Akt signaling and freezing survival in the wood frog, Rana sylvatica

Abstract

Background: The wood frog (Rana sylvatica) exhibits well-developed natural freeze tolerance supported by multiple mechanisms of biochemical adaptation. The present study investigated the role and regulation of the Akt signaling pathway in wood frog tissues (with a focus on liver) responding to freezing stress.

Methods: Immunoblotting was used to assess total and phospho-Akt levels, total and phospho-PDK1, PTEN protein level, as well as total and phospho-FOXO1 levels. RT-PCR was used to investigate transcript levels of PTEN and microRNAs.

Results: Akt was inhibited in skeletal muscle, kidney and heart after 24h freezing exposure with a reversal after thawing. The responses of the main kinase (PDK-1) and phosphatase (PTEN) that regulate Akt were consistent with freeze activation of Akt in liver; freezing exposure activated PDK-1 via enhanced Ser-241 phosphorylation whereas PTEN protein levels were reduced. Levels of three microRNAs (miR-26a, miR-126 and miR-217) that regulate pten expression were elevated in liver during freezing. One well-known role of Akt is in anti-apoptosis, mediated in part by Akt phosphorylation of Ser-256 on FOXO1. Freezing triggered an increase in liver phospho-FOXO1 Ser-256 content, suggesting that an important action of Akt may be apoptosis inhibition.

Conclusions: Akt activation in wood frog is stress and tissue specific, with multi-facet regulations (posttranslational and posttranscriptional) involved in supporting this specific signal transduction response.

General significance: This study implicates the Akt pathway in the metabolic reorganization of cellular metabolism in support of freezing survival.

Keywords: Apoptosis; FOXO1; Freeze tolerance; PDK-1; PTEN; miRNA.