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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In vitro Ras-GTP association and hydrolysis at 0°C. (a) Ras-GDP complex ...
In vitro Ras-GTP association and hydrolysis at 0°C. (a) Ras-GDP complex from E. coli was incubated with a molar excess of [α-32P]GTP at 30°C or at 0°C for up to 4 hours followed by precipitation and quantification of Ras-associated label. Values are expressed as percent of maximal association, as determined by equilibration in magnesium-free conditions. Values shown are averages of duplicate data points. (b) Ras from Sf9 cells was complexed with [α-32P]GTP and then incubated either with (+) or without (–) recombinant p120GAP at 0°C for 4 hours or at 30°C for 15 minutes. Ras-associated nucleotide was then precipitated and quantified. Lane S represents the starting material, substrate Ras-GTP complex that was precipitated immediately. The percent of total radiolabel that was present as GTP is shown above each lane. Chromatographic positions of GDP and GTP are shown on the right. (c) Ras-GDP from Sf9 cells was incubated at 0°C with a molar excess of [α-32P]GTP in the presence (p30) or absence (buffer) of the catalytic domain of Ras-GRF1 for 4 hours. Newly associated radiolabeled nucleotide was precipitated and quantified. Values are expressed as percent of maximal association and are the averages of triplicate data points with the standard deviation indicated. Note that when parallel reactions were performed at 30°C for 15 minutes, spontaneous and catalyzed association values of 4% and 43%, respectively, were observed.