| The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth proliferation and survival. The mTOR pathway is deregulated in human diseases such as cancer and type 2 diabetes. Recnet years, mTOR signaling have attracted broad scientific and clinical interest.Kinase acitivity of mTOR depends on the complexes formation of mTOR by combining with other molecules in cells, mTOR complex 1 (mTORC1) and mTORC2. Macrolides rapamycin specifically inhibits the activity of mTORC1 by combining with intracellular protein FK506-binding-protein 12 (FKBP12). Activated phosphoinositide 3-kinase (PI3-K)/Akt (protein kinase B, PKB) induced by growth factor and insulin stimulation may phosphorylate tuberous sclerosis complex 2 (TSC2), which will attenuate inhibitive effect on mTORCl. Activated mTORC1 will directly phosphorylate its downstream substrates,40S ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Phosphorylation at Thr389 site of S6K1 results in its activation and may phosphorylate and activate 40S ribosomal protein S6, which will initiate translation of 5'-terminal oligopyrimidine tract mRNA (5'-Top mRNAs). Phosphorylated 4E-BP1 by mTOR can no longer bind eIF4E, thus triggers a 5'-cap dependent protein translation. Consequently, protein synthesis begins.S6Ks belongs to the AGC family of Ser/Thr kinases. Mammalian cells express two forms of the kinase, S6K1 and S6K2 (also known as S6Kαand S6Kβrespectively), which are encoded by two different genes and share a high level of overall sequence homology. S6K1 has two isoforms produced from the same transcript by alternative translational start sites. The shorter form, which is largely cytoplasmic, was initially termed p70 S6K1, whereas the larger form, which appears to be exclusively nuclear, was referred to as p85 S6K1. In the case of p85 S6K1, the additional 23-amino-acid sequence residing at its amino terminus has been shown to function as a nuclear localization signal (NLS).S6K1 is the best characterized effector of mTOR, and its regulation serves as a model for mTOR signaling. A full and sustained S6K1 activation requires multiple growth factor-induced phosphorylation events. The phosphorylation of Thr389 by mTORC1 is particularly important because subsititution of the residue with alanine blocks the activation of the kinase domain. There is also evidence indicating that p70 S6K1 (T389) is directly phoshorylated by mTORC1 in vitro and rapamycin treatment of cells triggers the rapid dephosphorylation of Thr389 and inactivation of S6K1.Recent studies have addressed many important roles of S6Ks in cell size control, tumor invasiveness, motility and angiogenesis, insulin resistance and lifespan. Regulation of S6Ks by upstream signals and mechanisms through which S6Ks regulate cellular process, however, remains to be clarified. For lack of S6K1-or S6K-2-specific inhibitors which is useful in defining the effects of acute inhibition of S6Ks, most results for S6Ks functions come from inhibition of mTORC1 by rapamycin. But recent studies have addressed activation of S6K1 in rapamycin-resistant and mTORC1-independent manner. Moreover, mechanisms involved in rapamycin-resistance in rapamycin-based cancer therapy remain unclear. Defining mTORC1-independent mechanisms that regulate S6Ks is important for understanding the functions of S6Ks and its roles in human diseases.Findings to date would suggest that two isoforms of S6K1, p70 S6Kland p85 S6K1 are controlled in a similar manner. Most studies have been focused on cytoplasmic isoform p70 S6K1. Functions and regulation of nuclear isoform p85 S6K1 have been neglected and are poorly understood.TNF-αand reactive oxygen species (ROS) play important role in carcinogenesis and tumor progression. Previous studies have demonstrated that TNF-αand ROS are important upstream signals of mTORC1 and p70 S6K1. But their roles in regulation of p85 S6K1 have not been reported.In this study, we found novel and differential regulation and function of p70 and p85 S6K1. We demonstrated for the first time that p85 but not p70 S6K1 was activated by TNF-αand H2O2 through mTOR-independent, I (?)B-kinase (IKK)-dependent mechanism.1 Construction of mTOR shRNA and p70 S6K1, p85 S6K1 expression vectors and preparation of Glutathione-S-Transferase (GST)-S6 protein(1) N-terminal (69 bp,23 amino acids) of p85 S6K1 was added to rat p70-S6K1 cDNA (pRK7-p70 S6K1, from Addgene) by PCR, and both cDNAs of p70S6K1 and p85 S6K1 were subcloned into pcDNA3.1-Flag eukaryotic expression vectors, namely pcDNA3.1-Flag-p70 S6K1, pcDNA3.1-Flag-p85 S6K1. Notably, to avoid expression of p70 S6K1 in p85 S6K1 expression vector, the p70 translation initiate site (70-72th bp, ATG) was changed into TTG.(2) Human 40S ribosomal protein S6 cDNA was obtained from HEK293 cells by RT-PCR and was subcloned into prokaryotic expression vector pGEX-6P-1 (pGEX-6P-1-S6). GST-S6 protein was induced by isopropylβ-D-1-thiogalactopyranoside (IPTG) and purified using GST-agarose beads. Purified GST-S6 protein will be used as substrate for S6K1 in vitro kinase assay. (3) Recombinant mTOR shRNA expression vector (psiLV-H1-mTOR) was constructed and transfected into human breast cancer cell line MCF-7. Endogenous mTOR expression was significantly down-regulated after 72-96 h.2 mTOR-independent, IKK-dependent activation of p85 but not p70 S6K1 by tumor necrosis factor-α(TNF-α) and hydrogen peroxide (H2O2).(1) Different reaction of p70 S6K1 and p85 S6K1 to H2O2 and TNF-α.It has been demonstrated that phosphorylation of S6K1 (T389/412) and S6 (S235/236) stimulated by extracellular and intracellular signals is rapamycin-sensitive. We found that phosphorylation of p70 S6K1 (T389), p85 S6K1 (T412) and S6 (S235/236) stimulated by insulin and amino acid were blocked completely by 100 nM rapamycin in MCF-7 cells. When stimulated by H2O2 and TNF-α, however, p70 S6K1 (T389) but not p85 S6K1 (T412) was inhibited by rapamycin. Coordinate with the rapamycin-insensitive activation of p85 S6K1 (T412), phoshorylation of S6 (S235/236) was also elevated by H2O2 and TNF-αin the presence of rapamycin. Moreover, H2O2 stimulated phosphorylation of p85 S6K1 (T412) but inhibited p70 S6K1 (T389) in a variety of cells (MG63, osteoblasts, MCF-7 and HCT116). Taken together, it is suggested that p70 S6K1 and p85 S6K1 may react differently to some signals such as H2O2 and TNF-α.(2) Rapamycin and amino acid-independent phosphorylation of p85 S6K1 (T412) but not p70 S6K1 (T389) by H2O2.Previous studies suggest that amino acid-deprivation and rapamycin prevent mTORC1 activation by vairous upstream signals. In this study, we found that in MCF-7, HeLa and HCT116 cells, H2O2 could induce endogenous p85 S6K1 (T412) but not p70 S6K1 (T389) phosphorylation in the presence rapamycin or deprivation of amino acid. Moreover, overexpressed p85 S6K1 but not p70 S6K1 was also phosphorylated on Thr412 by H2O2 in rapamycin-insensitive manner.(3) Rapamycin-insensitive activation of p85 S6K1 by H2O2 and TNF-αis responsible for rapamycin-independent S6 (S235/236) phosphorylation.Phosphorylation of S6 on S235/236 is critical for its activation. We found that H2O2 and TNF-αstimulated phosphorylation of S6 (S235/236) in the presence of rapamycin in MCF-7 and HCT116 cells. Furthermore, overexpression of p85 S6K1 but not p70 S6K1 enhanced rapamycin-independent phosphorylation S6 (S235/236). Most importantly, in vitro kinase assay clearly showed that immuno-purified Flag-p85 S6K1 but not Flag-p70 S6K1 was responsible for H2O2 stimulated rapamycin-independent phosphorylation of S6 (S235/236). It is suggested that p85 S6K1 but not p70 S6K1 is responsible for rapamycin-independent activation of S6.(4) mTOR (mTORC1 and mTORC2) is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.Knockdown of endogenous mTOR expression by shRNA deceased constitutive phosphorylation of p70/p85 S6K1 (T389/412) and S6 (S235/236), but was unable to prevent H2O2 induced p85 S6K1 (T412) and S6 (S235/236) phosphorylation.(5) TSC2 is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.It has been shown that IKK-βactivated mTORC1 and S6K1 by phosphorylation and inhibition mTORC1 negative regulator TSC1/TSC2. It was found in this study that H2O2 induced p85 S6K1 (T412) but not p70 S6K1 (T389) phosphorylation both in TSC2+/+P53-/- mouse embryonic fibroblast (MEF) and in TSC2-/- P53-/-MEFs either in the presence or absence of rapamycin. This suggests that TSC2 is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.(6) IKK is required for mTOR-independent activation of p85 S6K1 and S6 by H2O2.To detect upstream signals involved in mTOR-independent activation of p85 S6K1 and S6, PI3-K and IKK specific inhibitors were used. It is revealed that IKK inhibitor, but not PI3-K and mTOR inhibitor blocks H2O2-induced p85 S6K1 (T412) and S6 (S235/236) phosphorylation. Further studies demonstrated that overexpressed myc-IKK-βassociated with Flag-p85 S6K1 and this association was enhanced by H2O2 treatment. These results demonstrate that IKK is required for mTOR-independent activation of p85 S6K1 and S6 by H2O2.In a summary, our study reveals a novel regulatory mechanism about S6K1. H2O2 and TNF-αactivate p85 but not p70 S6K1 by mTOR-independent, IKK-dependent pathway, which is responsible for rapamycin-insnesitive and mTORC1-independnet phosphorylation of S6 (S235/236). The finding will be helpful for us to understand the functions of p85 S6K1 and mechanisms involved in rapamycin-resistance in rapamycin-based cancer therapy.
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