Meeting Abstract

P1.31  Tuesday, Jan. 4  Cloning and expression of mTOR signaling components in the Y-organ of decapod crustaceans ABUHAGR, A.M.*; PITTS, N.L.; MACLEA, K.S.; CHANG, E.S.; MYKLES, D.L.; Colorado State Uinversity; UC Davis aliabuhagr@gmail.com

The metazoan target of rapamycin (mTOR) is a highly conserved protein kinase controlling cell growth in multicellular animals. Molting in crustaceans is regulated by ecdysteroids produced by a pair of molting glands (Y-organs, YO) located in the cephalothorax. During premolt, YOs hypertrophy and increase production of ecdysteroids. Here we focus on the role of mTOR signaling in YO growth. We hypothesize that up-regulation of mTOR signaling is necessary for YO hypertrophy and that molt-inhibiting hormone (MIH) down-regulates mTOR signaling in the YO of intermolt animals. cDNAs encoding Akt (protein kinase B), mTOR, Rheb, and p70 S6 kinase (S6K) were cloned from blackback land crab, Gecarcinus lateralis. and European green crab, Carcinus maenas. The G. lateralis clones were obtained by RT-PCR and RACE. Degenerate primers for G. lateralis were used in nested RT-PCR. We isolated partial cDNAs encoding 664bp of the mTOR kinase domain, 547bp of the Akt pleckstrin and kinase domains and 679bp of the S6K N-terminal kinase domain. Identity/similarity of each sequenced gene to its human ortholog was, for mTOR 75%/90%, for Akt 52%/73% and for S6K 76%/88%. The C. maenas clones were derived from ESTs. Partial cDNAs encoding 1624bp of the mTOR FAT domain, and an additional 1766bp of ORF and 3’UTR for mTOR, 728bp of the Akt pleckstrin and kinase domains and 869bp of the S6K N-terminal kinase domain. Identity/similarity of each sequenced gene was, for mTOR 55%/69% and 73%/82%, for Akt 56%/72% and for S6K 78%/88%. We are using real-time PCR (qPCR) to quantify the effects of molting induced by eyestalk ablation (ESA) or multiple leg autotomy (MLA) on expression of Akt, mTOR, Rheb, and S6K. Supported by NSF (IOS-0745224).