dS 6 K-regulated cell growth is dPKB / dPI ( 3 ) K-independent , but requires dPDK 1

Genetic studies in Drosophila melanogaster underscore the importance of the insulin-signalling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homologue), dPI(3)K, dPKB, dPTEN, and dS6K. Mutations in any of these components have a striking effect on cell size and number, with the exception of dS6K. Mutants in dS6K affect cell size but not cell number, seemingly consistent with arguments that dS6K is a distal effector in the signalling pathway, directly controlled by dTOR, a downstream effector of dPI(3)K and dPKB. Unexpectedly, recent studies showed that dS6K activity is unimpaired in chico-deficient larvae, suggesting that dS6K activation may be mediated through the dPI(3)K docking sites of the Drosophila insulin receptor. Here, we show genetically, pharmacologically and biochemically that dS6K resides on an insulin signalling pathway distinct from that of dPKB, and surprisingly also from that of dPI(3)K. More striking, despite dPKB-dPI(3)K-independence, dS6K activity is dependent on the Drosophila homologue of the phosphoinositide-dependent protein kinase 1, dPDK1, demonstrating that both dPDK1, as well as dTOR, mediated dS6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent. brief communications NATURE CELL BIOLOGY VOL 4 MARCH 2002 http://cellbio.nature. com 251 dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1 Thomas Radimerski*, Jacques Montagne*, Felix Rintelen†, Hugo Stocker†, Jeroen van der Kaay‡, C. Peter Downes‡, Ernst Hafen†, and George Thomas* *Friedrich Miescher Institute, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland †Zoology Institute, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland ‡Department of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK e-mail: gthomas@fmi.ch Published online: 25 February 2002, DOI: 10.1038/ncb763 Genetic studies in Drosophila melanogaster underscore the importance of the insulin-signalling pathway in controlling cell, organ and animal size1. Effectors of this pathway include Chico (the insulin receptor substrate homologue), dPI(3)K, dPKB, dPTEN, and dS6K2. Mutations in any of these components have a striking effect on cell size and number3–7, with the exception of dS6K8. Mutants in dS6K affect cell size but not cell number, seemingly consistent with arguments that dS6K is a distal effector in the signalling pathway, directly controlled by dTOR, a downstream effector of dPI(3)K and dPKB1,9,10. Unexpectedly, recent studies showed that dS6K activity is unimpaired in chicodeficient larvae, suggesting that dS6K activation may be mediated through the dPI(3)K docking sites of the Drosophila insulin receptor11. Here, we show genetically, pharmacologically and biochemically that dS6K resides on an insulin signalling pathway distinct from that of dPKB, and surprisingly also from that of dPI(3)K. More striking, despite dPKB-dPI(3)K-independence, dS6K activity is dependent on the Drosophila homologue of the phosphoinositidedependent protein kinase 1, dPDK1, demonstrating that both dPDK1, as well as dTOR, mediated dS6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)– independent. Insulin-induced dS6K activation has been depicted as being triggered by dPI(3)K activation, either through chico, or directly through multiple dPI(3)K-SH2 docking sites in the extended tail of the Drosophila insulin receptor. Activation of dPI(3)K induces increased levels of PIP3, which in turn functions as a second messenger to mediate dPDK1 and dPKB activation. Based on studies in mammalian systems, dPDK1 is thought to directly phosphorylate the activation loop site of dPKB, as well as that of dS6K. In contrast to dPDK1, dPKB is thought to exert its effects on dS6K indirectly, by mediating dTOR activation, which in turn controls the phosphorylation of key residues at the carboxyl tail of dS6K. However, recent studies have shown that dTOR lacks the PKB phosphorylation sites that were argued to mediate mammalian TOR (mTOR) activation. In addition, studies in mammalian systems have suggested that PKB may be part of a parallel pathway to that of S6K, instead of functioning as an upstream effector. To examine the function of dPDK1 and dPKB in the control of dS6K function, we employed a dS6K-dependent phenotype in the wing. Selective overexpression of an upstream activation sequence (UAS)-dS6K transgene under the control of the apterous-GAL4 driver (ap-GAL4) in the dorsal compartment of the wing causes a bending down of the wing, presumably because of an increased size of cells on the dorsal surface (Fig. 1a). Expression of dPDK1 or dPKB, driven by the same ap-GAL4-UAS system, has no effect on the wing (Fig. 1b). In contrast, co-expression of dS6K with dPDK1 strongly enhances the bent wing phenotype achieved with dS6K alone, whereas co-expression of dPKB with dS6K consistently led to a slight suppression of the dS6K phenotype (Fig. 1b), possibly