TRANSCRIPTIONAL ACTIVATION OF HEAT-SHOCK FACTOR HSF1 PROBED BY PHOSPHOPEPTIDE ANALYSIS OF FACTOR P-32 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 b y [P-32]orthophosphate, revealed four major phosphopeptides A to D. He at stress drastically increased phosphopeptide signals. To identify ta rget peptides and amino acids and to correlate phosphorylation and tra nsactivation function, phosphopeptide maps were produced of LexA-human HSF1 chimeras and mutant derivatives thereof, and transactivation act ivities of original and mutant chimeras were compared, LexA-HSF1 chime ras were previously shown to be regulated identically to HSF1, except that they transactivate promoters with LexA-binding sites instead of h sp promoters. The patterns of phosphopeptides of LexA-HSF1 and endogen ous HSF1 were similar. Analysis of single residue substitutions sugges ted that phosphopeptide C is peptide VKEEPPSPPQSPR (297-309) phosphory lated on Ser-307 but not Ser-303, Substitution of Ser-307 but not Ser- 303 caused deregulation of factor activity, Mapping of several constit utively active chimeras associated unphosphorylated peptide C with the transcriptionally active HSF1 conformation, suggesting that dephospho rylation of this peptide (at Ser-307) may either be an integral step i n the activation process or serve to maintain the active conformation of HSF1, Exploiting this correlation, indirect evidence was obtained t hat activation domains of HSF1 interact with the distantly located reg ulatory domain to maintain the factor in an inactive state.