PHOSPHORYLATION SITES IN THE AUTOINHIBITORY DOMAIN PARTICIPATE IN P70(S6K) ACTIVATION LOOP PHOSPHORYLATION

Here we have employed p70(s6k) truncation and point mutants to elucida te the role played by the carboxyl-terminal autoinhibitory domain <(S/ T)under bar>P phosphorylation sites in kinase activation. Earlier stud ies showed that truncation of the p70(s6k) amino terminus severely imp aired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr(22 9) phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). I n this study, substitution of acidic residues for the four autoinhibit ory domain <(S/T)under bar>P sites mimics the carboxyl-terminal deleti on largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr(229) phosph orylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr(389) phosphorylatio n, because substitution of an acidic residue at this position in the p 70(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 aci dic <(S/T)under bar>P and Thr(389) substitutions is an excellent in vi tro substrate for the newly identified Thr(229) kinase, phosphoinositi de-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Duf ner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279 , 707-710), whereas phosphoinositide-dependent kinase-l poorly utilize s 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) phos phorylation through an intrasteric mechanism.