Transcriptional activation of heat shock factor HSF1 probed by phosphopeptide analysis of factor 32P-labeled in vivo.
Abstract
Mapping of tryptic phosphopeptides of heat shock factor 1 (HSF1) from non-stressed or moderately heat-stressed HeLa cells, labeled in vivo by [32P]orthophosphate, revealed four major phosphopeptides A to D. Heat stress drastically increased phosphopeptide signals. To identify target peptides and amino acids and to correlate phosphorylation and transactivation function, phosphopeptide maps were produced of LexA-human HSF1 chimeras and mutant derivatives thereof, and transactivation activities of original and mutant chimeras were compared. LexA-HSF1 chimeras were previously shown to be regulated identically to HSF1, except that they transactivate promoters with LexA-binding sites instead of hsp promoters. The patterns of phosphopeptides of LexA-HSF1 and endogenous HSF1 were similar. Analysis of single residue substitutions suggested that phosphopeptide C is peptide VKEEPPSPPQSPR (297-309) phosphorylated on Ser-307 but not Ser-303. Substitution of Ser-307 but not Ser-303 caused deregulation of factor activity. Mapping of several constitutively active chimeras associated unphosphorylated peptide C with the transcriptionally active HSF1 conformation, suggesting that dephosphorylation of this peptide (at Ser-307) may either be an int...Continue Reading
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Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response
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