Glycogen synthase kinase 3beta negatively regulates both DNA-binding and transcriptional activities of heat shock factor 1.

Stress activation of heat shock factor (HSF1) involves the conversion of repressed monomers to DNA-binding homotrimers with increased transcriptional capacity and results in transcriptional up-regulation of the heat shock protein (hsp) gene family. Cells tightly control the activity of HSF1 through interactions with hsp90 chaperone complexes and through integration into a number of different signaling cascades. A number of studies have shown that HSF1 transcriptional activity is negatively regulated by constitutive phosphorylation in the regulatory domain by glycogen synthase kinase (GSK3) isoforms alpha/beta. However, previous studies have not examined the ability of GSK3 to regulate the DNA-binding activity of native HSF1 in vivo under heat shock conditions. Here we show that GSK3beta inhibits both DNA-binding and transcriptional activities of HSF1 in heat-shocked cells. Specific inhibition of GSK3 increased the levels of DNA binding and transcription after heat shock and delayed the attenuation of HSF1 during recovery. In contrast, the overexpression of GSK3beta resulted in significant reduction in heat-induced HSF1 activities. These results confirm the role of GSK3beta as a negative regulator of HSF1 transcription in cells during heat shock and demonstrate for the first time that GSK3beta functions to repress DNA binding.

Constitutive and heat-inducible heat shock element binding activities of heat shock factor in a group of filamentous fungi.

This study represents the initial characterization of the heat shock factor (HSF) in filamentous fungi. We demonstrate that HSFs from Beauveria bassiana, Metarhizium anisopliae, Tolypocladium nivea, Paecilomyces farinosus, and Verticillium lecanii bind to the heat shock element (HSE) constitutively (non-shocked), and that heat shock resulted in increased quantities and decreased mobility of HSF-HSE complexes. The monomeric molecular mass of both heat-induced and constitutive HSFs was determined to be 85.8 kDa by UV-crosslinking and the apparent molecular masses of the native HSF-HSE complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. Proteolytic band clipping assays using trypsin and chymotrypsin revealed an identical partial cleavage profile for constitutive and heat-induced HSF-HSE complexes. Thus, it appears that both constitutive and heat-inducible complexes are formed by trimers composed of the same HSF molecule which undergoes conformational changes during heat shock. The mobility difference between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation, indicating that the reduced mobility of the heat-induced HSF is probably due to a post-translational modification other than phosphorylation or glycosylation.

Production of listeriolysin O by Listeria monocytogenes (Scott A) under heat-shock conditions.

Early stationary phase cells of Listeria monocytogenes (Scott A) were examined to determine the effect of heat-shock on the production of listeriolysin O (LLO) during and after resuscitation at 37 degrees C. Cells were subjected to a heat-shock at 48 degrees C for 1 h. Intracellular and extracellular proteins of the heat-shocked cells were assayed for LLO using a microtiter plate hemolysis assay and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Our results showed that significant amounts of LLO are synthesized under heat-shock conditions that are not detected in the extracellular medium by a functional assay. This situation is evident by the absence of hemolytic activity immediately after heat-shock, and may be due to either a lack of excretion or inactivation of the LLO at 48 degrees C once outside the cell. By studying the intracellular and extracellular proteins using SDS-PAGE and immunoblots of the heat-shocked cells, we substantiated an absence of excretion as an operating mechanism. Heat-shocked cells resumed LLO production within 2-4 h of resuscitation at 37 degrees C, achieving an activity level 2-fold higher compared to the controls and 4-fold higher compared to cells immediately after heat-shock. Most likely, the LLO excreted must have been from LLO accumulated in the cells during heat-shock.