Phosphorylation Sites in the Autoinhibitory Domain Participate in p70s6k Activation Loop Phosphorylation

doi:10.1074/jbc.273.24.14845

Phosphorylation Sites in the Autoinhibitory Domain Participate in p70^s6k Activation Loop Phosphorylation*

From the Friedrich Miescher-Institut, Department of Growth Control, P. O. Box 2543, CH-4002, Basel, Switzerland

Abstract

Here we have employed p70^s6ktruncation 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 domainS/TP sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr²²⁹phosphorylation, 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 acidicS/TP and Thr³⁸⁹ substitutions is an excellentin 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.

Footnotes

↵* This work was supported in part by a grant from the Human Frontier Science Program Organization (to G. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Recipients of long term postdoctoral fellowships from the Human Frontier Science Program Organization.
↵§ Present address: Peter MacCallum Cancer Institute, Locked Bag No. 1, A′ Beckett St., Melbourne, Victoria 3000, Australia.
↵¶ To whom correspondence should be addressed. Tel.: 41-61-697-3012; Fax: 41-61-697-6681.
↵1 Y. Chen, C. D. Hoemann, G. Thomas, and S. C. Kozma, submitted for publication.
↵2 The abbreviations used are: PDK1, phosphoinositide-dependent kinase-1; FCS, fetal calf serum.
↵3 P. B. Dennis and G. Thomas, unpublished results.
↵4 P. B. Dennis, N. Pullen, R. B. Pearson, S. C. Kozma, and G. Thomas, unpublished data.
- Received January 22, 1998.
- Revision received March 16, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.