Identification of Bombyx mori Akt and its phosphorylation by bombyxin stimulation Shinji Nagata a, ⁎ , 1 , Fumihiko Hakuno b, ⁎ , 1 , Shin-Ichiro Takahashi b , Hiromichi Nagasawa a a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b Department of Animal Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan abstract article info Article history: Received 25 June 2008 Received in revised form 9 August 2008 Accepted 10 August 2008 Available online 12 August 2008 Keywords: Akt Bombyxin Bombyx mori Feeding Food intake Insulin-like signal Molecular cloning Phosphorylation Akt, a Ser/Thr protein kinase involved in insulin signaling, was identified from the silkworm, Bombyx mori. Bombyx Akt (BomAkt) is composed of 493 amino acid residues including regions conserved in other Akts: the Pleckstrin homology and kinase domains, and a dual phosphorylation site essential for kinase activation. Commercially available antibodies against mammalian Akt and phosphoAkt were able to recognize BomAkt and phosphorylated BomAkt in HEK293 cells expressing BomAkt. Additionally, phosphorylation of BomAkt was detectable in insulin-like growth factor (IGF)-I stimulated-HEK293 cells expressing BomAkt. RT-PCR and immunoblotting analyses revealed that BomAkt is expressed ubiquitously in Bombyx larvae. Phosphorylation of BomAkt was observed both in the isolated fat body after exposure to bombyxin, an endogenous insulin- like peptide, and in the larval fat body by refeeding a diet after starvation. These results suggest that dietary intake may activate the insulin signaling pathway, including Akt, through bombyxin action in B. mori. © 2008 Elsevier Inc. All rights reserved. 1. Introduction Insulin-like peptides are essential for biological function in various organisms, not only vertebrates but also invertebrates (Wu and Brown, 2006). In vertebrates, insulin-like peptides exert a variety of bioactivities, such as induction of proliferation and differentiation, inhibition of apoptosis in a variety of cells, and regulation of metabolism. In general, the signaling pathway of insulin-like peptides, such as insulin and insulin-like growth factors (IGFs), requires binding of such ligands to their receptors activating receptor tyrosine kinases that phosphorylate several intracellular substrates including insulin receptor substrates (IRSs) (White, 2002). The phosphorylated sub- strates are recognized by the Src-homology (SH) 2 domain of other signaling molecules, including a p85 regulatory subunit of phospha- tidylinositol (PI) 3-kinase (Backer et al., 1992). The binding between IRS and p85 activates a catalytic subunit of PI 3-kinase (p110), and consequently produces phosphoinositide-3,4,5-triphosphate (PIP 3 ) (White, 2002). PIP 3 is a key signaling molecule that recruits proteins possessing the Pleckstrin homology (PH) domain such as Akt to the plasma membrane (Bluem-Jensen and Hunter, 2001). Akt is a Ser/Thr protein kinase, originally identified in mouse (Jones et al., 1991; Scheid and Woodgett, 2001). Akt is phosphorylated at the plasma membrane by PIP 3 -activated phosphoinositide-dependent kinases (PDKs) at Thr 308 and Ser 473 , residues (human) that are conserved as a dual phosphorylation site (Meier and Hemmings, 1999). This phosphoryla- tion of Akt is known to be necessary for full activation of the kinase (Nicholson and Anderson, 2002). Akt activation is involved in various bioactivities regulated by insulin-like peptides in vertebrates. In invertebrates, the biological events modulated by insulin-like peptides have been extensively studied in a number of species. Abnormality in metabolism and the longevity of the nematode, Caenorhabditis elegans, is caused by a disorder in the insulin-like signaling molecules, DAF-2 (corresponding to the insulin receptor) and AGE-1 (corresponding to PI 3-kinase) (Hekimi et al., 1998). In the fruit fly, Drosophila melanogaster, dysfunction of the molecules in insulin signaling, including INR (corresponding to insulin receptor) (Tatar et al., 2003), Chico (corresponding to IRS) (Clancy et al., 2001) and PdIns-3-Ps (corresponding to PI 3-kinase) (Garofalo, 2002), results in biologically crucial phenotypes, such as morphological dwarfs and changes in the life span. Insect Akt is a Ser/Thr protein kinase that has been identified and characterized in three dipteran species, D. melanogaster (Franke et al., 1994; Andjelkovic et al., 1995), and two species of mosquitoes, Anopheles gambiae, and Aedes aegypti (Riehle and Brown, 2003). In D. melanogaster , it is reported that Akt regulates cell size, growth, proliferation (Scanga et al., 2000), and apoptosis (Liu and Lehemann, 2006; Staveley et al., 1998), possibly mediated by the somatic calorimetric control. In A. gambiae, oogenesis is strongly related to Akt activation in female ovary Comparative Biochemistry and Physiology, Part B 151 (2008) 355–360 ⁎ Corresponding authors. Nagata is to be contacted at Fax: +81 3 5841 8022. Hakuno, Fax: +81 3 5841 1310. E-mail addresses: anagashi@mail.ecc.u-tokyo.ac.jp (S. Nagata), ahakuno@mail.ecc.u-tokyo.ac.jp (F. Hakuno). 1 Equally contributing authors. 1096-4959/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2008.08.002 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb