Title

Human phosphoinositide-dependent protein kinase-1 (PDK1): Mechanism of autoactivation and substrate catalysis

Date of Award

2006

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Chemistry

First Committee Member

Thomas K. Harris, Committee Chair

Abstract

The human serine-threonine protein kinase PDK1 (phosphoinositide-dependent protein kinase-1) is widely recognized as a key point of interest in understanding molecular signal transduction pathways involved in regulation of cellular growth, survival, and proliferation. PDK1 has been termed the 'master kinase' in that it has been shown to phosphorylate the critical residue in the activation loops of many AGC kinase family members including PKB (protein kinase B). His6-PDK1 and His6-PKBbeta were expressed with improved yields from baculovirus-mediated Sf9 insect cells. The phosphorylation states of purified and phosphatase-treated His6-PDK1 and His6-PKBbeta were investigated. Mechanistic studies provide direct evidence that (i) the PH domain activates Ser-241 cis-autophosphorylation, (ii) the PH domain autoinhibits kinase catalytic activation of Ser-241 mono-phosphorylated PDK1, and (iii) autoinhibition of kinase activation is relieved upon binding of the PH domain to the PI(3,4,5)P3 second messenger, which facilitates trans-autophosphorylation of Thr-513 in the PH domain. Kinetic and chemical/solution perturbation studies aimed to establish the steady-state kinetic mechanism for PDK1-catalyzed trans-phosphorylation of PDK1-Tide were also performed. The results of these studies are best approximated a Rapid Equilibrium Random Bi Bi system, where chemical or conformational steps in the central ternary complex are largely rate determining.

Keywords

Biology, Molecular; Chemistry, Biochemistry; Chemistry, Inorganic

Link to Full Text

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