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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.