TI - Dual requirement for a newly identified phosphorylation site in p70s6k . AB - The activation of p70s6k 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 p70s6k activity , whereas similar mutations of S371 abolished kinase activity . Of these three sites and their surrounding motifs , only S371 is conserved in p70s6k 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 phosphorylation 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 p70s6k activity , indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity .