It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However

Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70 s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70 s6k or whether they reside on parallel pathways which bifurcate upstream of p70 s6k . Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70 s6k variants to distinguish between these possibilities. The rapamycin-resistant p70 s6k , which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block 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 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70 s6k constructs, which also blocked insulininduced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70 s6k activation, although only the kinase-dead p70 s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70 s6k revealed that the kinasedead p70 s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70 s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70 s6k , with the two pathways sharing a common rapamycin-sensitive activator.In insulin-responsive cells, hormonal stimulation provokes the coordinate activation of a complex network of signalling pathways which are involved in the regulation of specific metabolic processes (71). Critical among these affected metabolic processes is the activation and maintenance of high rates of protein synthesis, leading to both global and selective changes in the pattern of translation (14,23,56). Recent studies have suggested that in insulin-sensitive tissues, such as liver, heart, skeletal muscle, and adipose tissue, the increase in protein synthesis is triggered by ligand-induced activation of the receptor and propagated intracellularly through the phosphorylation of the insulin receptor substrate IRS-1 (66). IRS-1 is thought to mediate the insulin signal by inducing the activation of distinct kinases which then target specific components of the translational apparatus (45). In several cases, there is considerable knowledge concerning the functional roles of specific translational components in protein synthesis and of the effect of phosphorylation on their individual activities (48, 65). Much less is k...