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Inactivation of eIF2B and phosphorylation of PHAS-I in heat-shocked rat hepatoma cells
G C Scheper1, J Mulder, M Kleijn
1Department of Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands. g.scheper@biol.ruu.nl
Abstract:
Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.
Insights
Heat shock inhibits protein synthesis by regulating eIF2B activity, a guanine exchange factor crucial for translation initiation. This regulation occurs even at mild heat shock temperatures, impacting protein synthesis levels.
Area of Science:
- Molecular Biology
- Cellular Stress Response
Background:
- Heat shock inhibits protein synthesis through various mechanisms.
- Phosphorylation and inactivation of eukaryotic initiation factors (eIFs) like eIF2 and eIF4E are known responses.
- However, these changes in mammalian cells occur at higher temperatures than where protein synthesis inhibition is observed.
Purpose of the Study:
- To investigate the causes of protein synthesis inhibition at mild heat shock temperatures.
- To analyze the regulation of eIF2 and eIF4E by additional factors.
- To identify the primary mechanism controlling translation initiation during heat stress.
Main Methods:
- Measurement of eIF2B activity during and after heat shock.
- Analysis of eIF4E and PHAS-I phosphorylation in response to heat shock.
- Comparison of these changes across different cell lines.
Main Results:
- A strong correlation was observed between eIF2B activity and protein synthesis levels, even under mild heat shock.
- PHAS-I phosphorylation showed variable responses (increase or decrease) across different cell lines.
- Decreased eIF4E availability is not a universal mechanism for heat shock-induced protein synthesis inhibition.
Conclusions:
- Regulation of eIF2B activity is the primary mechanism controlling translation initiation after mild heat shock.
- The role of eIF4E and PHAS-I phosphorylation in this process is variable and not a general mechanism.
- eIF2B regulation is key to understanding cellular responses to heat stress.