RAPAMYCIN, WORTMANNIN, AND THE METHYLXANTHINE SQ20006 INACTIVATE P70(S6K) BY INDUCING DEPHOSPHORYLATION OF THE SAME SUBSET OF SITES

Activation of p70(s6k) in cells stimulated with serum correlates with the phosphorylation of seven sites. Pretreatment of Swiss 3T3 cells wi th the immunosuppressant rapamycin blocks phosphorylation of four of t hese sites (Thr(229), Thr(389), Ser(404), and Ser(411)), whereas phosp horylation proceeds in the remaining three sites (Ser(418), Thr(421), and Ser(424)). If rapamycin is added post-serum stimulation, the patte rn of phosphorylation is qualitatively similar except that Ser(411) is still highly phosphorylated. The inhibitory effect of rapamycin on se rum-induced p70(s6k) activation and the phosphorylation of Thr(229), T hr(389), Ser(404), and Ser(411) is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of r apamycin-FKBP12. Wortmannin treatment pre- or post-serum stimulation i nhibits phosphorylation of the same set of sites as rapamycin, support ing the argument that both agents act on the same pathway. Likewise, m ethylxan thine phosphodiesterase inhibitors block p70(s6k) activation and phosphorylation of the same set of sites as wortmannin and rapamyc in. However, other agents that raise intracellular cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory acti ons of methylxanthines on p70(s6k) activity are not through activating protein kinase A but through inhibition of an upstream kinase. Togeth er the results indicate that there are two kinase signaling pathways t hat must converge to activate p70(s6k) and that only one of these path ways is sensitive to rapamycin, wortmannin, and methylxanthine inhibit ion.