Phosphorylation sites in the autoinhibitory domain participate in p70s6k activation loop phosphorylation

Here we have employed p70(s6k) truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain <(S/T)under bar>P phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70(s6k) amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr(229) phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain <(S/T)under bar>P sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr(229) phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr(389) phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the <(S/T)under bar>P mutations leads to a large increase in basal Thr(229) phosphorylation and kinase activity. In contrast, an alanine substitution at Thr(389) blocks both responses. Consistent with these data, we, show that a mutant harboring the acidic <(S/T)under bar>P and Thr(389) substitutions is an excellent in vitro substrate for the newly identified Thr(229) kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-l poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the <(S/T)under bar>P sites, in cooperation with Thr(389) phosphorylation, controls Thr(229) phosphorylation through an intrasteric mechanism.