title: Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding.
abstract: 3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.
authors.affiliation: Division of Biological Chemistry and Molecular Microbiology and MRC Protein Phosphorylation Unit, MSI/WTB Complex, School of Life Sciences, University of Dundee, Scotland. david.komander@icr.ac.uk
authors.forename: David
authors.initials: D
authors.lastname: Komander
authors.forename: Gursant
authors.initials: G
authors.lastname: Kular
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D010455:Peptides; D012694:Serine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases