Transcriptional activation of heat shock factor HSF1 probed by phosphopeptide analysis of factor 32P-labeled in vivo.
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 integral step in the activation process or serve to maintain the active conformation of HSF1. Exploiting this correlation, indirect evidence was obtained that activation domains of HSF1 interact with the distantly located regulatory domain to maintain the factor in an inactive state.
Xia
W
W
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33136, USA.
Guo
Y
Y
Vilaboa
N
N
Zuo
J
J
Voellmy
R
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection; D014357:Trypsin; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D015500:Chloramphenicol O-Acetyltransferase; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D014357:Trypsin; D000099160:LexA Repressor Protein; C000619706:HSF1 protein, human