Involvement of Phosphorylation of Adenosine 5′-Monophosphate-Activated Protein Kinase in PTTH-Stimulated Ecdysteroidogenesis in Prothoracic Glands of the Silkworm, Bombyx mori

Bombyx mori. PLoS ONE 8(5): e63102. doi:10.1371/journal.pone.0063102 Involvement of Phosphorylation of Adenosine 59- Monophosphate-Activated Protein Kinase in PTTH- Stimulated Ecdysteroidogenesis in Prothoracic Glands of the Silkworm, Bombyx mori Shi-Hong Gu 0 Yun-Chin Hsieh 0 Shun-Chieh Young 0 Pei-Ling Lin 0 Immo A. Hansen, New Mexico State University, United States of America 0 Department of Zoology, National Museum of Natural Science , Taichung, Taiwan , Republic of China In this study, we investigated inhibition of the phosphorylation of adenosine 59-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm, Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPKa phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTHstimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPKa, b, and c by quantitative realtime PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis. - Funding: This study was supported by the National Science Council, Taipei, Taiwan (NSC99-2628-B-178-002-MY3 and NSC100-2313-B-001-MY3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Ecdysteroids are the insect steroid hormones that are synthesized and secreted by the prothoracic glands upon stimulation by the brain neurohormone, prothoracicotropic hormone (PTTH) [13]. PTTH was first purified and sequenced from the silkworm, Bombyx mori [4]. Due to structure similarities, it was proposed that PTTH resembles mammalian growth factors [5]. Recently, it was demonstrated that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila melanogaster, is a PTTH receptor [6]. PTTH appears to bind to its receptor to activate a signaling transduction network that up-regulates ecdysteroid synthesis [2,3,6] (Fig. 1). Cellular actions of PTTH are best understood for 2 lepidopteran insects, B. mori and Manduca sexta. Initial studies showed that cAMP and Ca2+ are intracellular second messengers involved in PTTH-stimulated ecdysteroidogenesis in both M. sexta [79] and B. mori [10,11]. Later, evidence indicated that the phosphorylation of p70S6 kinase (S6K) and ribosomal protein S6 are activated upon PTTH stimulation in M. sexta [1214]. Moreover, extracellular signal-regulated kinase (ERK) phosphorylation was demonstrated to be involved in PTTHs stimulation of ecdysteroidogenesis [1517]. It was recently further found that receptor tyrosine kinase is related to PTTH-stimulated ERK phosphorylation in B. mori prothoracic glands [17]. In addition, several other signaling pathways, such as tyrosine kinase, protein kinase C, the proteins involved in endosomal trafficking and constituents of the cytoskeleton, as well as the phosphorylation and translation of Halloween protein spook, appear to be involved in PTTH-stimulated ecdysteroidogenesis [1820]. Finally, our recent study showed that the signaling of phosphatidylinositol 3-kinase (PI3K)/the target of rapamycin (TOR) is involved in the stimulation of B. mori prothoracic glands by PTTH [21,22]. In eukaryotic cells, adenosine 59-monophosphate-activated protein kinase (AMPK) is a key regulatory enzyme of cellular energy homeostasis and is involved in regulating a diverse range of metabolic pathways [2326]. The AMPK protein complex consists of a catalytic a-subunit and regulatory b- and c-subunits [23,25]. AMPK activity is regulated allosterically by AMP and through phosphorylation at Thr172 in the activation loop of the asubunit. AMPK regulates carbohydrate, lipid, and protein metabolism via its effects on multiple signaling pathways and thereby suppresses ATP-demanding processes and activates ATPrepleting pathways [2326]. AMPK is activated during states of energy stress such as hypoxia, glucose starvation, and physical exercise. Protein synthesis, a major consumer of ATP in mammalian cells, is inhibited upon AMPK activation. Recent studies showed that one of the major downstream signaling pathways regulated by AMPK is the TOR signaling pathway [26]. The acute metabolic consequences of AMPK activation on protein homeostasis are mediated in part by the tuberous sclerosis complex (TSC)1 gene product and TSC2, which act upstream of the TOR pathways. Increased TSC2 phosphorylation and enhanced formation of the TSC1/TSC2 heterodimer both negatively regulate TOR activity [27]. In Drosophila, single genes encoding homologues of the a, b, and c subunits of mammalian AMPK were identified [28]. Drosophila AMPK is highly similar to mammalian AMPK, as it is formed via a heterotrimeric complex, is activated by AMP, and has many of the same targets, including acetyl-CoA carboxylase (ACC) [28]. Recently, it was demonstrated that reduced AMPK signaling in Drosophila through RNAi knockdown leads to hypersensitivity to starvation conditions as measured by lifespan and locomotor activity [29]. In the present study, we investigated the involvement of AMPK signaling in PTTH-stimulated ecdysteroidogenesis in B. mori prothoracic glands. Our results showed that PTTH inhibited the phosphorylation of AMPK both in vitro and in vivo. Moreover, PI3K appeared to be partially involved in PTTH-inhibited AMPK phosphorylation. The AMPK activator, 5-aminoimidazole-4carboxamide-1-b-D-ribofuranoside (AICAR), prevented PTTH (...truncated)