DUAL REQUIREMENT FOR A NEWLY IDENTIFIED PHOSPHORYLATION SITE IN P70(S6K)

The activation of p70(s6k) is associated with multiple phosphorylation s at two sets of sites. The first set, S-411, S-418, T-421, and S-424, reside within the autoinhibitory domain, and each contains a hydropho bic residue at -2 and a proline at +1. The second set of sites, T-229 (in the catalytic domain) and T-389 and S-404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues, Were w e describe the identification and mutational analysis of three new pho sphorylation sites, T-367, S-371, and T-447, all of which have a recog nition motif similar to that of the first set of sites, A mutation of T-367 or T-447 to either alanine or glutamic acid had no apparent effe ct on p70(s6k) activity, whereas similar mutations of S-371 abolished kinase activity. Of these three sites and their surrounding motifs, on ly S-371 is conserved in p70(s6k) homologs from Drosophila melanogaste r, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S -371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by au tophosphorylation, S-371 phosphorylation is regulated by an external m echanism, Phosphopeptide analysis of S-371 mutants further revealed th at the loss of activity in these variants was paralleled by a block in serum-induced T-389 phosphorylation, a phosphorylation sire previousl y shown to be essential for kinase activity. Nevertheless, the substit ution of an acidic residue at T-389, which mimics phosphorylation at t his site, did not rescue mutant p70(s6k) activity, indicating that S-3 71 phosphorylation plays an independent role in regulating intrinsic k inase activity.