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.
Highlights
Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome
We have previously reported that structurally diverse nuclear factor-erythroid 2 p45-related factor 2 (NRF2) activators, all of which react with sulfhydryl groups, induce the heat shock response, and demonstrated the essential requirement for HSF1 [30]
By use of mass spectrometry and protein sequencing, Guettouche et al [19] found that in cells subjected to heat shock, human HSF1 is phosphorylated at 12 serine residues: S121, S230, S292, S303, S307, S314, S319, S326, S344, S363, S419, and S444
Summary
The remaining pellet containing the nuclear fraction was washed twice with ice-cold 0.1% NP-40 (vol/vol) in PBS and dissolved in 1ϫ sample loading buffer (50 mM Tris-Cl [pH 6.8], 2% [vol/vol] SDS, 10% [vol/vol] glycerol, and 0.005% [wt/vol] bromophenol blue) and heated for 5 min at 100°C. The pellet containing the nuclear fraction was washed there times with the buffer A before dissolving it in 1ϫ sample loading buffer (50 mM Tris-Cl [pH 6.8], 2% [wt/vol] SDS, 10% [vol/vol] glycerol, and 0.005% [wt/vol] bromophenol blue). Protein G-Dynabeads (30 l slurry [Invitrogen]) were washed twice for 5 min with PBS and incubated with 1 g of mouse monoclonal HSF1 antibody (Santa Cruz) for 1 h at room temperature, after which the beads were washed three times every 10 min with PBS.
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