Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.
- mammalian target of rapamycin (mTOR)
- p70 ribosomal S6 kinase 1 (S6K1)
- phosphoinositide 3-kinase (PI3K)
- phosphoinositide-dependent protein kinase (PDK1)
- ribosomal S6 kinase 2 (S6K2)
↵1 These authors contributed equally to this work.
Abbreviations used: S6K2, ribosomal S6 kinase 2; S6K1, p70 ribosomal S6 kinase 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; PDK1, phosphoinositide-dependent protein kinase; MEK, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase; EGF, epidermal growth factor; HA, haemagglutinin; DTT, dithiothreitol; Ab, antibody.
- The Biochemical Society, London ©2003