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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Activation of Erk in response to various hypothermic conditions and in d...
Activation of Erk in response to various hypothermic conditions and in different cell types. (a) Rat2 cells were left untreated, stimulated with EGF at 37°C, or incubated on ice for various amounts of time followed by warming to 37°C for 12 minutes. When indicated, cells were given a pretreatment of the Mek inhibitor PD098059 (50 μM) for 90 minutes at 37°C. Erk activation was assessed using the mobility shift assay. The positions of inactive p42 and active pp42 Erk are indicated on the right. (b) Rat2 cells were exposed to various temperatures for 4 hours and then warmed to 37°C for 5 minutes. Top: Erk activation was determined with an immunoblot method that uses a primary antibody that recognizes the dually phosphorylated active forms of Erk. Bottom: Equivalence of protein loading was demonstrated by immunoblotting a parallel blot with an antibody that recognizes total Erk. The positions of doubly phosphorylated pp42 and pp44 Erk and total p42 and p44 Erk are shown on the right. (c) Rat2 cells, MDCK cells (canine kidney epithelial cells), KD cells (human primary fibroblasts), CEF (primary chicken embryo fibroblasts), and REC (primary rat embryo fibroblasts) were treated as described below, and Erk activation was assessed using the immunoblotting assay described in b. A, untreated; B, acute growth factor treatment for 5 minutes at 37°C; C, incubation on ice for 4 hours followed by warming to 37°C for 5 minutes; D, incubation on ice for 30 minutes followed by acute growth factor treatment on ice for 5 minutes. Acute growth factor stimulation was EGF (100 ng/mL) for all cell types except CEFs, which were stimulated with 10% FBS. (d) RECs and rat2 cells were left untreated, stimulated with EGF, or incubated on ice for 4, 16, or 24 hours and then warmed to 37°C for 10 minutes. When indicated, cells were given a pretreatment of the Mek inhibitor PD098059 (50 μM) for 90 minutes at 37°C. Erk activation was assessed using a mobility shift assay. Note that only in a and d were SDS-PAGE conditions designed to resolve phosphorylated and nonphosphorylated forms of Erk.