Reconstitution of modular PDK1 functions on trans-splicing of the regulatory PH and catalytic kinase domains.
The serine-threonine protein kinases PDK1 and PKB each contain a pleckstrin homology (PH) domain that binds the membrane-bound phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] second messenger and is required for PDK1-catalyzed phosphorylation and activation of PKB. While X-ray structures have been reported for the individual regulatory PH and catalytic kinase domain constructs of both PDK1 and PKB, diffraction quality crystals of full length constructs have yet to be obtained, likely due to conformational heterogeneity. In developing alternative approaches to understanding the potential role of conformational dynamics in regulating PKB phosphorylation by PDK1, an efficient in vitro method for protein trans-splicing was developed, which utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. For conjugating the regulatory PH domain to the catalytic kinase domain of PDK1, the recombinant trans-splicing fusion constructs KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH were designed, PCR assembled, overexpressed, and affinity purified. The cross-reacting (N)NpuDnaE and (C)SspDnaE inteins generated full length spliced-PDK1 with kobs = (2.8 +/- 0.3) x 10(-5) s(-1) and with < or =5% of any competing trans-cleavage reactions. Spliced-PDK1 was efficiently purified to > or =95% homogeneity from the reaction mixture by subsequent His6 affinity and ion exchange chromatography steps. In vitro kinase assays and phosphopeptide mapping studies confirmed that spliced-PDK1 retained the ability to colocalize and selectively phosphorylate Thr-309 of PKBbeta in a PI(3,4,5)P3-dependent manner. The high-level production and reconstitution of functional spliced-PDK1 establishes the feasibility of incorporating domain-specific biophysical probes for spectroscopic studies of regulatory PH domain mediated catalytic specificity.
Al-Ali
Hassan
H
Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, Florida 33101-6129, USA.
Ragan
Timothy J
TJ
Gao
Xinxin
X
Harris
Thomas K
TK
D064413:3-Phosphoinositide-Dependent Protein Kinases; D001798:Blood Proteins / Q000737:chemistry; D020134:Catalytic Domain; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D020040:Trans-Splicing
D001798:Blood Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases