Evidence for the reversible phosphorylation control of metabolism during prolonged environmental stress
I hypothesized that the environmental conditions endured during estivation in the spadefoot toad, Scaphiopus couchii, would dramatically affect metabolism and metabolic regulation in toad organs, and that reversible protein phosphorylation is important in the control of the necessary metabolic adaptations. Enzyme activities in tissues of control versus 2 month estivated toads revealed that glycolysis and ketone body metabolism dropped in liver, and that amino acid catabolism and NADPH production increased in brain and muscle. The influence of urea, which can build to 300 mM in estivating toads to aid desiccation resistance, was assessed on liver and muscle enzymes. Although known as a protein denaturant, urea had little effect on toad enzymes, being less disruptive of enzyme activities than 300 mM KCl. Estivation led to significant changes in the kinetic properties of both pyruvate kinase and phosphofructokinase that were mediated by reversible phosphorylation. To explore this mode of metabolic regulation, estivation effects on the activities of multiple protein kinases and protein phosphatases were studied. Protein kinase A (PKA) activities dropped in all tissues, and dormancy strongly affected type-1 and type-2 protein phosphatases. Estivation effects on the isozyme types, activities and distribution of protein tyrosine kinases (PTKs) and phosphatases (PTPs) revealed three major forms of PTKs and four PTPs in muscle and overall activities of PTKs decreased during estivation in the soluble fractions of liver, lung, and leg muscle, with the opposite pattern in heart. PTP activity in soluble fractions of gut, heart, and leg muscle also increased in estivation. Tissue specific changes in the enzyme activities and protein levels of mitogen-activated protein kinases (including Raf-1, ERK 1 & 2, MAPKAPK-1, JNK, p38) also occurred during estivation as did levels of various transcription factors (ATF-2, Elk-1, Egr-1, CREB). Involvement of these kinases and transcription factors implicates tissue-specific changes in gene expression during estivation and together with estivation-induced reversible phosphorylation of enzymes indicates that widespread, coordinated controls are used to readjust metabolism for the needs of the estivating state including metabolic rate depression, desiccation tolerance and long term starvation.