The target of rapamycin (TOR) is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP), including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1), we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

There is now compelling evidence to indicate a place for heat shock factor 1 (HSF1) in mammary carcinogenesis, tumor progression and metastasis. Here we have investigated a role for HSF1 in regulating the expression of the stem cell renewal factor β-catenin in immortalized human mammary epithelial and carcinoma cells. We found HSF1 to be involved in regulating the translation of β–catenin, by investigating effects of gain and loss of HSF1 on this protein. Interestingly, although HSF1 is a potent transcription factor, it was not directly involved in regulating levels of β-catenin mRNA. Instead, our data suggest a complex role in translational regulation. HSF1 was shown to regulate levels of the RNA binding protein HuR that controlled β-catenin translation. An extra complexity was added to this scenario when it was shown that the long non-coding RNA molecule lincRNA-p21, known to be involved in β-catenin mRNA (CTNNB1) translational regulation, was controlled by HSF1 repression. We have shown previously that HSF1 was positively regulated through phosphorylation by mTOR kinase on a key residue, serine 326 essential for transcriptional activity. In this study we found that mTOR knockdown not only decreased HSF1-S326 phosphorylation in mammary cells, but also decreased β-catenin expression through a mechanism requiring HuR. Our data point to a complex role for HSF1 in the regulation of HuR and β-catenin expression that may be significant in mammary carcinogenesis.

Heat shock proteins (HSP) are essential for intracellular protein folding during stress and protect cells from denaturation and aggregation cascades that can lead to cell death. HSP genes are regulated at the transcriptional level by heat shock transcription factor 1 (HSF1) that is activated by stress and binds to heat shock elements in HSP genes. The activation of HSF1 during heat shock involves conversion from an inert monomer to a DNA binding trimer through a series of intramolecular folding rearrangements. However, the trigger for HSF1 at the molecular level is unclear and hypotheses for this process include reversal of feedback inhibition of HSF1 by molecular chaperones and heat-induced binding to large non-coding RNAs. Heat shock also causes a profound modulation in cell signaling pathways that lead to protein kinase activation and phosphorylation of HSF1 at a number of regulatory serine residues. HSP genes themselves exist in an accessible chromatin conformation already bound to RNA polymerase II. The RNA polymerase II is paused on HSP promoters after transcribing a short RNA sequence proximal to the promoter. Activation by heat shock involves HSF1 binding to the promoter and release of the paused RNA polymerase II followed by further rounds of transcriptional initiation and elongation. HSF1 is thus involved in both initiation and elongation of HSP RNA transcripts. Recent studies indicate important roles for histone modifications on HSP genes during heat shock. Histone modification occurs rapidly after stress and may be involved in promoting nucleosome remodeling on HSP promoters and in the open reading frames of HSP genes. Understanding these processes may be key to evaluating mechanisms of deregulated HSP expression that plays a key role in neurodegeneration and cancer.