Crystal structures of S6K1 provide insights into the regulation mechanism of S6K1 by the hydrophobic motif

The p70 kDa ribosome protein subunit 6 kinase 1 (S6K1) belonging to the AGC family of kinases is an important downstream effector of the mTOR signaling pathway, and dysfunction of S6K1 has been related to tumorigenesis, metabolic diseases and developmental defects. The kinase activity of S6K1 is stimulated by multiple phosphorylation events, including phosphorylation of T229 in the activation loop and phosphorylation of T389 in the hydrophobic motif. Here we report six crystal structures of S6K1 with or without the hydrophobic motif in various forms, in complexes with a highly specific inhibitor, namely PF‐4708671. The structural and biochemical data reveal in vivo phosphorylation of T389 in the absence of phosphorylation of T229 and demonstrate the functional importance of two conserved residues of the N‐lobe, Q140 on strand β4 and R121 on helix αC, in the establishment of a hydrogen‐bonding network between the N‐lobe and the hydrophobic motif. Phosphorylation of T389 or introduction of a negatively charged group at the corresponding position leads to reinforcement of the network and stabilization of helix αC, particularly via interactions between the negatively charged group and Q140. Furthermore, sequence and structural comparisons of S6K1 with other AGC family kinases suggest that the divergences in both the hydrophobic motif and helix αC confer specificity on the kinase signaling cascades regarding their dependence on PDK1. Together, these results indicate that phosphorylation of the hydrophobic motif in S6K1 is independent of and probably, precedes and promotes phosphorylation of the activation loop by providing a docking site for PDK1.