Translational Thermotolerance Provided by Small Heat Shock Proteins Is Limited to Cap-dependent Initiation and Inhibited by 2-Aminopurine

Nicolette H. Lubsen,Carla Onnekink,Siebe T. van Genesen,Linda Doerwald,Wilfried W. de Jong

doi:10.1074/jbc.m302914200

Nicolette H. Lubsen, Carla Onnekink + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m302914200

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Abstract

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.

Highlights

Cells facing stress divert their resources to combating and surviving that stress
The leakage of the Hsp70 promoter used in these reporter constructs is very low, as the level of luciferase in non-heatshocked cells is only about 10% of that found in cells after recovery from a heat shock in the absence of an overexpressed heat shock proteins (Hsps)
The Hsp70 isoform Hsp72 has been shown to be associated with polysomes after a heat shock [27], and the effect of Hsp70 could well be on elongation as well as on initiation

Summary

The abbreviations used are

Heat shock protein; sHsp, small quired for optimal survival of heat (or other) stress. Heat shock proteins; eIF2␣, eukaryotic initiation factor 2␣; SG, stress granules; RT-PCR, reverse transcriptase-PCR; DMEM, Dulbecco’s modified Eagle’s medium; PKR, double-stranded RNA-activated kinase; IRES, internal ribosome entry site; FGF, fibroblast growth factor. In SGs is a highly dynamic process, and untranslated mRNAs are thought to be sorted and processed there for either reinitiation, degradation, or packaging into nonpolysomal messenger ribonucleoprotein complexes [39] This indicates that during and after stress, SGs are important checkpoints for initiation of translation. We show that 2-aminopurine, a kinase inhibitor that inhibits eIF2␣ kinases such as double-stranded RNA-activated kinase (PKR) [40, 41], blocks the establishment of translational tolerance by overexpression of an sHsp

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION