Journal of Molecular Biology
Volume 387, Issue 5, 17 April 2009, Pages 1106-1119
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Heat Shock Protein 90 Is Important for Sp1 Stability during Mitosis

https://doi.org/10.1016/j.jmb.2009.02.040Get rights and content

Abstract

Our previous study has revealed that heat shock protein (Hsp) 90 can interact with Sp1 to regulate the transcriptional activity of 12(S)-lipoxygenase. Herein, we further found that the interaction between Hsp90 and Sp1 occurred during mitosis. By geldanamycin (GA) treatment and knockdown of Hsp90, we found that this interaction during mitosis was involved in the maintenance of Sp1 stability, and that the phospho-c-Jun N-terminal kinase (JNK)-1 level also decreased. As the JNK-1 was knocked down by the shRNA of JNK-1, Sp1 was degraded through a ubiquitin-dependent proteasome pathway. In addition, for mutation of the JNK-1 phosphorylated residues of Sp1, namely, Sp1(T278/739A) and Sp1(T278/739D), the effect of GA on Sp1 stability was reversed. Finally, based on the involvement of Hsp90 in Sp1 stability, the transcriptional activities of p21WAF1/CIP1 and 12(S)-lipoxygenase under GA treatment were observed to have decreased. Taken together, Hsp90 is important for maintaining Sp1 stability during mitosis by the JNK-1-mediated phosphorylation of Sp1 to enable division into daughter cells and to regulate the expression of related genes in the interphase.

Introduction

Heat shock protein (Hsp) 90, a constituent molecular chaperone, is an abundant protein comprising 2% of the total cellular protein content under nonstress conditions. It is essential for many signal-transduction-regulating cellular proteins such as transcription factors, protein kinases, and nitric oxide synthase. It is also involved in various cellular processes such as cell proliferation, differentiation, and apoptosis.1, 2, 3, 4, 5 Unlike Hsp70, Hsp90 does not generally act on nascent protein folding; instead, it binds to its client proteins to stabilize protein folding.6, 7 The crystal structure of Hsp90 reveals that the N-terminal domain of Hsp90 binds adenosine 5′-triphosphate (ATP), which is consistent with the observation that ATP hydrolysis is required for conformational changes involved in the refolding of protein substrates or in the client proteins of Hsp90.8 Geldanamycin (GA), a benzoquinone ansamycin, and radicicol, a macrocyclic antifungal antibiotic, compete for the ATP-binding site on Hsp90 to inhibit its activity.9, 10 Hsp90 resides mostly in the cytoplasm to form a major functional component of an important cytoplasmic chaperone complex.11 However, it has also been found to be present inside the nucleus and outside the cells in stressed, unstressed, and cancer cells.12, 13, 14, 15 Most studies of nuclear Hsp90 focus on how Hsp90 shifts the glucocorticoid receptor into the nucleus and regulates its nuclear retention.15, 16 Our previous study17 also revealed that Hsp90 interacts with Sp1 to regulate 12(S)-lipoxygenase expression. However, because Hsp90 and Sp1 are mostly localized within the cytoplasm and the nucleus, respectively, the manner in which Hsp90 affects the proteins inside the nucleus remains unclear.

Sp1 was one of the first transcription factors to have been purified and cloned from mammalian cells.18, 19 It can recognize and specifically bind to GC-rich sites within the simian virus 40 promoter via three Cys2His2 zinc-finger motifs localized at its carboxyl-terminal region in order to regulate the transcription of its target genes.20, 21 In addition to the zinc-finger domain of the C-terminal region, the amino-terminal regions of Sp1 are much more variable and contain transcriptional activation or repression domains.22, 23 Sp1 is generally considered as a factor that primarily determines the core activity of the promoter (1) by direct interaction with the factors of the basal transcription machinery and (2) by cooperation with several transcriptional activators such as CRSP, p300/CBP, steroidogenic factor-1, vitamin D3 receptor, and TAFII130.24, 25, 26, 27, 28, 29 Recent studies have revealed that the transcriptional activity of Sp1 is mainly determined by at least three factors, namely, transactivational activity, DNA binding activity, and protein expression levels. Sp1 activates transcription from proximal and distal promoters; therefore, it possesses transactivational activity.30 Previous studies have shown that diverse kinase pathways phosphorylate different serine or threonine residues of Sp1; for example, serine 131 of Sp1 is phosphorylated by DNA-dependent protein kinase, thereby increasing Sp1 transcriptional activity.31 Threonine 579 of Sp1 is phosphorylated by casein kinase II (CKII), which attenuates its DNA binding ability.32 Rohlff et al. reported that a reduction in the level of Sp1 phosphorylation presents a proteolytic phenomenon. Protein kinase A phosphorylates the N-terminus of Sp1, and the up-regulated DNA binding activity increases the transcriptional activity of Sp1.33 In addition, acetylation of lysine 703 of Sp1 recruits HDAC1 and p300 to the 12(S)-lipoxygenase promoter.34 Therefore, the posttranslational modifications of Sp1 brought about by other factors are said to be important for regulating Sp1 activity.

c-Jun N-terminal kinases (JNKs) are a major group of the mitogen-activated protein kinase family that are responsive to stress stimuli such as ultraviolet irradiation, proinflammation, heat shock, and reactive oxygen species.35, 36 JNKs consist of three isoforms of which JNK-1 and JNK-2 are ubiquitously distributed and JNK-3 is found in neuronal tissues.37 JNK inactivation causes degradation of c-Jun, ATF2, JunB, and p53; however, phosphorylation of JNK would protect these proteins from ubiquitination.38, 39, 40 Thus, JNK activity may be important for the modulation of cell cycle progression and the regulation of the stability of some proteins. Recently, it has been shown that in bile-acid-inducible DR5/tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) R2, which is an apoptosis-inducing membrane receptor for TRAIL, expression is under the control of the JNK pathway, which targets the transcription factor Sp1.41 In addition, Sp1 drives transcription from the − 243/− 137-bp region of the DR5/TRAIL-R2 promoter, which contains two Sp1-binding sites (located at − 198/− 189 and − 152/− 143 bp).41 Interestingly, the JNK-1/2 inhibitor SP600125 reduces the formation of the Sp1-DR5/TRAIL-R2 promoter DNA complex, which has been demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) experiments.41 Our previous study found that Sp1 localized at Thr278 and Thr739 can be phosphorylated through JNK during the mitotic period.42 Moreover, we found that Sp1 phosphorylated during the mitotic stage can protect its stability from ubiquitination in a proteasome-dependent pathway.42

In this study, we found that Sp1 interacts with Hsp90 primarily in the mitotic stage. Hsp90 protects Sp1 from ubiquitination in order to stabilize it and then increases the transcriptional activities of the target genes regulated by Sp1. In addition, inactivation of JNK, GA, and shRNA-Hsp90 renders Sp1 unstable during mitosis. Taken together, our results indicate that Hsp90 modulates Sp1 phosphorylation during mitosis through JNK activation, regulates Sp1 stability, and mediates the transcriptional activities of the target genes regulated by Sp1.

Section snippets

Sp1 interacts with Hsp90 in the mitotic stage

Our previous findings17 indicate that Hsp90, owing to its interaction with Sp1, is important for 12(S)-lipoxygenase expression. Because Hsp90 is localized primarily in the cytoplasm and Sp1 is inside the nucleus, we examined the colocalization of Hsp90 and Sp1 in different cell cycle periods (Fig. 1). An immunofluorescence assay showed that, in the interphase, the genome was more relaxed and Sp1 was evenly distributed within the nucleus (Fig. 1A, a1 and b1); however, almost all of Hsp90

Discussion

Our previous finding revealed that the interaction between Sp1 and Hsp90 is involved in the transcriptional activity of 12(S)-lipoxygenase.17 This study further found that the interaction between Sp1 and Hsp90 occurred during mitosis, and it then maintained the Sp1 level by increasing Sp1 stability via facilitation of JNK-1-mediated phosphorylation of Sp1 to enable division into daughter cells for the regulation of its target genes (Fig. 7e).

The molecular chaperone Hsp90 has emerged as an

Materials

Polyclonal antibodies against ubiquitin, Hsp90, and JNK, and monoclonal antibodies against cyclin B1 and glutathione S-transferase (GST) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-JNK (Thr183/Thr185) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal antibody against GFP was obtained from BD Biosciences PharMingen (San Diego, CA). Monoclonal antibodies against Sp1 and p21WAF1/CIP1 were purchased from Upstate Biotechnology (Lake Placid,

Acknowledgements

This work was supported by the National Cheng Kung University Program for Promoting Academic Excellence and Developing World Class Research Centers and by grant NSC 97-2311-B-006-002-MY3 from the National Science Council of Republic of China.

References (63)

  • SuskeG.

    The Sp-family of transcription factors

    Gene

    (1999)
  • IwamotoI. et al.

    Differential effects of two C-terminal peptides of substance P on human neutrophils

    Neuropeptides

    (1990)
  • XiaoH. et al.

    p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor

    J. Biol. Chem.

    (2000)
  • SzpirerJ. et al.

    The Sp1 transcription factor gene (SP1) and the 1,25-dihydroxyvitamin D3 receptor gene (VDR) are colocalized on human chromosome arm 12q and rat chromosome 7

    Genomics

    (1991)
  • CoureyA.J. et al.

    Synergistic activation by the glutamine-rich domains of human transcription factor Sp1

    Cell

    (1989)
  • ArmstrongS.A. et al.

    Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity

    J. Biol. Chem.

    (1997)
  • RohlffC. et al.

    Modulation of transcription factor Sp1 by cAMP-dependent protein kinase

    J. Biol. Chem.

    (1997)
  • DavisR.J.

    Signal transduction by the JNK group of MAP kinases

    Cell

    (2000)
  • NagataY. et al.

    Activation of JNK signaling pathway by erythropoietin, thrombopoietin, and interleukin-3

    Blood

    (1997)
  • SabapathyK. et al.

    Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation

    Mol. Cell

    (2004)
  • FuchsS.Y. et al.

    c-Jun NH2-terminal kinases target the ubiquitination of their associated transcription factors

    J. Biol. Chem.

    (1997)
  • HiguchiH. et al.

    Bile acids up-regulate death receptor 5/TRAIL-receptor 2 expression via a c-Jun N-terminal kinase-dependent pathway involving Sp1

    J. Biol. Chem.

    (2004)
  • ZhangF. et al.

    Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6

    J. Biol. Chem.

    (2007)
  • MerchantJ.L. et al.

    Sp1 phosphorylation by Erk 2 stimulates DNA binding

    Biochem. Biophys. Res. Commun.

    (1999)
  • BenasciuttiE. et al.

    MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription

    Blood

    (2004)
  • PengC. et al.

    Inhibition of heat shock protein 90 prolongs survival of mice with BCR-ABL-T315I-induced leukemia and suppresses leukemic stem cells

    Blood

    (2007)
  • VicartA. et al.

    Increased chromatin association of Sp1 in interphase cells by PP2A-mediated dephosphorylations

    J. Mol. Biol.

    (2006)
  • SafeS. et al.

    Sp transcription factor family and its role in cancer

    Eur. J. Cancer

    (2005)
  • WangY.T. et al.

    Sumoylation of specificity protein 1 augments its degradation by changing the localization and increasing the specificity protein 1 proteolytic process

    J. Mol. Biol.

    (2008)
  • JascurT. et al.

    Regulation of p21(WAF1/CIP1) stability by WISp39, a Hsp90 binding TPR protein

    Mol. Cell

    (2005)
  • YoungJ.C. et al.

    Pathways of chaperone-mediated protein folding in the cytosol

    Nat. Rev. Mol. Cell Biol.

    (2004)
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