSFVAK, which was termed CTDtide, was phosphorylated with K(m) and V(max) values of approximately 140 microm and approximately 1 micromol/min/mg, respectively. Residues Leu at p -5 and Arg at p -3 are important for substrate recognition, but a hydrophobic residue at p +4 is not. RSK2 CTD is a much more selective peptide kinase than MAPK-activated protein kinase 2. CTDtide was used to probe regulation of hemagglutinin-tagged RSK proteins immunopurified from epidermal growth factor-stimulated BHK-21 cells. K100A but not K451A RSK2 phosphorylates CTDtide, indicating a requirement for the CTD. RSK2-(aa1-389) phosphorylates the S6 peptide, and this activity is inactivated by S386A mutation, but RSK2-(aa1-389) does not phosphorylate CTDtide. In contrast, RSK2-(aa373-740) containing only the CTD phosphorylates CTDtide robustly. Thus, CTDtide is phosphorylated by the CTD but not the NH(2)-terminal domain (NTD). Epidermal growth factor activates the CTD and NTD in parallel. Activity of the CTD for peptide phosphorylation correlates with Thr(577) phosphorylation. CTDtide activity is constrained in full-length RSK2. Interestingly, mutation of the conserved lysine in the ATP-binding site of the NTD completely eliminates S6 kinase activity, but a similar mutation of the CTD does not completely ablate kinase activity for intramolecular phosphorylation of Ser(386), even though it greatly reduces CTDtide activity. The standard lysine mutation used routinely to study kinase functions in vivo may be unsatisfactory when the substrate is intramolecular or in a tight complex.
authors.affiliation: Department of Pharmacology, Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Chrestensen
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Sturgill
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases