Control of cell number byDrosophilaFOXO: downstream and feedback regulation of the insulin receptor pathway

Abstract

The Drosophila insulin receptor (dInR) regulates cell growth and proliferation through the dPI3K/dAkt pathway, which is conserved in metazoan organisms. Here we report the identification and functional characterization of the Drosophila forkhead-related transcription factor dFOXO, a key component of the insulin signaling cascade. dFOXO is phosphorylated by dAkt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant dFOXO lacking dAkt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. dFOXO activation in S2 cells induces growth arrest and activates two key players of the dInR/dPI3K/dAkt pathway: the translational regulator d4EBP and the dInR itself. Induction of d4EBP likely leads to growth inhibition by dFOXO, whereas activation of dInR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of dFOXO in fly tissues regulates organ size by specifying cell number with no effect on cell size. Our results establish dFOXO as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation. During the development of multicellular organisms, growth is tightly regulated by controlling cell number and cell size so that each organ reaches its appropriate dimensions in relation to the size of the organism. Many studies indicate that growth and proliferation are coordinated but distinct processes and that cells progress through the cell cycle only when sufficient mass, size, and macromolecular biosynthesis have been reached. (Hartwell et al. 1974;Johnston et al. 1977;Weigmann et al. 1997). Organism growth is controlled by coordinating both cell cycle progression and survival, which is modulated by nutrient availability, growth factors, and temperature. Growth factors can stimulate cell division and survival by activating the insulin receptor, which in turn acts through two main signal transduction cascades: the Ras/MAP kinase (Lee and McCubrey 2002) and the PI3K/ Akt kinase pathways (Cantley 2002). Insulin-mediated activation of PI3K increases production of 3Ј-phosphorylated phosphoinositide lipids (PIP 3 ) that serve as second messengers to recruit Akt to the plasma membrane (Datta et al. 1999). Once properly localized in the membrane, Akt becomes activated by phosphorylation and in turn phosphorylates a number of downstream targets that ultimately regulate cell growth. For example, Akt stimulates protein synthesis through activation of the target of rapamycin (TOR) kinase, which subsequently phosphorylates and inactivates the translational repressor eukaryotic initiation factor 4E-binding protein (4EBP; Gingras et al. 2001).In addition to modulating translation, Akt regulates transcription through the forkhead-related FOXO family of transcription factors FOXO1, FOXO3a, and FOXO4 (Burgering and Kops 2002) by phosphorylating these proteins at three conserved serine/threonine residues. This leads to retention of FOXO transcription factor...

Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns

“…Several recent reports provide support for an involvement of fd88a (Foxo) in the insulin receptor pathway, including Junger et al (2003), Kramer et al (2003), and Puig et al (2003).…”

Long live FOXO: unraveling the role of FOXO proteins in aging and longevity

“…Without ligand binding at the insulin‐like receptor, dFoxO remains unphosphorylated and translocated to the nucleus, activating expression of factors that retard cell growth and proliferation (Junger et al ., 2003; Puig et al ., 2003). On the other hand, insulin treatment leads to dFoxO phosphorylation by dAKT, leading to cytoplasmic retention and inhibition of its transcriptional activity.…”

“…On the other hand, insulin treatment leads to dFoxO phosphorylation by dAKT, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant dFoxO lacking dAKT phosphorylation sites does not respond to insulin inhibition and is constitutively active in the nucleus (Puig et al ., 2003). dFoxO activation induces growth arrest and increases the expression of two key players of the dInR/dPI3K/dAKT pathway: the translational regulator d4E‐BP and the dInR itself.…”

“…dFoxO activation induces growth arrest and increases the expression of two key players of the dInR/dPI3K/dAKT pathway: the translational regulator d4E‐BP and the dInR itself. Interestingly, targeted expression of dFoxO in fly tissues regulates organ size by specifying cell number with no effect on cell size (Puig et al ., 2003). …”

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

“…In contrast, when the genes that encode Drosophila insulin-like peptides (Dilps) are overexpressed, the flies show increased body size (Brogiolo et al, 2001). Furthermore, the overexpression of dFOXO, which is normally repressed through phosphorylation by PKB/Akt, decreases organ size and increases relative amounts of transcript of both Drosphila insulin receptor and 4EBP (Puig et al, 2003). …”