Pb. Dennis et al., PHOSPHORYLATION SITES IN THE AUTOINHIBITORY DOMAIN PARTICIPATE IN P70(S6K) ACTIVATION LOOP PHOSPHORYLATION, The Journal of biological chemistry, 273(24), 1998, pp. 14845-14852
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.