THE PRINCIPAL RAPAMYCIN-SENSITIVE P70(S6K) PHOSPHORYLATION SITES, T-229 AND T-389, ARE DIFFERENTIALLY REGULATED BY RAPAMYCIN-INSENSITIVE KINASE KINASES

Mitogen-induced activation of p70(s6k) is associated with the phosphor ylation of specific sites which are negatively affected by the immunos uppressant rapamycin, the fungal metabolite wortmannin, and the methyl xanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with t he observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortma nnin. Mere we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflec ted in the phosphorylation state of the enzyme, Mutation of the princi pal rapamycin-targeted phosphorylation site, T-389, to an acidic resid ue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observa tion that T-389 was a common target of all three inhibitors, Truncatio n of the first 54 residues of the amino terminus blocks the serum-indu ced phosphorylation of three rapamycin-sensitive sites, T-229 in the a ctivation loon and T-389 and S-404 in the linker region, This correlat es with a severe reduction in the ability of the kinase to be activate d by serum. However, loss of mitogen activation conferred by the remov al of the amino terminus is reversed by additional truncation of the c arboxy-terminal domain, with the resulting mutant demonstrating phosph orylation of the remaining two rapamycin-sensitive sites, T-229 and T3 89. In this double-truncation mutant, phosphorylation of T-229 occurs in tile basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation, The phosphorylation of b oth sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites a re not inhibited by the macrolide, In contrast, activation of the doub le-truncation mutant is blocked in the presence of wortmannin or SQ200 06, and these agents completely block the phosphorylation of T-389 whi le having only a marginal effect on T-229 phosphorylation, Wen the T-3 89 site is mutated to an acidic residue in the double-truncation backg round, the activation of the resulting mutant is insensitive to the wo rtmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of ra pamycin. These data are consistent with the hypothesis that T-359 is t he principal regulatory phosphorylation site, which, in combination wi th hyperphosphorylation of the autoinhibitory domain S/TP sites, is ac utely regulated by external effectors, whereas T-229 phosphorylation i s regulated primarily by internal mechanisms.