The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylationdeficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.The heat shock response is an evolutionarily well-conserved cellular stress response mechanism that is characterized by the elevated synthesis and accumulation of heat shock proteins (Hsps). Hsps are molecular chaperones involved in protein folding and maintenance of protein homeostasis, and a robust increase in Hsp levels is essential for survival when cells are exposed to various protein-damaging stresses (for reviews see references 15 and 34). The stress-inducible expression of Hspencoding genes is regulated by a family of heat shock transcription factors (HSFs), which bind to the heat shock element (HSE) in the promoter of heat shock genes and stimulate their transcription (for reviews see references 37 and 62). Three members of the HSF family, HSF1, HSF2, and HSF4, have been identified in mammalian species (41,47,54,56). HSF1, the vertebrate homologue of the single HSF found in the yeast and the fly, is the major stress-responsive family member, since no other HSF is able to functionally substitute for HSF1 or to rescue the heat shock response in HSF1-deficient cells or mice (35,44,64).The elevated synthesis of Hsps upon heat shock is caused by a multistep activation of HSF1 (for a review see reference 45). Under normal growth conditions, inactive HSF1 is distributed throughout the cell as a monomer that is constitutively phosphorylated on certain serine residues. Upon activation, HSF1 undergoes several modifications, such as trimerization and localization to specific nuclear structures, called nuclear stress granules (for a review s...

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/calmodulindependent 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 ®rst evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.