Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

Mitogens induce the coordinated activation of a number of anabolic events which culminate in cell growth and division (41). Recent studies have defined the distinction between growth and proliferation, demonstrating the dominance of growth in this process (42, 58). An important component of the growth response is the generation of new translational machinery, required to accommodate the increased demand for additional proteins (56). The enhanced expression of protein synthetic components, most notably ribosomal proteins and elongation factors, is largely controlled at the translational level (5, 39). The transcripts for ribosomal proteins and elongation factors are characterized by an oligopyrimidine tract, termed 5′TOP, at their translational start site (5, 39). More importantly, it has been shown that the translational upregulation of these transcripts is mediated, in part, by the activation of the 40S ribosomal protein S6 kinase (S6K1) (32), presumably through the increased phosphorylation of S6 (33, 34). The importance of S6K1 in cell growth was initially inferred from the microinjection of neutralizing antibodies into cells (38, 50) and the use of the immunosuppressant rapamycin, each inhibiting mitogen-induced S6K1 activation and impeding cell growth (33, 34). Recently, the significance of S6K1 in cell growth has been emphasized by the generation of S6K1-deficient mice, which are significantly reduced in size (55), and by the discovery of a new, highly homologous S6 kinase, S6K2 (28, 55). The signal transduction pathway which mediates S6K1 activation has received considerable attention because of its implied importance in the growth response (25, 47). Early studies demonstrated that mitogen-induced S6K1 activation is initiated at a specific growth factor receptor docking site distinct from that utilized by the mitogen-activated protein kinase-Ras signaling pathway (40). The use of the inhibitory fungal metabolite wortmannin and platelet-derived growth factor (PDGF) receptor mutants led to the identification of phosphatidylinositide-3OH kinase (PI3K) as the effector which initiates downstream signaling from the receptor (17). The activation of S6K1 appears to be mediated in a hierarchical manner, initiated by the phosphorylation of a set of sites in its autoinhibitory domain (24) that facilitate subsequent phosphorylation at T389 in the adjacent linker domain. These two sets of initial phosphorylation events act in a synergistic manner to regulate T229 phosphorylation in the catalytic domain and thus kinase activation (24). Except for the recent identification of phosphoinositide-dependent protein kinase 1 (PDK1) as the S6K1-T229 kinase (4, 49), little is known concerning the identity of the other kinases which regulate the additional phosphorylation of S6K1 (49). The key step in the activation process is T389 phosphorylation, which unlike the phosphorylation of T229, appears to be positively regulated by a wortmannin-sensitive PI3K-dependent input (23). Although the S6K1-T389 kinase has yet to be identified, recent studies have suggested that this step is mediated by PI3K through the activation of protein kinase B (PKB) (15). Like S6K1, PKB activation is mediated by PI3K through the increased phosphorylation of T308 and S473, the sites homologous to T229 and T389 in S6K1, respectively (1). The dependence of PKB activation on PI3K as well as its wortmannin sensitivity can be circumvented by constitutively anchoring PKB to the membrane (6). This leads to increased levels of T308 and S473 phosphorylation, highly activates PKB (6), and induces S6K1 activation (15). These findings have led to the hypothesis that PKB mediates PI3K-induced S6K1 activation. Membrane-targeted alleles of PKB also induce the phosphorylation of two additional signaling components which regulate the translational machinery. One is glycogen synthase kinase 3 (GSK-3), whose direct phosphorylation by PKB leads to its inactivation (21). A key substrate of GSK-3 is the ɛ subunit of protein synthesis initiation factor eIF2B (65), whose phosphorylation suppresses eIF2B GDP-GTP exchange activity, inhibiting translation (65). The inhibition of GSK-3 activity by PKB increases eIF2B GDP-GTP exchange activity, raising the amount of active eIF2-GTP and leading to increased rates of global translation (19). The second downstream target of PKB is eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) (27, 36, 53, 60). The phosphorylation of 4E-BP1 disrupts its interaction with the mRNA m7G cap-binding protein eIF4E, allowing eIF4E to form a productive initiation complex (44). Unlike GSK-3, the effects of PKB on increased 4E-BP1 phosphorylation are indirect and may be mediated by a second kinase, possibly mTOR, the target of rapamycin (14, 53), or a kinase tightly bound to mTOR (43). Indeed, 4E-BP1 is thought to reside on the same rapamycin-sensitive PI3K-dependent signaling pathway as S6K1 (64). Although highly activated alleles of PKB lead to S6K1 activation, the same is also true for activated oncogenic alleles of Ras (10). However, the activation of wild-type Ras by mitogens is neither sufficient nor necessary to bring about S6K1 activation (40). This raised the possibility that highly activated alleles of PKB or constitutive membrane localization may not reflect wild-type PKB signaling. To test the role of PKB signaling in S6K1 activation we have compared the abilities of specific variants of PKB to activate and confer wortmannin resistance on S6K1, as well as the ability of interfering mutants to block this response. In parallel, we have used GSK-3β inactivation and 4E-BP1 phosphorylation to independently monitor the effects of the different PKB alleles on rapamycin-sensitive and -insensitive PKB downstream signaling. The results demonstrate that constitutive membrane localization of active PKB is essential for its ability to signal S6K1 and that the pathway leading to 4E-BP1 phosphorylation, but surprisingly not S6K1 activation, is dominantly regulated by PKB.