Dual requirement for a newly identified phosphorylation site in p70(s6k)

The activation of p70(s6k) is associated with multiple phosphorylations at two sets of sites. The first set, S₄₁₁, S₄₁₈, T₄₂₁, and S₄₂₄, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T₂₂₉ (in the catalytic domain) and T₃₈₉ and S₄₀₄ (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T₃₆₇, S₃₇₁, and T₄₄₇, all of which have a recognition motif similar to that of the first set of sites. A mutation of T₃₆₇ or T₄₄₇ to either alanine or glutamic acid had no apparent effect on p70(s6k) activity, whereas similar mutations of S₃₇₁ abolished kinase activity. Of these three sites and their surrounding motifs, only S₃₇₁ is conserved in p70(s6k) homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S₃₇₁ phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S₃₇₁ phospholrylation is regulated by an external mechanism. Phosphopeptide analysis of S₃₇₁ mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T₃₈₉ phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T₃₈₉, which mimics phosphorylation at this site, did not rescue mutant p70(s6k) activity, indicating that S₃₇₁ phosphorylation plays an independent role in regulating intrinsic kinase activity.