The Transcriptional Activation Function of the HIF-like Factor Requires Phosphorylation at a Conserved Threonine

Gradin, Katarina; Takasaki, Chikahisa; Fujii‐Kuriyama, Yoshiaki; Sogawa, Kazuhiro

doi:10.1074/jbc.m201307200

Cited by 89 publications

(72 citation statements)

References 26 publications

(33 reference statements)

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“…Mutations such as R806A and Q810A had no significant effect on the transactivation function of C-TAD, whereas the mutants N807A and R816A showed an increased transactivation function at hypoxia when compared with the wild type C-TAD. The S805A mutation seemed to have no effect per se on the transactivation function of C-TAD either at normoxia or hypoxia, in agreement with a recent report where mutation of residue Ser 857 in HLF (corresponding to Ser 805 in mHIF-1␣) does not affect transactivation mediated by the C-TAD (36). The expression levels of all of the mutants tested in gene reporter assays were investigated by immunoblot analysis and proven to be very comparable (data not shown).…”

Section: Two Predicted ␣-Helices Within the C-tad Are Critical For Thsupporting

confidence: 78%

Functional Analysis of Hypoxia-inducible Factor-1α-mediated Transactivation

Ruas¹,

Poellinger

Pereira³

2002

Journal of Biological Chemistry

101

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The hypoxia-inducible factor-1␣ (HIF-1␣) is a key regulator of adaptive responses to hypoxia. HIF-1␣ has two independent transactivation domains (TADs). Whereas the N-terminal TAD (N-TAD) also constitutes a degradation box, the C-terminal TAD (C-TAD) functions in a strictly hypoxia-inducible fashion. Oxygen-dependent hydroxylation of an asparagine residue has recently been reported to regulate C-TAD function by disrupting the interaction with the CH1 domain of the p300/CBP coactivator at normoxia. Here we have performed alanine-scanning mutagenesis of a predicted ␣-helix within the C-TAD of mouse HIF-1␣ to identify residues important for transactivation and interaction of the C-TAD with transcriptional coactivators. We observed that several hydrophobic residues, Ile 802 , Leu 808 , Leu 814 , Leu 815 , and Leu 818 , were critical for transactivation and binding to the CH1 domain of CBP in hypoxic cells. Moreover, E812A/E813A and D819A mutations impaired hypoxiadependent transactivation without disrupting binding to CH1. In the context of full-length HIF-1␣, mutation of the leucine residues conferred conformational changes to the protein and significantly reduced the transactivation function as well as functional interaction with the transcriptional coactivators CBP and SRC-1. These mutations also affected intranuclear redistribution of HIF-1␣ in the presence of CBP, indicating that the integrity of the C-TAD is critical for intracellular localization of mouse HIF-1␣.

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Section: Two Predicted ␣-Helices Within the C-tad Are Critical For Thsupporting

confidence: 78%

Functional Analysis of Hypoxia-inducible Factor-1α-mediated Transactivation

Ruas¹,

Poellinger

Pereira³

2002

Journal of Biological Chemistry

101

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“…For example, phosphorylation of hHIF-1a at Thr796 by casein kinase II was first postulated by Gradin et al 51 , and substitution of an aspartic acid residue at this position to mimic phosphorylation increased the interaction of the CAD with p300 within cells. It was subsequently shown that a peptide phosphorylated at this position could not be hydroxylated by FIH-1, 52 and the aspartic acid mutation significantly decreased hydroxylation efficiency in vitro.…”

Section: Other Modifications Affecting Hif-driven Transcriptionmentioning

confidence: 99%

Turn me on: regulating HIF transcriptional activity

2008

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The hypoxia-inducible factors (HIFs) are critical for cellular adaptation to limiting oxygen and regulate a wide array of genes when cued by cellular oxygen-sensing mechanisms. HIF is able to direct transcription from either of two transactivation domains, each of which is regulated by distinct mechanisms. The oxygen-dependent asparaginyl hydroxylase factor-inhibiting HIF-1a (FIH-1) is a key regulator of the HIF C-terminal transactivation domain, and provides a direct link between oxygen sensation and HIFmediated transcription. Additionally, there are phosphorylation and nitrosylation events reported to modulate HIF transcriptional activity, as well as numerous transcriptional coactivators and other interacting proteins that together provide cell and tissue specificity of HIF target gene regulation. The maintenance of oxygen homeostasis is a crucial physiological requirement that involves coordinated regulation of a plethora of genes. The hypoxia-inducible transcription factors (HIFs) are responsible for a major genomic response to hypoxia, where cellular oxygen demand exceeds supply. The HIFs directly regulate the transcription of more than 70 genes involved in cellular processes that act to directly address this deficit by decreasing oxygen dependence and consumption by cells, and by increasing the efficiency of oxygen delivery to cells. These processes include vasculogenesis and angiogenesis, metabolism, vasodilation, cell migration, signalling and cell fate decisions. As such, the HIFs are fundamental to embryonic development and the pathophysiology of many serious human diseases, and are therefore subject to strict regulatory mechanisms that act to limit transcriptional activity to periods of cellular oxygen stress. The HIF proteins are subject to oxygen-dependent regulation, both at the level of protein stability (reviewed in this issue by R Bruick) and transcriptional activity, rendering them almost inactive at normoxia, but potently inducible in hypoxia. The regulation of the transcriptional activity of the HIF proteins, both via oxygen-dependent and oxygen-independent mechanisms, will be discussed in this review. The HIFsHIF is assembled from a-and b-subunits to become a transcriptionally active heterodimer. 1 Both subunits are members of the bHLH/PAS (basic helix-loop-helix/Per-ArntSim homology) family of transcription factors, and both contain transactivation domains (Figure 1). 2,3 Whereas HIF1b, also known as the aryl hydrocarbon receptor nuclear translocator (Arnt1), is a constitutively expressed nuclear protein, the a-subunit is strictly regulated in an oxygendependent manner. There are three paralogues of the HIF-a subunit, HIF-1a, HIF-2a and HIF-3a, and three paralogues of HIF-1b (Arnt1, Arnt2 and Arnt3), with either HIF-1a or HIF-2a being able to heterodimerize with HIF-1b to form the functional HIF transcription factor complexes responsible for the hypoxic shift in gene expression. HIF-3a has no known role as an active transcription factor, and is instead postulated to behave as a negative reg...

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“…We also addressed the question whether HIF-1 would be directly phosphorylated by CK2. Gradin et al 20 indicated that while T796 is phosphorylated in vivo and is a putative CK2 phosphorylation site, the TAD-C domain is not directly phosphorylated by CK2 in vitro. Computer analysis indicated that S551, S581, T700 and S787 in the human HIF-1a are also putative CK2 phosphorylation site with T700 having a higher score.…”

Section: Discussionmentioning

confidence: 99%

“…17,18 However, the C-terminal transactivation domain (TAD-C) of HIF-1a is not directly phosphorylated by ERKs 19 but a negative charge on threonine 796, located in the C-terminal transactivation domain of HIF-1a, seems to be essential for the interaction with CBP/p300 and hence for the activity of HIF-1. 20 These results suggest that kinases other than ERK1/2 could be involved. The minimal consensus CK2 phosphorylation site is S/T-X-X-E/D and acidic amino acids around are favorable.…”

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confidence: 91%

Role for casein kinase 2 in the regulation of HIF‐1 activity

Mottet

Ruys

Demazy

et al. 2005

Intl Journal of Cancer

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Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in vivo. The HIF-1a subunit is highly sensible to oxygen and is rapidly degraded by the proteasome 26S in normoxia. Activation in hypoxia occurs through a multistep process including inhibition of HIF-1a degradation, but also increase in the transactivation activity of HIF-1. Several data indicate that phosphorylation could play a role in this regulation. In this report, we investigated the role of casein kinase 2 (CK2), an ubiquitous serine/ threonine kinase, in the regulation of HIF-1 activity. Hypoxia was capable of increasing the expression of the b subunit of CK2, of inducing a relocalization of this subunit at the plasma membrane, of inducing nuclear translocation of the a subunit and of increasing CK2 activity. Three inhibitors of this kinase, DRB (5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole), TBB (4,5,6,7-tetrabromotriazole) and apigenin, as well as overexpression of a partial dominant negative mutant of CK2a, were shown to inhibit HIF-1 activity as measured by a reporter assay and through hypoxiainduced VEGF and aldolase expression. This does not occur at the stabilization process since they did not affect HIF-1a protein level. DNA-binding activity was also not inhibited. We conclude that CK2 is an important regulator of HIF-1 transcriptional activity but the mechanism of this regulation remains to be determined. Since HIF-1 plays a major role in tumor angiogenesis and since CK2 has been described to be overexpressed in tumor cells, this new pathway of regulation can be one more way for tumor cells to survive. ' 2005 Wiley-Liss, Inc.Key words: casein kinase 2; HIF-1; neoangiogenesis HIF-1 (hypoxia-inducible factor-1) is a key regulator of cell response to reduced oxygen level. In response to hypoxic conditions, HIF-1 increases the expression of downstream target genes such as erythropoietin (EPO), vascular endothelial growth factor (VEGF) and glycolytic enzymes (aldolase A, enolase-a) and mediates adaptation of cells to decreased oxygen level. 1 The role of HIF-1 in the regulation of tumor growth by hypoxia via the initiation of angiogenesis is certainly the best example of such an adaptive response. 2 Oncogene activation and tumor-suppressor inactivation result in deregulated cellular proliferation. However, most tumors grown larger than 1 mm 3 contain regions of low oxygen tension (hypoxia) due to an imbalance between oxygen supply and consumption. Formation of new blood vessels or ''neoangiogenesis'' is thus essential for further tumor growth. It is also important to allow tumor cell dissemination at distant sites, i.e., metastasis.Several studies using HIF-1 mutant cells have shown that HIF-1 has a profound effect on tumor biology. For example, tumors grown from HIF-1a-defective embryonic stem cells display abnormal vascularity and reduced growth rate. 3 Moreover, HIF-1 is upregulated in a broad r...

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The Transcriptional Activation Function of the HIF-like Factor Requires Phosphorylation at a Conserved Threonine

Cited by 89 publications

References 26 publications

Functional Analysis of Hypoxia-inducible Factor-1α-mediated Transactivation

Functional Analysis of Hypoxia-inducible Factor-1α-mediated Transactivation

Turn me on: regulating HIF transcriptional activity

Role for casein kinase 2 in the regulation of HIF‐1 activity

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