Mechanism of PDK1-catalyzed Thr-229 Phosphorylation of the S6K1 Protein Kinase

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/KmS6K1 = 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.