TY - JOUR
T1 - Reconstitution of modular PDK1 functions on trans-splicing of the regulatory PH and catalytic kinase domains
AU - Al-Ali, Hassan
AU - Ragan, Timothy J.
AU - Gao, Xinxin
AU - Harris, Thomas K.
N1 - Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2007/7
Y1 - 2007/7
N2 - 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) × 10-5 s_1 and with ≤5% of any competing trans-cleavage reactions. Spliced-PDK1 was efficiently purified to ≥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 PKBβ1 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.
AB - 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) × 10-5 s_1 and with ≤5% of any competing trans-cleavage reactions. Spliced-PDK1 was efficiently purified to ≥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 PKBβ1 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.
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U2 - 10.1021/bc070055r
DO - 10.1021/bc070055r
M3 - Article
C2 - 17500509
AN - SCOPUS:34547207073
VL - 18
SP - 1294
EP - 1302
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
SN - 1043-1802
IS - 4
ER -