Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress

BACKGROUND: Heat shock factor (HSF/HSF1) not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs). HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser(303), Ser(307 )and Ser(363), which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser(230 )contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells. RESULTS: An alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser(121), Ser(230), Ser(292), Ser(303), Ser(307), Ser(314), Ser(319), Ser(326), Ser(344), Ser(363), Ser(419), and Ser(444). Phosphorylation of Ser(326 )but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser(326 )increased rapidly during heat stress as shown by experiments using a pSer(326 )phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type factor. CONCLUSION: Twelve Ser residues but no Thr or Tyr residues were identified that were phosphorylated in heat-activated HSF1. Mutagenesis experiments and functional studies suggested that phosphorylation of HSF1 residue Ser(326 )plays a critical role in the induction of the factor's transcriptional competence by heat stress. PhosphoSer(326 )also contributes to activation of HSF1 by chemical stress. To date, no functional role could be ascribed to any of the other newly identified phosphoSer residues

CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70

Exposure of cells to various stresses often leads to the induction of a group of proteins called heat shock proteins (HSPs, molecular chaperones)1,2. Hsp70 is one of the most highly inducible molecular chaperones, but its expression must be maintained at low levels under physiological conditions to permit constitutive cellular activities to proceed3,4. Heat shock transcription factor 1 (HSF1) is the transcriptional regulator of HSP gene expression5, but it remains poorly understood how newly synthesized HSPs return to basal levels when HSF1 activity is attenuated. CHIP (carboxy terminus of Hsp70-binding protein), a dual-function co-chaperone/ubiquitin ligase, targets a broad range of chaperone substrates for proteasomal degradation6–11. Here we show that CHIP not only enhances Hsp70 induction during acute stress but also mediates its turnover during the stress recovery process. Central to this dual-phase regulation is its substrate dependence: CHIP preferentially ubiquitinates chaperone-bound substrates, whereas degradation of Hsp70 by CHIP-dependent targeting to the ubiquitin–proteasome system occurs when misfolded substrates have been depleted. The sequential catalysis of the CHIP-associated chaperone adaptor and its bound substrate provides an elegant mechanism for maintaining homeostasis by tuning chaperone levels appropriately to reflect the status of protein folding within the cytoplasm

The Identification of Protein Kinase C Iota as a Regulator of the Mammalian Heat Shock Response Using Functional Genomic Screens

BACKGROUND: The heat shock response is widely used as a surrogate of the general protein quality control system within the cell. This system plays a significant role in aging and many protein folding diseases as well as the responses to other physical and chemical stressors. METHODS/PRINCIPAL FINDINGS: In this study, a broad-based functional genomics approach was taken to identify potential regulators of the mammalian heat shock response. In the primary screen, a total of 13724 full-length genes in mammalian expression vectors were individually co-transfected into human embryonic kidney cells together with a human HSP70B promoter driving firefly luciferase. A subset of the full-length genes that showed significant activation in the primary screen were then evaluated for their ability to hyper-activate the HSP70B under heat shock conditions. Based on the results from the secondary assay and gene expression microarray analyses, eight genes were chosen for validation using siRNA knockdown. Of the eight genes, only PRKCI showed a statistically significant reduction in the heat shock response in two independent siRNA duplexes compared to scrambled controls. Knockdown of the PRKCI mRNA was confirmed using quantitative RT-PCR. Additional studies did not show a direct physical interaction between PRKCI and HSF1. CONCLUSIONS/SIGNIFICANCE: The results suggest that PRKCI is an indirect co-regulator of HSF1 activity and the heat shock response. Given the underlying role of HSF1 in many human diseases and the response to environmental stressors, PRKCI represents a potentially new candidate for gene-environment interactions and therapeutic intervention

Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin

DAXX stimulates the cellular stress protein response by activating the transactivation competence of heat shock transcription factor HSF1

HSF1 is the essential transcription factor that is required for the stress-induced expression of a heterogeneous class of proteins known as heat shock proteins (HSPs). Monomeric HSF1 is released from a HSP90-containing chaperone complex when the folding aides (chaperones) are needed to prevent intracellular aggregation of un- or missfolded proteins. Unbound HSF1 monomers form a homo-trimeric complex that accumulates in the nucleus and acquires the ability to bind to specific DNA sequences known as heat shock elements (HSEs) in promoters of genes that encode heat shock proteins.DAXX, a protein initially characterized as an enhancer of Fas-induced apoptosis, binds to the trimeric HSF1 complex---thereby stimulating the transcriptional competence of HSF1. This activation of trimeric HSF1 can be achieved in the absence of stress by overexpression of DAXX. DAXX fragments exert a dominant-negative effect on HSF1 activation by different types of stress. Interference with the expression of the DAXX protein by antisense oligonucleotides against the DAXX mRNA significantly interferes with the heat-induced HSF1 activity. The complete loss of daxx results in a defective stress protein response. These findings identify DAXX as the key mediator of activation of HSF1 transcriptional competence.The latter activation event appears to be associated with nuclear domain ND10 (PML oncogenic domain), which is known to be involved in a variety of cellular activities like aging, apoptosis, the cell cycle, stress response, hormone signaling, transcriptional regulation and development.Of the two kinases that are known to bind to DAXX only FIST/HIPK3 but not ASK1/MEKK5 co-mediates the activation of HSF1 by DAXX. The target of the protein kinase FIST is probably neither HSF1 nor DAXX but acts downstream of these two proteins

CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70

Exposure of cells to various stresses often leads to the induction of a group of proteins called heat shock proteins (HSPs, molecular chaperones)(1,2). Hsp70 is one of the most highly inducible molecular chaperones, but its expression must be maintained at low levels under physiological conditions to permit constitutive cellular activities to proceed(3,4). Heat shock transcription factor 1 (HSF1) is the transcriptional regulator of HSP gene expression(5), but it remains poorly understood how newly synthesized HSPs return to basal levels when HSF1 activity is attenuated. CHIP (carboxy terminus of Hsp70-binding protein), a dual-function co-chaperone/ubiquitin ligase, targets a broad range of chaperone substrates for proteasomal degradation(6–11). Here we show that CHIP not only enhances Hsp70 induction during acute stress but also mediates its turnover during the stress recovery process. Central to this dual-phase regulation is its substrate dependence: CHIP preferentially ubiquitinates chaperone-bound substrates, whereas degradation of Hsp70 by CHIP-dependent targeting to the ubiquitin–proteasome system occurs when misfolded substrates have been depleted. The sequential catalysis of the CHIP-associated chaperone adaptor and its bound substrate provides an elegant mechanism for maintaining homeostasis by tuning chaperone levels appropriately to reflect the status of protein folding within the cytoplasm

Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin