Hypothermic stress leads to activation of Ras-Erk signaling
Edmond Y.W. Chan, … , Drell A. Bottorff, James C. Stone
Edmond Y.W. Chan, … , Drell A. Bottorff, James C. Stone
Published May 1, 1999
Citation Information: J Clin Invest. 1999;103(9):1337-1344. https://doi.org/10.1172/JCI5474.
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Article

Hypothermic stress leads to activation of Ras-Erk signaling

Abstract

The small GTPase Ras is converted to the active, GTP-bound state during exposure of vertebrate cells to hypothermic stress. This activation occurs more rapidly than can be accounted for by spontaneous nucleotide exchange. Ras–guanyl nucleotide exchange factors and Ras GTPase–activating proteins have significant activity at 0°C in vitro, leading to the hypothesis that normal Ras regulators influence the relative amounts of Ras-GTP and Ras-GDP at low temperatures in vivo. When hypothermic cells are warmed to 37°C, the Raf-Mek-Erk protein kinase cascade is activated. After prolonged hypothermic stress, followed by warming to physiologic temperature, cultured fibroblasts assume a rounded morphology, detach from the substratum, and die. All of these biologic responses are attenuated by pharmacologic inhibition of Mek. Previously, it had been found that low temperature blocks acute growth factor signaling to Erk. In the present study, we found that this block occurs at the level of Raf activation. Temperature regulation of Ras signaling could help animal cells respond appropriately to hypothermic stress, and Ras-Erk signaling can be manipulated to improve the survival of cells in cold storage.

Authors

Edmond Y.W. Chan, Stacey L. Stang, Drell A. Bottorff, James C. Stone

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Figure 4

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Low temperature blocks acute growth factor activation of Raf and Erk but...
Low temperature blocks acute growth factor activation of Raf and Erk but not Shc. (a) Rat2 cells were left untreated, stimulated with EGF at 37°C, incubated on ice for 4 hours followed by warming to 37°C for 5 minutes, or chilled on ice for 15 minutes followed by EGF stimulation on ice. Cellular proteins were then analyzed using a phosphotyrosine immunoblot method. The positions of pp42 Erk and the different tyrosine-phosphorylated Shc species are shown on the right. (b) Rat2 cells overexpressing wild-type Raf-1 were left untreated, chilled on ice for 30 minutes, and then stimulated with EGF on ice, or stimulated with EGF at 37°C. Raf was immunoprecipitated and assayed for catalytic activity (at 30°C), as described in Figure 3a. Triplicate samples shown are from 3 independent plates of cells. As negative and positive controls, respectively, no antibody was used to immunoprecipitate Raf from EGF-treated cells, and recombinant GST-Mek1 (Q56P) (mutationally activated) was used in place of Raf1 and wild-type GST-Mek1.