Characterization of phosphatidylinositol 3-kinase-dependent phosphorylation of the hydrophobic motif site Thr(389) in p70 S6 kinase 1.
Phosphorylation of the highly conserved hydrophobic motif site in AGC kinases is necessary for phosphotransferase activity. Phosphorylation of this motif (FLGFT389Y) in p70 S6 kinase (S6K1) is both rapamycin- and wortmannin-sensitive, suggesting a role for both mammalian target of rapamycin- and phosphatidylinositol 3-kinase-dependent pathways. We report here that co-expression of phosphoinositide-dependent kinase-1 (PDK1) and the phosphatidylinositol 3-kinase-regulated atypical protein kinase Czeta cooperate to increase both phosphorylation of the hydrophobic motif site Thr(389), as well as the activation loop site Thr(229). Interestingly, although PDK1 alone can promote an increase in Thr(389) phosphorylation in both wild type S6K1 and a kinase-inactive mutant of S6K1, the cooperative effect between PDK1 and protein kinase Czeta required S6K1 activity. Furthermore, Akt, another phosphatidylinositol 3-kinase effector and regulator of S6K1, also increased Thr(389) phosphorylation in a S6K1 activity-dependent manner. Consistent with this, epidermal growth factor-induced Thr(389) phosphorylation in wild type S6K1 persisted for up to 120 min, whereas kinase-inactive mutants of S6K1 displayed only a reduced and transient increase in Thr(389) phosphorylation. We conclude that S6K1 activity is required for maximal Thr(389) phosphorylation by mitogens and by multiple phosphatidylinositol 3-kinase-dependent inputs including PDK1, PKCzeta, and Akt, and we propose that autophosphorylation is an important regulatory mechanism for phosphorylation of the hydrophobic motif Thr(389) site in S6K1.
Romanelli
Angela
A
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. angela.romanelli@serono.com
Dreisbach
Valley C
VC
Blenis
John
J
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D019893:Ribosomal Protein S6 Kinases / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D013912:Threonine; D019869:Phosphatidylinositol 3-Kinases; D019893:Ribosomal Protein S6 Kinases