Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1.
Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.
Holmberg
C I
CI
Turku Centre for Biotechnology, University of Turku, Abo Akademi University, Finland.
Hietakangas
V
V
Mikhailov
A
A
Rantanen
J O
JO
Kallio
M
M
Meinander
A
A
Hellman
J
J
Morrice
N
N
MacKintosh
C
C
Morimoto
R I
RI
Eriksson
J E
JE
Sistonen
L
L
D000906:Antibodies / Q000276:immunology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D000076249:Heat Shock Transcription Factors; D006358:Hot Temperature; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000276:immunology; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D010748:Phosphopeptides; D011994:Recombinant Proteins; D014157:Transcription Factors; D012694:Serine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases