부산대학교 도서관

The activation of p70s6kis 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 T389and 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 T367or T447to either alanine or glutamic acid had no apparent effect on p70s6kactivity, whereas similar mutations of S371abolished kinase activity. Of these three sites and their surrounding motifs, only S371is conserved in p70s6khomologs from Drosophila melanogaster, Arabidopsis thaliana,and Saccharomyces cerevisiae,as well as many members of the protein kinase C family. Serum stimulation increased S371phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389phosphorylation, 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 p70s6kactivity, indicating that S371phosphorylation plays an independent role in regulating intrinsic kinase activity.