RAS/ERK Signaling Promotes Site-specific Ribosomal Protein S6 Phosphorylation via RSK and Stimulates Cap-dependent Translation

Abstract

Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser235/236 in vitro and in vivo using an mTOR-independent mechanism. Mutation of rpS6 at Ser235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTOR-independent pathway linking the Ras/ERK signaling cascade to the translational machinery. Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser235/236 in vitro and in vivo using an mTOR-independent mechanism. Mutation of rpS6 at Ser235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTOR-independent pathway linking the Ras/ERK signaling cascade to the translational machinery. In eukaryotic cells, the main rate-limiting step of translation is initiation, which is controlled by an array of proteins that respond to signaling cascades activated by extracellular signals (reviewed in Refs. 1Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 2Clemens M.J. Oncogene. 2004; 23: 3180-3188Crossref PubMed Scopus (187) Google Scholar, 3Richter J.D. Sonenberg N. Nature. 2005; 433: 477-480Crossref PubMed Scopus (745) Google Scholar). The mammalian target of rapamycin, mTOR, 4The abbreviations used are: mTOR, mammalian target of rapamycin; RSK, p90 ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; S6K1, ribosomal S6 kinase 1; 4E-BP, 4E-binding proteins; rpS6, ribosomal protein S6; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; siRNA, short interfering RNA; PMA, phorbol 12-myristate 13-acetate; fmk, fluoromethylketone; GST, glutathione S-transferase; wt, wild type; EGF, epidermal growth factor; HA, hemagglutinin. 4The abbreviations used are: mTOR, mammalian target of rapamycin; RSK, p90 ribosomal S6 kinase; ERK, extracellular signal-regulated kinase; S6K1, ribosomal S6 kinase 1; 4E-BP, 4E-binding proteins; rpS6, ribosomal protein S6; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; siRNA, short interfering RNA; PMA, phorbol 12-myristate 13-acetate; fmk, fluoromethylketone; GST, glutathione S-transferase; wt, wild type; EGF, epidermal growth factor; HA, hemagglutinin. is a conserved serine/threonine kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate mammalian cell growth (reviewed in Refs. 4Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3377) Google Scholar, 5Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1036) Google Scholar, 6Richardson C.J. Schalm S.S. Blenis J. Semin. Cell Dev. Biol. 2004; 15: 147-159Crossref PubMed Scopus (115) Google Scholar, 7Gingras A.C. Raught B. Sonenberg N. Curr. Top Microbiol. Immunol. 2004; 279: 169-197Crossref PubMed Google Scholar, 8Averous J. Proud C.G. Oncogene. 2006; 25: 6423-6435Crossref PubMed Scopus (164) Google Scholar). Under conditions of nutrient and energy sufficiency and insulin or mitogen stimulation, mTOR stimulates two important translational regulators, the ribosomal S6 kinases (S6K1 and S6K2) and the eukaryotic initiation factor 4E (eIF4E). eIF4E is crucial for ribosome recruitment as it binds to the 7-methylguanosine cap structure (m7GpppN, where N is any nucleotide) at the 5′-end of nearly all transcribed mRNAs to initiate cap-dependent translation (reviewed in Ref. 7Gingras A.C. Raught B. Sonenberg N. Curr. Top Microbiol. Immunol. 2004; 279: 169-197Crossref PubMed Google Scholar). When mTOR is active, eIF4E nucleates the assembly of the translation preinitiation complex through recruitment of numerous initiation factors, resulting in association of the ribosomal subunits to the mRNA. S6K1 and S6K2 are serine/threonine kinases directly stimulated by mTOR which in turn, phosphorylate substrates involved in cell and body size (5Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1036) Google Scholar, 6Richardson C.J. Schalm S.S. Blenis J. Semin. Cell Dev. Biol. 2004; 15: 147-159Crossref PubMed Scopus (115) Google Scholar). S6K1 phosphorylates several substrates located in the cytoplasm and the nucleus, including the ribosomal protein (rp) S6 (reviewed in Ref. 9Ruvinsky I. Meyuhas O. Trends Biochem. Sci. 2006; 31: 342-348Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). Ribosomal protein S6 is one of 33 proteins that comprise the 40 S ribosomal subunit and represents the most extensively studied substrate of S6K1 (10Thomas G. Siegmann M. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3952-3956Crossref PubMed Scopus (107) Google Scholar). Because the initial discovery that liver-derived rpS6 was phosphorylated (11Gressner A.M. Wool I.G. J. Biol. Chem. 1974; 249: 6917-6925Abstract Full Text PDF PubMed Google Scholar), mitogenic stimulation of cells was found to correlate with phosphorylation of rpS6 on serines, which suggested that rpS6 may control mRNA translation in dividing cells (12Bandi H.R. Ferrari S. Krieg J. Meyer H.E. Thomas G. J. Biol. Chem. 1993; 268: 4530-4533Abstract Full Text PDF PubMed Google Scholar). rpS6 phosphorylation sites have been mapped to five clustered residues that are conserved in metazoans, consisting of Ser235, Ser236, Ser240, Ser244, and Ser247, located at the C-terminal part of the protein (13Krieg J. Hofsteenge J. Thomas G. J. Biol. Chem. 1988; 263: 11473-11477Abstract Full Text PDF PubMed Google Scholar). Two classes of protein kinases were found to phosphorylate rpS6 in vitro, the S6K1/2 and the p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases (reviewed in Refs. 14Roux P.P. Blenis J. Microbiol. Mol. Biol. Rev. 2004; 68: 320-344Crossref PubMed Scopus (1880) Google Scholar and 15Hauge C. Frodin M. J. Cell Sci. 2006; 119: 3021-3023Crossref PubMed Scopus (151) Google Scholar). Subsequent studies determined that rpS6 phosphorylation was largely sensitive to the mTOR inhibitor rapamycin, indicating that S6K1/2 were the main physiological rpS6 kinases operating in somatic cells (16Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1012) Google Scholar, 17Blenis J. Chung J. Erikson E. Alcorta D.A. Erikson R.L. Cell Growth Differ. 1991; 2: 279-285PubMed Google Scholar, 18Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (117) Google Scholar). The RSK family members, in contrast, are not affected by rapamycin as they are activated via the classical mitogen-activated protein kinase (MAPK) signaling pathway. The contribution of S6K1/2 in rpS6 phosphorylation was recently addressed using S6K1/S6K2 double knock-out animals, which were found to display no phosphorylation of rpS6 at Ser240/244, but persistent phosphorylation at Ser235/236 (19Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar). Phosphorylation of Ser235/236 was found to require extracellular signal-regulated kinase (ERK) signaling, suggesting that RSK or other kinases downstream of ERK, such as the mitogen- and stress-activated kinases (MSK1/2), contribute to rpS6 phosphorylation upon mitogen stimulation. The functional importance of rpS6 in animals was underscored by conditional ablation of rpS6 in the liver (20Volarevic S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (312) Google Scholar). In these mice, hepatocytes failed to proliferate after partial hepatectomy due to a blockage in ribosome biogenesis and cell cycle progression. In vivo and in vitro studies have suggested that rpS6 phosphorylation exerts an effect on translation at the level of mRNA binding; initial chemical protection studies and cross-linking experiments localized rpS6 to the mRNA/tRNA binding site junction between the small and large ribosomal subunits (21Nygard O. Nilsson L. J. Biol. Chem. 1990; 265: 6030-6034Abstract Full Text PDF PubMed Google Scholar). Consistent with this finding, highly phosphorylated ribosomes were found to bind and utilize both synthetic and natural mRNA more efficiently in vitro than unphosphorylated counterparts (22Burkhard S.J. Traugh J.A. J. Biol. Chem. 1983; 258: 14003-14008Abstract Full Text PDF PubMed Google Scholar). More recently, the role of rpS6 phosphorylation was addressed through the generation of viable and fertile knock-in mice containing alanine substitutions of all five phosphorylatable serine residues in rpS6 (rpS6P–/–) (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google Scholar). These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose an of protein synthesis and cell from these animals are than the size of was not upon rapamycin that rpS6 is a mTOR regulating cell size (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google Scholar). is in to data from S6K1 and S6K2 which that S6K2 rpS6 phosphorylation are involved in the growth of or cells (19Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar, S. Stewart M.J. Ledermann B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (312) Google Scholar). it rpS6 phosphorylation a role in cell In the of all regulating rpS6 and this regulated to protein In this we demonstrate that agonists of the signaling pathway rpS6 phosphorylation using an mTOR-independent pathway that RSK We found that activation of all RSK stimulates cap-dependent indicating that RSK provides an and linking the signaling pathway to the of translation initiation. of phosphorylation of rpS6 that Ras/ERK signaling promotes translation initiation by assembly of the preinitiation complex. S6K1, and were P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). rpS6 was in with a The was by and been M. C. F. Mol. Cell Biol. PubMed Scopus Google Scholar). The was from and in with a protein was P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar). The used in this were using the Cell and and cells were in with and and using P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google or the were for and of where for cells were with rapamycin or a inhibitor C. J. Science. 2005; PubMed Scopus Google Scholar), and stimulated with insulin epidermal growth factor or the small interfering with two were from The which for S6K1, and control were were P.P. Blenis J. Curr. Biol. 2005; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, P.P. Mieulet V. Cohen Raught B. J. Blenis J. M. Sonenberg N. J. 2006; 25: PubMed Scopus Google Scholar). cells were using and was determined to than using a cells were for and stimulated with or The inhibitor was as C. J. Science. 2005; PubMed Scopus Google Scholar). and were using 1 1 1 of of as P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar). were with the for by with protein or protein for 1 cap binding were with for were in and with cell were to and as P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar, P.P. Blenis J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). were from were by of was A. A. Blenis J. Mol. Cell Biol. 1999; 19: PubMed Google Scholar). were by were from Cell with the of the RSK which was from and were P.P. Blenis J. Mol. Cell Biol. 23: PubMed Scopus Google Scholar). and were from and from were in and in kinase were with as substrate and all were to of or was determined by or using a with or cells were with a M. C. F. Mol. Cell Biol. PubMed Scopus Google Scholar), which cap-dependent translation of the and translation of the was with the and were using a and a or were in and are as from the was to the from the ribosome stimulation of was to the were in with and in and were on for and at for at The resulting was on a and in a at for at the was and using a to a Ras/ERK rpS6 Phosphorylation on Ser235/236 an mTOR-independent but mTOR-independent pathways to rpS6 cells were stimulated with a Phosphorylation of rpS6 was using two that rpS6 phosphorylated on Ser235/236 or We of stimulation, rpS6 is phosphorylated at all sites phosphorylation at of stimulation The of rpS6 phosphorylation was between the sites with phosphorylation of Ser235/236 with than Ser240/244, suggesting that phosphorylation of these sites is regulated by signaling When cells were with rapamycin, which S6K1 and phosphorylation at its not rpS6 phosphorylation at Ser235/236 phosphorylated in cells with rapamycin, indicating the of an mTOR-independent pathway to rpS6 phosphorylation at these the signaling pathway was to Ser235/236 cells were with the inhibitor stimulation with phosphorylation of was not affected by at a that efficiently phosphorylation and RSK we found that Ser235/236 phosphorylation was by the in a of the of Ser235/236 phosphorylation suggesting a between signaling and the initial of rpS6 phosphorylation upon stimulation. rapamycin and were rpS6 phosphorylation was indicating that Ser235/236 phosphorylation through and phosphorylation is regulated via an mechanism. the role of signaling in rpS6 we the of rpS6 phosphorylation in cells with of the Ras/ERK signaling cascade. of cells with the phorbol PMA, which stimulates Ras/ERK but not signaling in cells, in a in and RSK and rpS6 phosphorylation Ser235/236 phosphorylation was by and by rapamycin, the more important contribution by the signaling cascade these we determined oncogenic Ras rpS6 phosphorylation at We found that oncogenic Ras but not a rpS6 phosphorylation at Ser235/236 stimulation was by indicating that signaling is required for rpS6 phosphorylation mTOR-independent Growth and Ribosomal S6 Phosphorylation via data that a kinase activated by Ras/ERK signaling regulates rpS6 phosphorylation at and the kinases of the RSK and the of RSK in rpS6 we used and the RSK in and of S6K1 rpS6 phosphorylation at all sites upon stimulation we found that of or rpS6 phosphorylation at and to a and was both RSK were indicating that and are involved in rpS6 phosphorylation upon and growth factor stimulation. inhibitor was recently and as an kinase inhibitor of RSK C. J. Science. 2005; PubMed Scopus Google Scholar). We the of this inhibitor and found that of cells and by while S6K1 and not to the data using of RSK using rpS6 phosphorylation at but not phosphorylation RSK was more at of stimulation which with the of RSK and as by phosphorylation at and These the RSK in the of rpS6 phosphorylation at as suggested by the of the inhibitor RSK is required for rpS6 phosphorylation by other mitogenic cells were with stimulation with serum, PMA, EGF, or insulin of RSK rpS6 phosphorylation at Ser235/236 in to all Ras/ERK pathway Consistent with the for Ras/ERK signaling, with no effect on rpS6 phosphorylation by insulin, which is not a of RSK in these cells P.P. Blenis J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and S6K1 rpS6 in and in with phosphorylation sites on rpS6 are located a highly C-terminal of the protein The of residues at the and and these phosphorylation for both RSK family and S6K1 Cohen Cohen P. PubMed Scopus Google Scholar). and are not by such residues indicating that they are not RSK or S6K1 phosphorylation the of and S6K1 rpS6, of these kinases were to in vitro kinase with a protein containing the C-terminal of rpS6 as of kinase were as and S6K1 were to phosphorylate rpS6 on Ser235/236 as with we found that S6K1, but not or was of rpS6 on while and stimulated in to levels by S6K1, of this was found to These that and have a for residues at the and and that the RSK not directly phosphorylate in the kinase of RSK rpS6 in cells were with wt, and kinase of cells were for and for rpS6 phosphorylation at Ser235/236 and of or a not rpS6 phosphorylation in of rpS6 phosphorylation at The level of Ser235/236 phosphorylation was to that in cells, but phosphorylation at was not by We have found that RSK modulate mTOR signaling through the phosphorylation and of P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). we found that activated rpS6 phosphorylation was to rapamycin indicating that RSK regulates rpS6 phosphorylation using mTOR-independent activated stimulated rpS6 phosphorylation cells were with or rapamycin for and for rpS6 phosphorylation We have that activation of is to these Blenis J. Mol. Cell Biol. PubMed Scopus Google and as rpS6 phosphorylation stimulated by was not affected by of mTOR, or These data that the RSK directly target the translational by promoting rpS6 phosphorylation at Ser235/236 of mTOR Phosphorylation of rpS6 to the the function of rpS6 phosphorylation at we rpS6 with alanine or substitutions at and These were in cells and cell were for and rpS6 phosphorylation the while rpS6 phosphorylation was stimulated by phosphorylation of Ser235/236 was in the and phosphorylation of rpS6 its recruitment to the mRNA cap complex, we the of the rpS6 to bind 7-methylguanosine cap We found both in the and of serum, binding of the rpS6 was impaired with protein binding of the rpS6 to cap was more than protein in the of serum, indicating that phosphorylation of Ser235/236 promotes rpS6 binding to the 7-methylguanosine cap complex. Under conditions where rpS6 is phosphorylated in to serum, both and the rpS6 were found to to cap with we determined signaling agonists require phosphorylation of Ser235/236 to rpS6 to the 7-methylguanosine complex. rpS6 was to the 7-methylguanosine cap complex in a we found that of Ser235/236 to alanine residues impaired binding of rpS6 to cap Phosphorylation of Ser235/236 to important for recruitment of rpS6, as the of rpS6 to the mRNA cap complex in the of stimulation. These that rpS6 phosphorylation at Ser235/236 rpS6 recruitment to the mRNA complex, indicating that phosphorylation of rpS6 may assembly of the translation initiation complex. The RSK and the RSK are required for translation initiation, we used a that the between cap-dependent and translation initiation M. C. F. Mol. Cell Biol. PubMed Scopus Google Scholar, Blenis J. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). this we that and both cap-dependent translation and which was by rapamycin of the of eIF4E function RSK is required for cap-dependent or cells were in and for with of the inhibitor Under conditions that RSK we found that cap-dependent translation was in a with to a in translation initiation level was to the effect of rapamycin suggesting that RSK is important for translation initiation. this we the effect of RSK on cap-dependent in of or cap-dependent translation or of cap-dependent that kinase is to cap-dependent all RSK stimulated cap-dependent were in the we found that all RSK stimulated cap-dependent translation in cells translation initiation was found to sensitive to rapamycin, which is with the that mTOR is required for eIF4E and of the translation initiation complex. these data demonstrate that RSK to cap-dependent directly the for RSK in ribosomal recruitment to we the of ribosomes from cells with the RSK The at was the resulting to which two by the of and in stimulation in with cells that were of both rapamycin and for 1 the level of by stimulation indicating that mTOR and RSK are important to these data that RSK provides a between the Ras/ERK signaling cascade and the translational by promoting recruitment of rpS6 and ribosomal subunits to the translation preinitiation complex We have an important between the Ras/ERK signaling cascade and the translational which of the mechanisms by which of this cascade protein We found that serum, growth factors, oncogenic and phorbol rpS6 phosphorylation at Ser235/236 using an mTOR-independent pathway that RSK Phosphorylation of Ser235/236 was found to regulate the of rpS6 for the 7-methylguanosine cap complex, indicating that RSK signaling to the assembly of the translation initiation complex. We that all RSK cap-dependent translation in cells to and phorbol and that RSK was required for recruitment of ribosomes to indicating that RSK family are of translation initiation. the RSK of Ras signaling, and the of mTOR and signaling, modulate protein was in as a serine kinase that phosphorylated ribosomal protein S6 in vitro E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). S6K1 and S6K2 were found to the rpS6 kinases operating in somatic cells (16Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1012) Google Scholar, H. Thomas G. Nature. 1991; PubMed Scopus Google Scholar), RSK family were no to involved in rpS6 phosphorylation and translation initiation. in that S6K1 and S6K2 were the rpS6 but that a kinase phosphorylated rpS6 at Ser235/236 (19Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar). Consistent with these we found that all RSK phosphorylated rpS6 at Ser235/236 but not in vitro and in with the that S6K1/2 and the RSK on rpS6 to modulate mRNA We have that phosphorylate and the tumor a of mTOR signaling P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). was found to regulate L. H. P. P.P. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), indicating that Ras/ERK signaling modulate rpS6 phosphorylation through the of mTOR Here, we that rapamycin not rpS6 phosphorylation at indicating that RSK rpS6 phosphorylation using and by Meyuhas and (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google a knock-in (rpS6P–/–) with alanine substitutions of all five phosphorylatable the of These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose indicating that rpS6 phosphorylation is required for the synthesis or function of proteins that (23Ruvinsky I. Sharon N. Lerer T. Cohen H. Stolovich-Rain M. Nir T. Dor Y. Zisman P. Meyuhas O. Genes Dev. 2005; 19: 2199-2211Crossref PubMed Scopus (458) Google Scholar). association and protein synthesis were found to in and from these mice, indicating that rpS6 phosphorylation regulates mRNA The of protein synthesis and were found to in but this from in protein to In are than wild indicating that rpS6 phosphorylation to the of cell size via an mechanism. on rpS6 phosphorylation to regulated by at two of the RSK and We found that the RSK phosphorylate Ser235/236 but not Ser240/244, suggesting that the sites of rpS6 phosphorylation may molecular The of the may the of a of molecular by phosphorylation of We that rpS6 is to the 7-methylguanosine cap binding complex more efficiently in cells with activated Ras/ERK signaling, suggesting a by which phosphorylation of rpS6 by RSK proteins promotes its recruitment to the translation preinitiation complex. These are in with that phosphorylated ribosomes with both synthetic and natural mRNA more efficiently than unphosphorylated counterparts (22Burkhard S.J. Traugh J.A. J. Biol. Chem. 1983; 258: 14003-14008Abstract Full Text PDF PubMed Google Scholar). that RSK is important for and a for the between the and Ras/ERK signaling cascades in translation initiation. Because these pathways have activation may provide cells with a for of molecular between these pathways was to contribute to by the recruitment of mRNAs to ribosomes A. M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the mRNAs most affected are proteins that regulate growth and suggesting a by which Ras/ERK and signaling to by the of mRNAs with of both pathways in mRNA translation been in of A. S. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), where of and mTOR signaling was found to translation initiation as well as phosphorylation of important regarding the of rpS6 as for activation of the pathway from tumor as in phosphorylation of these sites may regulated by signaling, are for in with activated Ras or where phosphorylated Ser235/236 may regulated by data to the the of signaling In the may on the of such as or to activation and rpS6 to In the of rapamycin in may in the of due to the by the pathway. Under these in this pathway of RSK in cell and P.P. Blenis J. Microbiol. Mol. Biol. Rev. 2004; 68: 320-344Crossref PubMed Scopus (1880) Google Scholar), RSK may to in the of We for of the