title: Mouse 3-phosphoinositide-dependent protein kinase-1 undergoes dimerization and trans-phosphorylation in the activation loop.
abstract: Activation of mouse 3-phosphoinositide-dependent protein kinase-1 (mPDK1) requires phosphorylation at a conserved serine residue, Ser244, in the activation loop. However, the mechanism by which mPDK1 is phosphorylated at this site remains unclear. We have found that kinase-defective mPDK1 (mPDK1KD), but not a kinase-defective mPDK1 in which Ser244 was replaced with alanine (mPDK1KD/S244A), is significantly phosphorylated in intact cells and is a direct substrate of wild-type mPDK1 fused to the yellow fluorescence protein. Phosphoamino acid analysis and phosphopeptide mapping studies revealed that mPDK1 trans-autophosphorylation occurred mainly on Ser244. On the other hand, Ser399 and Thr516, two recently identified autophosphorylation sites of mPDK1, are phosphorylated primarily through a cis mechanism. In vivo labeling studies revealed that insulin stimulated both mPDK1KD and mPDK1KD/S244A phosphorylation in Chinese hamster ovary cells overexpressing the insulin receptor. However, Western blot analysis using a phosphospecific antibody revealed no increase in insulin-stimulated phosphorylation of Ser244 in these cells overexpressing mPDK1. mPDK1 undergoes dimerization in cells and this self-association is enhanced by kinase inactivation. Deletion of the extreme C terminus disrupts mPDK1 dimerization and Ser244 trans-phosphorylation, suggesting that dimerization is important for mPDK1 trans-phosphorylation. Taken together, our results show that mPDK1 autophosphorylation occurs at multiple sites through both cis and trans mechanisms and suggest that dimerization and trans-phosphorylation may serve as mechanisms to regulate PDK1 activity in cells.
authors.affiliation: Departments of Pharmacology, Biochemistry, and Cellular & Structural Biology, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wick
authors.forename: Fresnida J
authors.initials: FJ
authors.lastname: Ramos
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Quon
authors.forename: Lily Q
authors.initials: LQ
authors.lastname: Dong
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D019606:Phosphoamino Acids / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry
chemical_list: D007328:Insulin; D019606:Phosphoamino Acids; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases