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.

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Available from: Bernd Bussian, Sep 28, 2015
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    • "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). "
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    Reviews of Physiology, Biochemistry and Pharmacology 01/2014; 166:43-95. DOI:10.1007/112_2013_17 · 6.27 Impact Factor
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    • "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. "
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    PLoS ONE 10/2013; 8(10):e78979. DOI:10.1371/journal.pone.0078979 · 3.23 Impact Factor
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    • "We found that the inhibition of IGF-1R by AG1024 had no effect on radiation-induced phosphorylation of Akt S473 (unpublished results). To determine whether IGF-1R signaling is upstream of mTOR activation following radiation exposure, we examined the effect of AG1024 on phosphorylation of S6 ribosomal protein (pS6), a known target of S6 kinase which is downstream of activated mTOR [48]. AG1024 treatment significantly reduced the phosphorylation of S6 ribosomal protein (Ser 235/236, Figure 5) suggesting that intact mTOR activity is required for radiation-induced IGF-1R-mediated accelerated senescence. "
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