THE INSULIN-INDUCED SIGNALING PATHWAY LEADING TO S6 AND INITIATION-FACTOR 4E BINDING-PROTEIN-1 PHOSPHORYLATION BIFURCATES AT A RAPAMYCIN-SENSITIVE POINT IMMEDIATELY UPSTREAM OF P70(S6K)

Employing specific inhibitors end docking-site mutants of growth facto r receptors, recent studies have indicated that the insulin-induced in crease in 40S ribosomal protein S6 and initiation factor 4E binding pr otein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70(s6k) signal transduction pathway. However, it has not been resolved whethe r the phosphorylation of both proteins is mediated by p70(s6k) or whet her they reside on parallel pathways which bifurcate upstream am of p7 0(s6k). Here me have used either rapamycin-resistant, kinase-dead, or wild-type p76(s6k) variants to distinguish between these possibilities . The rapamycin-resistant p70(s6k), which has high constitutive activi ty, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced black of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70(s6k) inhibited insulin-induced 4E-BP1 pho sphorylation in a dose-dependent manner, This effect was mimicked by t he kinase-dead and mild-type p70(s6k) constructs, which also blocked i nsulin-induced dissociation of 4E-BP1 front initiation factor 4E, Both the kinase-dead and mild-type constructs also blocked reporter p70(s6 k) activation, although only the kinase-dead p70(s6k) had a dominant-i nterfering effect on S6 phosphorylation. Analysis of phosphopeptides f rom reporter 4E-BP1 and p70(s6k) revealed that the kinase-dead p70(s6k ) affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70(s6k) medi ates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalli ng pathway that bifurcates immediately upstream of p70(s6k), with the two pathways sharing a common rapamycin-sensitive activator.