Structural Basis of Human p70 Ribosomal S6 Kinase-1 Regulation by Activation Loop Phosphorylation

Tomoko Sunami,Noel Byrne,Ronald E Diehl,Kaoru Funabashi,Dawn L Hall,Mari Ikuta,Sangita B Patel,Jennifer M Shipman,Robert F Smith,Ikuko Takahashi,Joan Zugay-Murphy,Yoshikazu Iwasawa,Kevin J Lumb,Sanjeev K Munshi,Sujata Sharma

doi:10.1074/jbc.m109.040667

Abstract

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3'-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3'-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The most studied substrate is the 40 S ribosomal protein S6, a major component of the machinery involved in protein synthesis in mammalian cells, suggesting that p70 ribosomal S6 kinase (p70S6K) plays a role in regulating translation
Cloning and Expression—A PCR product encoding residues 75–399 of human p70S6K1 was amplified from full-length p70S6K1 cDNA (Open Biosystems; NM_003161) and ligated into a custom destination vector designed for baculovirus

Summary

To whom correspondence may be addressed

P70S6K is a downstream kinase of insulin receptor-mediated signaling and is a potential therapeutic target for the management of obesity and diabetes as shown by enhanced metabolic rate and insulin sensitivity in p70S6K knock-out mice (4, 5). The activation of p70S6K requires multiple phosphorylation events in both the kinase and autoinhibitory domains (Fig. 1). The C-terminal autoinhibitory domain, which is believed to block phosphorylation within the hydrophobic motif and the activation loop, is phosphorylated by upstream kinases such as ERK (6, 7). Other activating phosphorylation events occur at Thr-412 in the hydrophobic motif by mTOR (mammalian target of rapamycin) and at Thr-252 in the activation loop by PDK1 (8, 9). Given the potential of p70S6K as a therapeutic target, we determined crystal structures of both the unphosphorylated and activation-loop phosphorylated p70S6K1 kinase domains bound to staurosporine. The structures represent the first crystal structures of any member of the p70S6K family and provide a platform for the structure-guided design of selective p70S6K inhibitors

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION