Akt and its downstream targets play key roles in mediating dormancy in land snails Christopher J. Ramnanan a, ⁎ , Amy G. Groom b , Kenneth B. Storey b a Vanderbilt University School of Medicine, Department of Molecular Physiology, 710 Robinson Research Building, 2200 Pierce Avenue, Nashville, TN 37232, USA b Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 Received 4 May 2007; received in revised form 4 June 2007; accepted 5 June 2007 Available online 12 June 2007 Abstract Estivation, a state of aerobic dormancy, facilitates survival during adverse environmental conditions and is characterized at the molecular level by regulatory protein phosphorylation. The Akt (protein kinase B) signaling pathway regulates diverse responses in cells and the present study analyzes its role in the estivating desert snail Otala lactea. Kinetic analysis (maximal velocity, substrate affinities) determined that Akt was activated in tissues of estivating snails and Western blotting and in vitro incubations promoting changes to Akt phosphorylation state both confirmed that higher amounts of active (phosphorylated Ser473) Akt were present during estivation. Akt protein stability was also enhanced during estivation as assessed from urea denaturation studies. Multiple downstream targets of Akt were differentially regulated during estivation. Estivating animals showed elevated levels of phosphorylated FOXO3a (Ser253) and BAD (Ser136), no change in mTOR (Ser2481 and Ser2448), and reduced amounts of phosphorylated glycogen synthase kinase-3 (GSK-3) β subunit (Ser9). Kinetic analysis of GSK-3 showed 1.5–1.7 fold higher activities in estivating snails coupled with increased GSK-3 substrate affinities in hepatopancreas. The data suggest an active role for Akt signaling during estivation emphasizing anti-apoptotic actions but uncoupling growth/proliferation actions to help achieve life extension on a limited energy budget. © 2007 Elsevier Inc. All rights reserved. Keywords: Akt; Anti-apoptosis; Estivation; Forkhead box class O transcription factor (FOXO); Glycogen synthase kinase-3 (GSK3); Mammalian target of rapomycin (mTOR); Metabolic rate depression; Otala lactea 1. Introduction Estivation is a form of aerobic dormancy that is used by many types of organisms to survive periods of harsh environ- mental conditions, typically low water or low food availability. Terrestrial snail species have been frequent models for estivation research and have provided multiple insights into the molecular regulatory mechanisms that control hypometa- bolism (Brooks and Storey, 1997; Bishop and Brand, 2000; Storey, 2002; Ramos-Vasconcelos and Hermes-Lima, 2003). One of these is Otala lactea (Mollusca, Gastropoda); a native of the seasonally arid lands of the Mediterranean region. The snails are typically active for only a few weeks of the year and, during that time, they must accumulate sufficient body fuel reserves to endure many months of dormancy. Strong metabolic rate depression is an integral part of estivation; metabolic rates of estivating snails are commonly b 30% of the resting rate in nonestivating animals (Rees and Hand, 1993; Brooks and Storey, 1997; Bishop and Brand, 2000). Studies by our laboratory have identified reversible protein phosphorylation as a major mechanism regulating the suppression of metabolic functions in O. lactea during estivation. Changes in phosphorylation state underlie the suppression of multiple enzymes during estivation including enzymes of carbohydrate catabolism (e.g. phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase), the pentose phosphate pathway (glucose-6- phosphate dehydrogenase), and sodium pumping (Na + K + ATPase) (Whitwam and Storey, 1990, 1991; Brooks and Storey, 1992, 1997; Ramnanan and Storey, 2006a, 2006b). However, relatively little is known to date about the role of protein kinases in O. lactea estivation. The cyclic AMP-dependent protein kinase A (PKA) Available online at www.sciencedirect.com Comparative Biochemistry and Physiology, Part B 148 (2007) 245 – 255 www.elsevier.com/locate/cbpb ⁎ Corresponding author. Tel.: +1 615 322 7014; fax: +1 615 343 0490. E-mail address: chris.ramnanan@vanderbilt.edu (C.J. Ramnanan). 1096-4959/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2007.06.002