State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Biochem J. 2013 Aug 15;454(1):39-47. doi: 10.1042/BJ20121863.
The activity of S6K1 (p70 ribosomal protein subunit 6 kinase 1) is stimulated by phosphorylation of Thr389 in the hydrophobic motif by mTORC1 (mammalian target of rapamycin complex 1) and phosphorylation of Thr229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1); however, the order of the two events is still ambiguous. In the present paper we report six crystal structures of the S6K1 kinase domain alone or plus the hydrophobic motif in various forms, in complexes with a highly specific inhibitor. The structural data, together with the biochemical data, reveal in vivo phosphorylation of Thr389 in the absence of Thr229 phosphorylation and demonstrate the importance of two conserved residues, Gln140 and Arg121, in the establishment of a hydrogen-bonding network between the N-lobe (N-terminal lobe) and the hydrophobic motif. Phosphorylation of Thr389 or introduction of a corresponding negatively charged group leads to reinforcement of the network and stabilization of helix αC. Furthermore, comparisons of S6K1 with other AGC (protein kinase A/protein kinase G/protein kinase C) family kinases suggest that the structural and sequence differences in the hydrophobic motif and helix αC account for their divergence in PDK1 dependency. Taken together, the results of the present study indicate that phosphorylation of the hydrophobic motif in S6K1 is independent of, and probably precedes and promotes, phosphorylation of the activation loop.
S6K1（核糖体蛋白 S6 激酶 1 亚基 p70）的活性受 mTORC1（雷帕霉素靶蛋白复合物 1）对 Thr389 位疏水模体的磷酸化和 PDK1（磷酸肌醇依赖性激酶 1）对 Thr229 位激活环的磷酸化的刺激；然而，这两个事件的顺序仍然不清楚。在本文中，我们报告了 S6K1 激酶结构域单独或以各种形式与疏水模体形成复合物的六个晶体结构，这些复合物与一种高度特异性抑制剂结合。结构数据与生化数据一起，揭示了 Thr389 在 Thr229 磷酸化缺失的情况下在体内的磷酸化，并证明了两个保守残基 Gln140 和 Arg121 在 N-结构域（N-端结构域）和疏水模体之间氢键网络的建立中的重要性。Thr389 的磷酸化或引入相应的带负电荷的基团会导致网络的增强和αC 螺旋的稳定。此外，S6K1 与其他 AGC（蛋白激酶 A/蛋白激酶 G/蛋白激酶 C）家族激酶的比较表明，疏水模体和αC 中的结构和序列差异解释了它们在 PDK1 依赖性上的差异。总之，本研究的结果表明，S6K1 疏水模体的磷酸化独立于、可能先于并促进激活环的磷酸化。
