Holmberg C I, Hietakangas V, Mikhailov A, Rantanen J O, Kallio M, Meinander A, Hellman J, Morrice N, MacKintosh C, Morimoto R I, Eriksson J E, Sistonen L
Turku Centre for Biotechnology, University of Turku, Abo Akademi University, Finland.
EMBO J. 2001 Jul 16;20(14):3800-10. doi: 10.1093/emboj/20.14.3800.
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.
热休克因子1（HSF1）是应激反应中一种富含丝氨酸的组成型磷酸化介质。在应激状态下，HSF1形成DNA结合三聚体，重新定位于核颗粒，经历诱导性磷酸化并获得反式激活因子的特性。HSF1在多个位点被磷酸化，但其位点及其功能仍是一个谜。在此，我们分析了人类HSF1内源性磷酸化位点，并开发了一种磷酸肽抗体以鉴定Ser230为一个新的体内磷酸化位点。Ser230位于HSF1的调节结构域内，并促进诱导性转录活性的强度。Ser230位于钙/钙调蛋白依赖性蛋白激酶II（CaMKII）的共有位点内，CaMKII的过表达增强了体内Ser230的磷酸化水平以及HSF1的反式激活作用。当在hsf1（-/-）细胞中表达时，S230A HSF1突变体相对于野生型HSF1表现出明显降低的活性，这进一步证实了Ser230的重要性。我们的研究提供了首个证据，即磷酸化对于HSF1的转录活性至关重要，因此对于热休克反应的诱导也至关重要。
