Protein kinase C in the wood frog,Rana sylvatica: reassessing the tissue-specific regulation of PKC isozymes during freezing

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).

A practical guide to buildde-novoassemblies for single tissues of non-model organisms: the example of a Neotropical frog

“…Another essential domain for frogs is the ‘Protein kinase domain’ that we found as the third more abundant (643 hits; 2.74%). This domain is supposed to play an important role in frogs in freezing tolerance during cold winters, likely inducing the transcription of antioxidant response genes ( Dieni & Storey, 2014 ). Although freezing winters are not common within the current range of O. cruralis , the relative abundance of protein kinase domains could have been important in the evolutionary history of Oreobates , a genus that may have originated at high altitude in the Andes ( Padial, Chaparro & De la Riva, 2008 ).…”

Peer Review #2 of "A practical guide to build de-novo assemblies for single tissues of non-model organisms: the example of a Neotropical frog (v0.1)"

“…Another essential domain for frogs is 421 the 'Protein kinase domain' that we found as the third more abundant (643 hits; 2.74%). This 422 domain is supposed to play an important role in frogs in freezing tolerance during cold winters, 423 likely inducing the transcription of antioxidant response genes (Dieni & Storey 2014). Although 424 freezing winters are not common within the current range of O. cruralis, the relative abundance 425 of protein kinase domains could have been important in the evolutionary history of Oreobates, a 426 genus that may have originated at high altitude in the Andes (Padial et al 2008).…”

Reversible Enzyme Phosphorylation as a Mechanism for Metabolic Adaptation to Dehydration in the Skeletal Muscle of the African Clawed Frog, Xenopus laevis

“…The PKC family of kinases is known to participate in many biological responses including the regulation of insulin sensitivity and lipid metabolism (Nishizuka 1995). Furthermore, it is has been shown to be strongly reduced in response to freezing in another frog species (Dieni and Storey 2014) In this thesis, in silico methods of predicting phosphorylation sites concluded that all enzymes isolated in this thesis were likely regulated by PKC in some capacity. Kinetic alterations were demonstrated after incubations that stimulated endogenous PKC that resulted in decreased substrate Km values for aldolase, CK and IDH.…”