Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

Abstract

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.

Regulation of the mammalian heat shock factor 1

“…The phosphorylation of serine 326 of HSF1 allows DAXX to bind to HSF1 to enhance its transcription , and mutations of serine 326 have been reported to reduce the transcriptional activity of HSF1 . Several kinases have been implicated in phosphorylating HSF1 at serine 326, including the mechanistic target of rapamycin , the MAPK kinase MEK and the p38 MAPK . Notably, among the p38 MAPK family members, p38δ MAPK is a particularly efficient catalyst of this phosphorylation, whereas p38γ MAPK is the most specific .…”

WITHDRAWN: Abrogation of heat shock factor 1 (Hsf1) phosphorylation deregulates its activity and lowers activation threshold, leading to obesity in mice

“…As presented in Figure 3F, exposure of MEFs to 40°C for 5 minutes was sufficient for Hsf1 303A/307A protein appearance in the nuclear fraction while significantly higher stress was required for WT. Hsf1 phosphorylation of serine residue at S326 has been shown to represent activated Hsf1 (26,27). We also investigated the Hsf1 pS326 phosphorylation status when cells were incubated at 37°C, 40°C or 42°C for 30 minutes using WT or Hsf1 303A/307A MEFs.…”

Phenethyl Isothiocyanate, a Dual Activator of Transcription Factors NRF2 and HSF1

“…Within minutes, PEITC induced the phosphorylation of HSF1 at S326 as well as the activation of the p38 mitogen‐activating protein kinases (MAPKs) 56. By the use of p38 MAPK inhibitors, we found that the p38 gamma isoform was, in part, responsible for the phosphorylation of HSF1 pS326 upon PEITC exposure.…”

“…In cervical cancer HeLa cells stably transfected with the HSP70.1 promoter fused upstream of the luciferase gene, PEITC robustly induced the promoter activity in a time‐ and dose‐dependent manner 56. HSP70, the prototypic marker of activation of the HSF1‐mediated transcription, was induced by PEITC and the induction was absent in MEF cells deficient in HSF1, suggesting that PEITC induced the HSR by activating HSF1 56. In agreement with our findings, PEITC treatment in murine hepatoma Hepa1c1c7 cells induced the HSP70 gene expression 47a.…”

“…Exposure to PEITC causes the activation of stress‐activated MAPKs such as c‐Jun N‐terminal kinase (JNK),77a p38 MAPKs,56, 82 and extracellular signal‐regulated kinase1/2 (ERK1/2) 83. It has been shown by several groups that the activation of these kinases by PEITC correlates with activation of the caspase‐dependent apoptotic pathway, and that chemical inhibition of these kinases blocks this process.…”