Article

Mitogen-activated 70 K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites

Friedrich Miescher Institute, Basel, Switzerland.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/1991; 266(33):22770-5.
Source: PubMed

ABSTRACT

Recently we purified and cloned the mitogen/oncogene-activated Mr 70,000 (70K) S6 kinase from the livers of rats treated with cycloheximide (Kozma, S. C., Lane, H. A., Ferrari, S., Luther, H., Siegmann, M., and Thomas, G. (1989) EMBO J. 8, 4125-4132; Kozma, S. C., Ferrari, S., Bassand, P., Siegmann, M., Totty, N., and Thomas, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7365-7369). Prior to determining the ability of this kinase to phosphorylate the same sites observed in S6 in vivo, we established the effects of different cations and autophosphorylation on kinase activity. The results show that the 70K S6 kinase is dependent on Mg2+ for activity and that this requirement cannot be substituted for by Mn2+. Furthermore, 50-fold lower concentrations of Mn2+ block the effect of Mg2+ on the kinase. This effect is not limited to Mn2+ but can be substituted for by a number of cations, with Zn2+ being the most potent inhibitor, IC50 approximately 2 microM. In the presence of optimum Mg2+ concentrations the enzyme incorporates an average of 1.2 mol of phosphate/mol of kinase and an average of 3.7 mol of phosphate/mol of S6. The autophosphorylation reaction appears to be intramolecular and leads to a 25% reduction in kinase activity toward S6. In the case of S6 all of the sites of phosphorylation are found to reside in a 19-amino acid peptide at the carboxyl end of the protein. Four of these sites have been identified as Ser235, Ser236, Ser240, and Ser244, equivalent to four of the five sites previously observed in vivo (Krieg, J., Hofsteenge, J., and Thomas, G. (1988) J. Biol. Chem. 263, 11473-11477). A fifth mole of phosphate is incorporated at low stoichiometry into the peptide, but the amino acid which is phosphorylated cannot be unequivocally assigned. The low level of phosphorylation of the fifth site in vitro is discussed with regard to known results and to a potential three-dimensional model for the carboxyl terminus of S6.

Download full-text

Full-text

Available from: Bernd Bussian
  • Source
    • "Moreover, the contribution of Protein Kinase A (PKA) and Protein Kinase C (PKC) in the control of rpS6 phosphorylation (Valovka et al., 2003; Valjent et al., 2011; Gangarossa and Valjent, 2012; Biever et al., 2015) is also unlikely since the phosphorylation of the AMPA subunit GluA1 at S845 and S831 sites, targeted by PKA and PKC respectively is unchanged following MES (Table 2). Combined with the fact that p70S6K is the only kinase able to trigger rpS6 phosphorylation at both S235/236 and S240/244 sites (Ferrari et al., 1991), these results strongly suggest that MES-induced rpS6 phosphorylation depends on p70S6K. By controlling the state of phosphorylation of 4E-BP1 and eIF4G, mTORC1 activity promotes cap-dependent mRNA translation (Wang et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Exacerbated hippocampal activity has been associated to critical modifications of the intracellular signaling pathways. We have investigated rapid hippocampal adaptive responses induced by maximal electroshock (MES) stimulation. Here, we demonstrate that abnormal and exacerbated hippocampal activity induced by MES triggers specific and temporally distinct patterns of phosphorylation of extracellular signal-related kinase (ERK), mTORC and Akt/GSK3 pathways in the mouse hippocampus. While the ERK pathway is transiently activated, the mTORC1 cascade follows a rapid inhibition followed by a transient activation. This rebound of mTORC1 activity leads to the selective phosphorylation of p70S6K, which is accompanied by an enhanced phosphorylation of the ribosomal subunit S6. In contrast, the Akt/GSK3 pathway is weakly altered. Finally, MES triggers a rapid upregulation of several plasticity-associated genes as a consequence exacerbated hippocampal activity. The results reported in the present study are reminiscent of the one observed in other models of generalized seizures, thus defining a common molecular footprint induced by intense and aberrant hippocampal activities.
    Full-text · Article · Nov 2015 · Neuroscience
  • Source
    • "As mentioned above, another target of p70 S6K1 with the potential to regulate protein synthesis and cell size is rpS6. rpS6 is predominantly phosphorylated on 5 residues in response to serum stimulation: S235, S236, S240, S244 and S247 (Krieg et al. 1988; Ferrari et al. 1991; Bandi et al. 1993). As the phosphorylation of these sites is almost completely inhibited by rapamycin, mTORC1-activated p70 S6K1 is considered the predominant kinase responsible for rpS6 phosphorylation (Blenis et al. 1991; Chung et al. 1992). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Skeletal muscle plays a fundamental role in mobility, disease prevention, and quality of life. Skeletal muscle mass is, in part, determined by the rates of protein synthesis, and mechanical loading is a major regulator of protein synthesis and skeletal muscle mass. The mammalian/mechanistic target of rapamycin (mTOR), found in the multi-protein complex, mTORC1, is proposed to play an essential role in the regulation of protein synthesis and skeletal muscle mass. The purpose of this review is to examine the function of mTORC1 in relation to protein synthesis and cell growth, the current evidence from rodent and human studies for the activation of mTORC1 signaling by different types of mechanical stimuli, whether mTORC1 signaling is necessary for changes in protein synthesis and skeletal muscle mass that occur in response to different types of mechanical stimuli, and the proposed molecular signaling mechanisms that may be responsible for the mechanical activation of mTORC1 signaling.
    Full-text · Article · Jan 2014 · Reviews of Physiology, Biochemistry and Pharmacology
  • Source
    • "The experiments described here have identified phosphorylation of ribosomal protein S6 at its S240 and S235 residues as specific readouts of mTOR hyperactivity vs. combined mTOR and MAPK hyperactivity, respectively. Early test tube studies using purified rat S6K1 and ribosomes concluded that S6K1 can phosphorylate both sites on S6 [47]. Subsequent studies in cells [48], [49] found that RSK is primarily responsible for S6(S235) phosphorylation and perusal of the data in these reports suggested to us that S235 phosphorylation is dependent upon or greatly enhanced by prior S240 phosphorylation by S6K1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5' untranslated regions (5'-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5'-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC.
    Full-text · Article · Oct 2013 · PLoS ONE
Show more