Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity.
In addition to its catalytic domain, phosphoinsositide-dependent protein kinase-1 (PDK1) contains a C-terminal pleckstrin homology (PH) domain, which binds the membrane-bound phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P3] second messenger. Here, we report in vitro kinetic, phosphopeptide mapping, and oligomerization studies that address the role of the PH domain in regulating specific autophosphorylation events, which are required for PDK1 catalytic activation. First, 'inactive' unphosphorylated forms of N-terminal His6 tagged full length (His6-PDK1) and catalytic domain constructs [His6-PDK1(Delta PH)] were generated by treatment with Lambda protein phosphatase (lambda PP). Reconstitution of lambda PP-treated His6-PDK1(Delta PH) catalytic activity required activation loop Ser-241 phosphorylation, which occurred only upon trans-addition of 'active' PDK1 with an apparent bimolecular rate constant of (app)k1(S241) = 374+/-29 M(-1) s(-1). In contrast, full length lambda PP-treated His6-PDK1 catalyzed Ser-241 cis-autophosphorylation with an apparent first-order rate constant of (app)k1(S241) = (5.0+/-1.5) x 10(-4) s(-1) but remained 'inactive'. Reconstitution of lambda PP-treated His(6)-PDK1 catalytic activity occurred only when autophosphorylated in the presence of PI(3,4,5)P3 containing vesicles. PI(3,4,5)P3 binding to the PH domain activated apparent first-order Ser-241 autophosphorylation by 20-fold [(app)k1(S241) = (1.1+/-0.1) x 10(-2) s(-1)] and also promoted biphasic Thr-513 trans-autophosphorylation [(app)k2(T513) = (4.9+/-1.1) x 10(2) M(-1) s(-1) and(app)k3(T513) = (1.5+/-0.2) x 10(3) M(-1) s(-1)]. The results of mutagenesis studies suggest that Thr-513 phosphorylation may cause dissociation of autoinhibitory contacts formed between the contiguous regulatory PH and catalytic kinase domains.
Gao
Xinxin
X
Department of Biochemistry and Molecular Biology, University of Miami, Miller School of Medicine, P.O. Box 016129, Miami, FL 33101-6129, USA.
Harris
Thomas K
TK
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D002384:Catalysis; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014461:Ultracentrifugation
D010748:Phosphopeptides; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases