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 S6K1 alpha II (the 70-kDa 40 S ribosomal protein S6 kinase-1 alpha II isoform), and Thr-229 phosphorylation is synergistic with C-terminal Thr-389 phosphorylation to activate S6K1 alpha II regulatory functions in protein translation preinitiation complexes. Unlike its common AGC kinase subfamily member S6K1 alpha II, PDK1 does not contain the synergistic C-terminal phosphorylation site, and it has been proposed that phosphorylated Thr-389 in S6K1 alpha II 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 (K-d(S6K1) = 1.2 mu M and k(cat) = 1.1 s(-1)) and the phosphomimicking T389E mutant S6K1 alpha II (K-d(S6K1) = 1.5 mu M and k(cat) = 1.2 s(-1)), although similar to 2-fold enhanced specificity was displayed for the T389E mutant (k(cat)/K-m(S6K1) = 0.08 mu M-1 s(-1) compared with 0.04 mu M-1 s(-1)). Considering that transient kinetic binding studies showed all nucleotide and S6K1 alpha II substrates and products to rapidly associate with PDK1 (k(on) = 1-6 mu M-1 s(-1)), it was concluded that positioning a negative charge at residue Thr389 reduced similar to 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 S6K1 alpha II kinase and (ii) initial binding of S6K1 alpha II precludes ATP binding to PDK1.