Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) 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/TP 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 Thr229 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/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr229 phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr389 phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the S/TP mutations leads to a large increase in basal Thr229 phosphorylation and kinase activity. In contrast, an alanine substitution at Thr389 blocks both responses. Consistent with these data, we show that a mutant harboring the acidic S/TP and Thr389 substitutions is an excellent in vitro substrate for the newly identified Thr229 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-1 poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the S/TP sites, in cooperation with Thr389 phosphorylation, controls Thr229 phosphorylation through an intrasteric mechanism.