Mechanism of PDK1-catalyzed Thr-229 phosphorylation of the S6K1 protein kinase

Malik M. Keshwani, Xinxin Gao, Thomas K. Harris

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11 Scopus citations


PDK1 (phosphoinositide-dependent protein kinase-1) catalyzes (phosphorylation of Thr-229 in the T-loop of S6K1αII (the 70-kDa 40 S ribosomal protein S6 kinase-1 αII isoform), and Thr-229 phosphorylation is synergistic with C-terminal Thr-389 phosphorylation to activate S6K1αII regulatory functions in protein translation preinitiation complexes. Unlike its common AGC kinase subfamily member S6K1αII, PDK1 does not contain the synergistic C-terminal phosphorylation site, and it has been proposed that phosphorylated Thr-389 in S6K1αII may initially serve to trans-activate PDK1-catalyzed Thr-229 phosphorylation. Herein, we report direct binding and kinetic studies that showed PDK1 to exhibit nearly equal binding affinities and steady-state kinetic turnover numbers toward native (KdS6K1 = 1.2 μM and kcat = 1.1 s-1) and the phosphomimicking T389E mutant S6K1αII (KdS6K1=1.5 μM and kcat = 1.2 s-1), although ∼2-fold enhanced specificity was displayed for the T389E mutant (kcat/KmS6KI = 0.08 μM-1 s-1 compared with 0.04 μM-1 s-1). Considering that transient kinetic binding studies showed all nucleotide and S6K1αII substrates and products to rapidly associate with PDK1 (kon = 1-6 μM-1 s-1), it was concluded that positioning a negative charge at residue Thr-389 reduced ∼2-fold the occurrence of nonproductive binding events that precede formation of a reactive ternary complex for Thr-229 phosphorylation. In addition, steady-state kinetic data were most simply accommodated by an Ordered Bi Bi mechanism with competitive substrate inhibition, where (i) the initially formed PDK1-ATP complex phosphorylates the nucleotide-free form of the S6K1αII kinase and (ii) initial binding of S6K1αII precludes ATP binding to PDK1.

Original languageEnglish (US)
Pages (from-to)22611-22624
Number of pages14
JournalJournal of Biological Chemistry
Issue number34
StatePublished - Aug 21 2009

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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