Justin A. MacDonald, Ph.D. Biology, 1998

Enzyme thermal adaptations and signal transduction involvement in ground squirrel hibernation



Regulatory enzymes are integral to the regulation of metabolism, and reversible protein phosphorylation by kinases and phosphatases controls most aspects of cell life. Brown adipose (BAT) PKA, liver PP-1 and muscle phosphofructokinase (PFK) from the ground squirrel were purified to homogeneity; physical and kinetic properties of these were assessed with respect to function at low temperature. An assessment of PKA catalytic subunit function via in vitro incubations of ground squirrel BAT extracts with 32P-ATP revealed differences in the patterns of phosphorylated proteins between euthermic and hibernating animals and between 37° and 5°C. The addition of 10% (w/v) polyethylene glycol reversed all negative effects of cold temperature, low pH and urea on PFK stability. A reduction in assay temperature from 37° to 5°C had numerous effects on ground squirrel enzymes including changes in pH optima, decreases in Km values for substrates, and reduced inhibition by salts. The percentage of membrane associated PKC increased during hibernation in liver, but the % active PKA was unaffected by hibernation in any tissue. Changes in PP-1 and -2 activities in tissues of euthermic and hibernating animals showed that phosphatases are regulated in hibernation. The subcellular localization of PP-1 in liver and muscle was affected by hibernation. The mitogen-activated protein kinase (MAPKs) MAPK-activated protein kinase (MAPKAPK-1 and -2) activities were affected by hibernation. p70S6Kkinase activity decreased in kidney during hibernation. c-jun kinase activity increased in 4 hibernating tissues but decreased slightly in brain. p38 was activated in hibernating muscle and heart. The responses of immediate-early gene products c-jun, egr-1, and c-myc were tissue specific with no apparent overall pattern. Maintenance of muscle energy status at the expense of the total adenylate pool was accompanied by a 60% decrease in the Na+K+-ATPase activity. Enzyme activity was reduced in vitro when incubated with protein kinase activators and was relieved by addition of alkaline phosphatase. A change in the ATP dependency of the enzyme also occurred in hibernation. Overall, protein phosphorylation is key to the regulation of hibernating metabolism in ground squirrels, and hibernator enzymes demonstrate functional adaptations to cold temperature.