Abstract
The wood frog, Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites—in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study indicate a potential important role for PKC in cerebral protection during wood frog freezing. Our findings also call for a reassessment of the previously-inferred importance of PKC in other tissues, particularly in liver; a more thorough investigation is required to determine whether PKC activity in this physiological situation is indeed dependent on phosphorylation, or whether it deviates from the generally-accepted model and can be “overridden” by exceedingly high levels of second messengers, as has been demonstrated with certain PKC isozymes (e.g., PKCδ).
Highlights
For animals living in boreal climates, cold temperatures, sustained periods of subzero temperatures for months at a time, present a challenge to survival
Conventional protein kinase C (PKC) isozymes bind to the membrane via two specific bridging interactions: C1 domains that bind to DAG and C2 domains that bind to calcium-phospholipid complexes
For Conventional PKC isozymes (cPKC), all three criteria of DAG, calcium, and upstream phosphorylation are often necessary for full activity; this is complicated for novel PKC isozymes and atypical PKC isozymes. nPKCs are calcium-independent, but still rely on DAG for activity; aPKCs rely on neither calcium nor DAG, or any other phospholipids, though they possess other unique domains such as the phox-bem1 domain, which suggests that protein–protein interactions with cytosolic partners may be necessary for activity (Gomperts, Kramer & Tatham, 2009)
Summary
For animals living in boreal climates, cold temperatures, sustained periods of subzero temperatures for months at a time, present a challenge to survival. Intracellular freezing and any resulting irreparable damage to cellular contents is prevented by natural cryoprotection; liver glycogen stores undergo extensive hydrolysis (causing a decrease in liver mass by approximately 45%), and glucose is exported and systemically distributed, accumulating in some tissues at levels up to 40–60 times higher than euglycemic levels (Storey & Storey, 1985; Costanzo, Lee & Lortz, 1993) Such a broad reorganization requires numerous modulations at several levels of the signaling and metabolic hierarchy of glucose metabolism, including: (1) phosphorylation and sustained activation of liver glycogen phosphorylase (Crerar, David & Storey, 1988; Mommsen & Storey, 1992); (2) adaptations to plasma membranes in order to facilitate glucose transport and distribution (King, Rosholt & Storey, 1993); (3) tissue-specific adjustment of anabolic and catabolic signaling pathways (e.g., the insulin/Akt pathway, and the adenosine monophosphate-activated protein kinase or AMPK pathway) to optimize glucose production, distribution, uptake, and utilization as a cryoprotectant (Rider et al, 2006; Dieni, Bouffard & Storey, 2012; Zhang & Storey, 2013; do Amaral, Lee & Costanzo, 2013), and; (4) suppression of metabolic pathways that would otherwise divert glucose away from cryoprotection (e.g., pentose phosphate pathway, glycolysis; Dieni & Storey, 2010; Dieni & Storey, 2011), among others. Following the return of warmer temperatures and the arrival of spring, frogs thaw and resume their natural life cycle with no apparent debilitating results of the freeze-thaw process
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