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Regulation of Ribosomal S6 Kinase 2 by Mammalian Target of Rapamycin*

  • In-Hyun Park
    Affiliations
    From the Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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  • Rebecca Bachmann
    Affiliations
    From the Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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  • Haider Shirazi
    Affiliations
    From the Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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  • Jie Chen
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell and Structural Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Ave. B107, Urbana, IL 61801.
    Affiliations
    From the Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant GM58064.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:June 26, 2002DOI:https://doi.org/10.1074/jbc.M204080200
      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 cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.
      S6K
      S6 kinase
      mTOR
      mammalian target of rapamycin
      PI3K
      phosphatidylinositol 3-kinase
      NLS
      nuclear localization signal
      Erk
      extracellular signal-regulated kinase
      PA
      phosphatidic acid
      LMB
      leptomycin B
      HA
      hemagglutinin
      HEK
      human embryonic kidney
      FBS
      fetal bovine serum
      FITC
      fluorescein isothiocyanate
      NES
      nuclear export sequence
      One of the critical events involved in mitogenic stimulation of mammalian cell growth and proliferation is the increased translation initiation of 5′-terminal oligopyrimidine tract-containing mRNAs, which encode components of the protein synthesis machinery (
      • Meyuhas O.
      • Hornstein E.
      ). Phosphorylation of the ribosomal S6 subunit is correlated with 5′-terminal oligopyrimidine tract-dependent translation initiation, and the 70-kDa S6 kinase 1 (S6K1)1 is a serine/threonine protein kinase responsible for mitogen-stimulated S6 phosphorylation (
      • Fumagalli S.
      • Thomas G.
      ). 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 (
      • Harada H.
      • Andersen J.S.
      • Mann M.
      • Terada N.
      • Korsmeyer S.J.
      ) and RNA processing (
      • Wilson K.F., Wu, W.J.
      • Cerione R.A.
      ). 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 (
      • Fumagalli S.
      • Thomas G.
      ,
      • Gingras A.C.
      • Raught B.
      • Sonenberg N.
      ). 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 (
      • Hara K.
      • Yonezawa K.
      • Weng Q.P.
      • Kozlowski M.T.
      • Belham C.
      • Avruch J.
      ,
      • Iiboshi Y.
      • Papst P.J.
      • Kawasome H.
      • Hosoi H.
      • Abraham R.T.
      • Houghton P.J.
      • Terada N.
      ,
      • Wang X.
      • Campbell L.E.
      • Miller C.M.
      • Proud C.G.
      ,
      • Xu G.
      • Kwon G.
      • Marshall C.A.
      • Lin T.A.
      • Lawrence Jr., J.C.
      • McDaniel M.L.
      ).
      mTOR is a serine/threonine protein kinase that belongs to the family of phosphatidylinositol kinase-related kinases (
      • Keith C.T.
      • Schreiber S.L.
      ). The kinase activity of mTOR is required, but not sufficient, for signaling to downstream effectors including S6K1 (
      • Brown E.J.
      • Beal P.A.
      • Keith C.T.
      • Chen J.
      • Shin T.B.
      • Schreiber S.L.
      ,
      • Brunn G.J.
      • Hudson C.C.
      • Sekulic A.
      • Williams J.M.
      • Hosoi H.
      • Houghton P.J.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ). Most recently we have found that phosphatidic acid, likely produced by phospholipase D, directly mediates mitogenic stimulation of mTOR signaling to S6K1 (
      • Fang Y.
      • Vilella-Bach M.
      • Bachmann R.
      • Flanigan A.
      • Chen J.
      ). Thus, mTOR appears to regulate S6K1 by integrating nutrient and mitogen signals. We have also reported that mTOR is a cytoplasmic-nuclear shuttling protein (
      • Kim J.E.
      • Chen J.
      ), and this shuttling is involved in S6K1 regulation. Specifically, the constant nuclear entry and exit of mTOR is necessary for mitogenic activation of S6K1 (
      • Kim J.E.
      • Chen J.
      ), although the shuttling itself does not seem to be regulated by any known upstream signals.
      J. E. Kim, R. Bachmann, and J. Chen, unpublished observation.
      2J. E. Kim, R. Bachmann, and J. Chen, unpublished observation.
      Targeted gene disruption of S6K1 in mice led to a reduced animal size, implicating S6K1 in cell growth and cell size regulation (
      • Shima H.
      • Pende M.
      • Chen Y.
      • Fumagalli S.
      • Thomas G.
      • Kozma S.C.
      ), but S6 phosphorylation and 5′-terminal oligopyrimidine tract-dependent translation were normal in the S6K1-deficient cells (
      • Shima H.
      • Pende M.
      • Chen Y.
      • Fumagalli S.
      • Thomas G.
      • Kozma S.C.
      ), suggesting the existence of a redundant kinase(s). Indeed, a homologue of S6K1 has been identified and named S6K2 (
      • Shima H.
      • Pende M.
      • Chen Y.
      • Fumagalli S.
      • Thomas G.
      • Kozma S.C.
      ,
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ,
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ,
      • Saitoh M.
      • ten Dijke P.
      • Miyazono K.
      • Ichijo H.
      ). S6K1 and S6K2 are highly homologous in the kinase domain and adjacent regulatory region, and sequence diversity occurs mainly in the N and C termini. The most notable difference between these two proteins is their subcellular localization. Alternative splicing at the N terminus gives rise to two isoforms for both S6K1 (p70 S6K1αII and p85 S6K1αI) and S6K2 (p60 S6K2βI and p54 S6K2βII). p70 S6K1 is cytosolic, whereas p85 S6K1 is nuclear due to the unique N-terminal nuclear localization signal (NLS) (
      • Reinhard C.
      • Fernandez A.
      • Lamb N.J.
      • Thomas G.
      ). On the other hand, both S6K2 isoforms are predominantly nuclear due to an NLS in the C termini of the proteins (
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ). The differential subcellular localization of S6K1 (p70, αII) and S6K2 suggests potentially distinct regulatory mechanisms and/or diverse downstream effectors.
      Like S6K1, the activation of S6K2 requires both the PI3K pathway and the mTOR pathway (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ,
      • Martin K.A.
      • Schalm S.S.
      • Richardson C.
      • Romanelli A.
      • Keon K.L.
      • Blenis J.
      ) and also involves the mitogen-activated protein kinase Erk (
      • Martin K.A.
      • Schalm S.S.
      • Romanelli A.
      • Keon K.L.
      • Blenis J.
      ,
      • Wang L.
      • Gout I.
      • Proud C.G.
      ). While the PI3K and Erk pathways upstream of S6K2 have been characterized recently (
      • Martin K.A.
      • Schalm S.S.
      • Richardson C.
      • Romanelli A.
      • Keon K.L.
      • Blenis J.
      ,
      • Martin K.A.
      • Schalm S.S.
      • Romanelli A.
      • Keon K.L.
      • Blenis J.
      ,
      • Wang L.
      • Gout I.
      • Proud C.G.
      ), the mTOR pathway has not been fully examined in relation to S6K2. Here we report the investigation of S6K2 regulation by mTOR. We show that S6K2 activation requires the kinase activity of mTOR, is dependent on amino acid sufficiency, and involves phosphatidic acid (PA). Furthermore, our data suggest that an optimal rate of mTOR cytoplasmic-nuclear shuttling gives rise to maximal activation of S6K2.

      DISCUSSION

      The rapamycin sensitivity of S6K2 has been a controversial issue. While several groups reported a complete blockage of S6K2 activity by low concentrations of rapamycin (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ,
      • Saitoh M.
      • ten Dijke P.
      • Miyazono K.
      • Ichijo H.
      ), one group observed a partial resistance of S6K2 activity to rapamycin at concentrations up to 200 nm (
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ,
      • Minami T.
      • Hara K.
      • Oshiro N.
      • Ueoku S.
      • Yoshino K.
      • Tokunaga C.
      • Shirai Y.
      • Saito N.
      • Gout I.
      • Yonezawa K.
      ), and persistence of S6K2 activity upon amino acid withdrawal (
      • Minami T.
      • Hara K.
      • Oshiro N.
      • Ueoku S.
      • Yoshino K.
      • Tokunaga C.
      • Shirai Y.
      • Saito N.
      • Gout I.
      • Yonezawa K.
      ). We have found that in HEK293 and CV-1 cells S6K2 (βII isoform) activity was completely inhibited by 100 nm rapamycin (Fig. 1A and data not shown). In addition, amino acid withdrawal abolished S6K2 activation by serum, contrary to the report by Minami et al.(
      • Minami T.
      • Hara K.
      • Oshiro N.
      • Ueoku S.
      • Yoshino K.
      • Tokunaga C.
      • Shirai Y.
      • Saito N.
      • Gout I.
      • Yonezawa K.
      ), and readdition of amino acids restored serum stimulation of S6K2 (Fig. 2). Furthermore, we have demonstrated that the activation of S6K2 is dependent on mTOR kinase activity (Fig. 1B). The difference in amino acid requirement may be attributed to the two different isoforms of S6K2 that we (βII) and Minami et al.(βI, Ref.
      • Minami T.
      • Hara K.
      • Oshiro N.
      • Ueoku S.
      • Yoshino K.
      • Tokunaga C.
      • Shirai Y.
      • Saito N.
      • Gout I.
      • Yonezawa K.
      ) studied. However, it is not clear what gave rise to the discrepancy in the effect of rapamycin on S6K2. The βII isoform with an optimal Kozak sequence surrounding the start codon (
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ) was examined by all groups (
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ,
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ). In addition, all reported assays (
      • Gout I.
      • Minami T.
      • Hara K.
      • Tsujishita Y.
      • Filonenko V.
      • Waterfield M.D.
      • Yonezawa K.
      ,
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ,
      • Minami T.
      • Hara K.
      • Oshiro N.
      • Ueoku S.
      • Yoshino K.
      • Tokunaga C.
      • Shirai Y.
      • Saito N.
      • Gout I.
      • Yonezawa K.
      ), as well as ours, were carried out with transiently expressed S6K2 in HEK293 cells. Thus, no obvious explanation can be found for the discrepancy in rapamycin sensitivity of S6K2; subtle differences in cell culture and/or assay conditions may be responsible but are not assessable from the published information.
      The recent finding that PA mediates mitogenic activation of mTOR signaling to S6K1 and 4E-BP1 has uncovered a previously unexpected regulatory mode for mTOR (
      • Fang Y.
      • Vilella-Bach M.
      • Bachmann R.
      • Flanigan A.
      • Chen J.
      ). We now report that S6K2 is also regulated by PA as S6K2 was inhibited by a low concentration of butanol in serum-stimulated cells and activated by exogenous PA in serum-starved cells (Fig. 3). Although implicated by the effect of 1-butanol, the involvement of phospholipase D in S6K2 and S6K1 activation is yet to be definitively proven and is currently under investigation.
      The cytoplasmic-nuclear shuttling of mTOR, both the nuclear entry and subsequent nuclear exit, appears to be required for the activation of S6K1 and 4E-BP1 (
      • Kim J.E.
      • Chen J.
      ). The predominantly nuclear localization of S6K2 (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ) (Fig. 4B), as opposed to the cytoplasmic localization of S6K1 and 4E-BP1, might suggest a distinct requirement for mTOR localization. Increased mTOR nuclear import (NLS-mTOR) led to enhanced S6K2 activation, whereas increased mTOR nuclear export (NES-mTOR) resulted in reduced S6K2 activity (Fig. 5), which may simply reflect a correlation between nuclear mTOR and the activation of nuclear S6K2. However, S6K2 activation was inhibited by LMB (Fig.4A), suggesting that the nuclear export of an upstream component is required. Strong evidence for the critical role of mTOR shuttling came from the observations that while two copies of NLS tagged to mTOR further enhanced S6K2 activation in vivo, additional increase of nuclear entry by tagging four copies of NLS to mTOR reduced S6K2 activation (Fig. 7). Similar results were also obtained with S6K1 (data not shown). It is thus likely that a balanced distribution of mTOR between the cytoplasm and nucleus, or an optimal shuttling rate for mTOR, may be essential for maximal activation of downstream signaling. The fact that mTOR with two exogenous copies of NLS is most active does not necessarily suggest that nature has designed a suboptimal mTOR for downstream signaling. Since these experiments rely on overexpression of recombinant proteins, the stoichiometry of various components in the pathway may be different from that of the endogenous proteins. Nevertheless, the outcome of the multiple NLS tagging experiments has proven as a principle the importance of mTOR shuttling in activating downstream signaling.
      Lee-Fruman et al. (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ) reported that S6K2βII was in a detergent-soluble fraction, whereas S6K2βI stayed in the particulate fraction, suggesting that the two isoforms may be localized to different nuclear compartments. Interestingly, the localization of S6K2βII appears identical to that of S6K1αI (p85s6k) (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Reinhard C.
      • Fernandez A.
      • Lamb N.J.
      • Thomas G.
      ), the activation of which is also dependent on mTOR shuttling (data not shown). It would be intriguing to examine the regulation of S6K2βI in the context of mTOR localization. It remains a puzzle why activation of S6K2, a nuclear protein, requires the cytoplasmic-nuclear shuttling (and not just nuclear entry) of mTOR, a predominantly cytoplasmic protein. One simple possibility would be that upon activation of S6K2 in the nucleus mTOR is inactivated, and it is necessary for mTOR to be reactivated in the cytoplasm to allow maximal S6K2 activation. However, this hypothesis is not supported by the observation that nuclear entry of mTOR is much slower than the rate of full S6K2 activation in the cell: while S6K2 is maximally activated at 30 min (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ) (data not shown), mTOR nuclear entry, as indicated by sequestration by LMB, required more than 3 h to complete (Fig. 4C). It is not known whether activation of S6K2 occurs in the cytoplasm or nucleus or in both as a multistep process. Both PI3K and Akt, upstream regulators of S6K2 (
      • Lee-Fruman K.K.
      • Kuo C.J.
      • Lippincott J.
      • Terada N.
      • Blenis J.
      ,
      • Koh H.
      • Jee K.
      • Lee B.
      • Kim J.
      • Kim D.
      • Yun Y.H.
      • Kim J.W.
      • Choi H.S.
      • Chung J.
      ,
      • Martin K.A.
      • Schalm S.S.
      • Richardson C.
      • Romanelli A.
      • Keon K.L.
      • Blenis J.
      ), have been found to translocate into the nucleus upon stimulation (e.g. see Refs.
      • Neri L.M.
      • Milani D.
      • Bertolaso L.
      • Stroscio M.
      • Bertagnolo V.
      • Capitani S.
      and
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ), and it cannot be ruled out that S6K2 itself may also shuttle between the two compartments. Therefore, many possibilities exist for the activation process of S6K2. The regulation of mTOR is also a complex process; the relationship between PA binding (presumably in the intracellular membranes) and nuclear translocation of mTOR is currently unclear and awaits future investigations.

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