title: Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA.
abstract: The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP- and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF-binding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.
authors.affiliation: Divison of Signal Transduction Therapy, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. rbiondi@bad.dundee.ac.uk
authors.forename: R M
authors.initials: RM
authors.lastname: Biondi
authors.forename: P C
authors.initials: PC
authors.lastname: Cheung
authors.forename: A
authors.initials: A
authors.lastname: Casamayor
authors.forename: M
authors.initials: M
authors.lastname: Deak
authors.forename: R A
authors.initials: RA
authors.lastname: Currie
authors.forename: D R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: C061570:protein kinase N; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C