Regulation of Ribosomal S6 Kinase 2 by Mammalian Target of Rapamycin

Abstract

Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmicnuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.One of the critical events involved in mitogenic stimulation of mammalian cell growth and proliferation is the increased translation initiation of 5Ј-terminal oligopyrimidine tract-containing mRNAs, which encode components of the protein synthesis machinery (1). Phosphorylation of the ribosomal S6 subunit is correlated with 5Ј-terminal oligopyrimidine tractdependent translation initiation, and the 70-kDa S6 kinase 1 (S6K1) 1 is a serine/threonine protein kinase responsible for mitogen-stimulated S6 phosphorylation (2). In addition to playing an essential role in regulating cell growth, S6K1 appears to be a multifunctional protein involved in other cellular processes such as anti-apoptosis (3) and RNA processing (4). Two parallel pathways are both required for activation of S6K1, and they are mediated by the phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), respectively (2, 5). While the PI3K pathway transduces mitogenic signals to S6K1, the mTOR pathway is believed to sense amino acid sufficiency and play a permissive role to govern S6K1 activation by PI3K signals (6 -9). mTOR is a serine/threonine protein kinase that belongs to the family of phosphatidylinositol kinase-related kinases (10). The kinase activity of mTOR is required, but not sufficient, for signaling to downstream effectors including S6K1 (11, 12). Most recently we hav...

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