Regulation of metabolism in estivating land snails: role of reversible protein phosphorylation.
Estivation is a state of aerobic dormancy used by animals such as the desert land snail, Otala lactea, to endure harsh environmental conditions. Metabolic rate depression is key to survival during estivation and requires coordinated suppression of ATP generating and ATP-consuming cellular functions by stable regulatory mechanisms. Current studies examined the role of reversible protein phosphorylation in metabolic arrest in snail organs focusing on a major ATP-consuming process, the Na+/K+-ATPase; a key enzyme that produces NADPH for antioxidant defense, glucose-6-phosphate dehydrogenase (G6PDH); and selected protein kinases and protein phosphatases that could regulate the process. Studies documented a decrease in foot muscle and hepatopancreas Na+/K+-ATPase activity, and an increase in hepatopancreas G6PDH activity during estivation, as indicated by changes in kinetic parameters (e.g. maximal velocity, substrate affinity, Arrhenius activation energy). Furthermore, in vitro incubations stimulating specific endogenous kinases and phosphatases implicated roles for PKG and PP1 in estivation-dependent changes in Na+/K+-ATPase and G6PDH. Ion exchange chromatography of G6PDH revealed two enzyme forms – a high phosphate, high activity form and a low phosphate, low activity form – whose proportions changed in dormant snails. The peak profiles of G6PDH from active and estivating snails were also interconverted after incubations promoting PKG or PP1 activities. Profiles of protein phosphatases in O. lacteatissues revealed a general suppression of activity during estivation. For PP1 and PP2A the differential activity in estivation was linked to altered enzyme elution profiles from gel filtration chromatography, indicating that differential association into phosphatase holoenzymes is partly responsible for reduced phosphatases activities in estivation. Type-1 and type-2 phosphatases were purified and analyzed; the data generally indicated that the mammalian phosphatase classification system was applicable to O. lacteaphosphatases. Examination of several protein kinases, utilizing a relatively novel assay method with P81 paper array/phosphor imaging, revealed increased activities of AMPK, PKB, and PKG in estivating O. lactea. Increased activities of AMPK and PKB were related to changes in their phosphorylation state and confirmed by changes in activities and/or phosphorylation status of downstream targets. Overall, these studies confirm the integral role of reversible protein phosphorylation in the suppression and reorganization of metabolism during estivation.