Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

Abstract

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...

Ha-rasval12 induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-rasval12-transformed cells

“…It seems, therefore, that Ha-ras val12 expression does not have a significant effect on Hsf1 phosphorylation. The fact that Ras properly regulated HSF1 S303A rules out the possibility that Ras affect SUMO-1 modification, because Ser303 phosphorylation is a prerequisite for this modification (Hietakangas et al, 2003). In order to evaluate a possible effect of Ras on Hsf1 redox state, we measured the in vivo redox state of Hsf1 upon cotransfection with Ha-ras val12 .…”

Sumoylated RGS-Rz Proteins Act as Scaffolds for Mu-Opioid Receptors and G-Protein Complexes in Mouse Brain

“…Phosphorylation could also account for the coexistence of the nonsumoylated and sumoylated forms of RGSZ1 and GAIP. Indeed, proteins like heat stress factor 1 must be phosphorylated to undergo efficient sumoylation (Hietakangas et al, 2003), although the contrary has been observed for the transcription factor c-Jun, the sumoylation of which is downregulated after phosphorylation (Müller et al, 2000). As sumoylation can be regulated by phosphorylation, it is possible that phosphorylation of critical residues on RGSZ1 impairs SUMO conjugation.…”

Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line

“…MEFs derived from an Hsf1 Ϫ/Ϫ or Hsf1 ϩ/ϩ mouse (19) were maintained as in ref. 37. For heat shock treatments, the plates were immersed in a water bath set to the indicated temperature (Ϯ0.1°C).…”