title: Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo.
abstract: The AGC family of protein kinases, which includes isoforms of protein kinase B (also known as Akt), ribosomal S6 protein kinase (S6K), and serum- and glucocorticoid-induced protein kinase (SGK) are activated in response to many extracellular signals and play key roles in regulating diverse cellular processes. They are activated by the phosphorylation of the T loop of their kinase domain by the 3-phosphoinositide-dependent protein kinase-1 and by phosphorylation of a residue located C-terminal to the kinase domain in a region termed the hydrophobic motif. Recent work has implicated the NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) as the enzyme that phosphorylates the hydrophobic motif of S6K1 in vivo. Here we demonstrate that in addition to phosphorylating S6K1 and SGK1 at their hydrophobic motif, NEK6 also phosphorylates S6K1 at two other sites and phosphorylates SGK1 at one other site in vitro. Employing the Jerini pepSTAR method in combination with kinetic analysis of phosphorylation of variant peptides, we establish the key substrate specificity determinants for NEK6. Our analysis indicates that NEK6 has a strong preference for Leu 3 residues N-terminal to the site of phosphorylation. Its mutation to either Ile or Val severely reduced the efficacy with which NEK6-phosphorylated peptide substrates, and moreover, mutation of the equivalent Leu residue in S6K1 or SGK1 prevented phosphorylation of their hydrophobic motifs by NEK6 in vitro. However, these mutants of S6K1 or SGK1 still became phosphorylated at their hydrophobic motif following insulin-like growth factor-1 stimulation of transfected 293 cells. This study provides the first description of the basis for the substrate specificity of NEK6 and indicates that NEK6 is unlikely to be responsible for the IGF1-induced phosphorylation of the hydrophobic motif of S6K, SGK, and protein kinase B isoforms in vivo.
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom. j.m.lizcano@dundee.ac.uk
authors.forename: Jose M
authors.initials: JM
authors.lastname: Lizcano
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: Nick
authors.initials: N
authors.lastname: Morrice
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kieloch
authors.forename: C James
authors.initials: CJ
authors.lastname: Hastie
authors.forename: Liying
authors.initials: L
authors.lastname: Dong
authors.forename: Mike
authors.initials: M
authors.lastname: Schutkowski
authors.forename: Ulf
authors.initials: U
authors.lastname: Reimer
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D000072379:NIMA-Related Kinases; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D005938:Glucocorticoids; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; C494918:AKT1 protein, human; C459456:NEK6 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases