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

Katarina Gradin, Chikahisa Takasaki, Yoshiaki Fujii-Kuriyama, Kazuhiro Sogawa
2002 Journal of Biological Chemistry  
The hypoxia-inducible factor (HIF)-1␣ and the HIFlike factor (HLF) transcription factors are regulated at multiple levels including protein stabilization, nuclear import, and activation of transactivation, resulting in recruitment of coactivators such as the cAMP-response element-binding protein (CREB)-binding protein (CBP)/ p300 and SRC-1. During low oxygen tension these proteins modulate a network of genes that are necessary for angiogenesis, erythropopoiesis, and glycolysis. We report here
more » ... at the C-terminal transactivation domain of HLF is phosphorylated on multiple sites and that phosphorylation on threonine 844 of HLF is necessary for the transcriptional activation function of the protein independently of the hypoxia condition. Importantly, using the mammalian two-hybrid system we demonstrate that a substitution of threonine 844 to an alanine decreased the enhanced transcriptional activation function mediated by CBP/p300. Low oxygen tension (hypoxia) plays an important role in diseases such as cancer, cerebral and myocardial ischemia, and chronic lung and heart diseases (reviewed in Ref. 1). Oxygen tension is also an important factor for regulation of mammalian genes that are involved in angiogenesis, vasculogenesis, glucose metabolism, and apoptosis (for a review see Ref. 1). These oxygen-sensitive genes are regulated by the hypoxia-inducible factor (HIF)-1␣, 1 (2, and reviewed in Ref. 3) or the homologue protein HIF-like factor (HLF) (4), which also is termed EPAS1, HIF-2␣, HRF, or MOP2 (reviewed in Ref. 5). The mechanism of activation of oxygen-regulated genes has been determined mainly by studies of HIF-1␣; however, the important functional domains in HIF-1␣ are highly conserved in HLF (4). Moreover, HIF-1␣ appears to have a general role by conferring oxygendependent regulation to the transcription machinery in all cells, whereas HLF is cell type-restricted and plays a more specialized role, according to analysis of knock-out mice of HIF-1␣ and HLF (6 -8). The N-terminal half of these proteins contains a basic helix-loop-helix domain (bHLH) and a PER-ARNT-SIM (PAS) homology domain. The basic domain is necessary for DNA-binding, and both the helix-loop-helix and the PER-ARNT-SIM homology domain are necessary for dimerization of HIF-1␣ and HLF to their partner factor, the aryl hydrocarbon nuclear translocator (ARNT) protein. Under hypoxic conditions the heterodimer binds to specific consensus DNA sequences and up-regulates genes such as erythropoietin, vascular endothelial growth factor, and glycolytic enzymes (for a review see Ref. 1). The stimulatory effect of hypoxia is caused by stabilization of the oxygen-sensitive HIF-1␣ and HLF. At normoxia these two proteins are rapidly degraded through the ubiquitin-proteasome machinery that involves the von Hippel-Lindau tumor suppressor (pVHL) E3 ubiquitin ligase complex (9 -11). pVHL interacts with the HIF proteins through a short peptide motif containing a hydroxylated proline (12-15). The prolyl hydroxylases that are necessary for post-translational modification are oxygen-dependent, resulting in lower levels of pVHL binding and thus stabilization of HIF-1␣ (12-15). In the C-terminal half of both HIF-1␣ and HLF two transactivation domains, the N-terminal transactivation domain and C-terminal transactivation domain (CAD) are located, and it has been shown that these transactivation domains interact with coactivators such as the histone acetyltransferase cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300, SRC-1, and TIF-2 (16 -18). It also has been reported that CAD is a direct target of redox regulation because the interaction of CBP/p300 and SRC-1 with HIF-1␣ and HLF is increased by the redox factor 1 (REF-1) and by thioredoxin (17, 18) . In addition, Lando et al. (19) recently have shown that hypoxic induction of CAD activity is regulated by hydroxylation of a conserved asparagine. Recent studies have shown that HLF and HIF-1␣ are phosphoproteins and that the proteins are phosphorylated in the hypoxic cell (20 -22). The involvement of protein kinases has mainly been proposed using inhibitors for tyrosine and serine/ threonine kinases that block the transactivation function of HLF and HIF-1␣ (20, 23). More recently, it has been reported that the Ser/Thr kinase Akt regulates HIF-1␣ stability (22), and some reports also suggest that mitogen-activated protein kinases phosphorylate HIF-1␣ and increase the transcriptional activity of the protein (21). However, the sites for phosphorylation and the mechanism behind the enhanced transactivation function have not yet been elucidated. In this study we were interested to investigate the role of phosphorylation in modulating the transactivation function of HLF. To understand the role of phosphorylation of the Cterminal transactivation domain in a cellular context, we have
doi:10.1074/jbc.m201307200 pmid:11983697 fatcat:jffdtpfihvfdxgdv6vxbwyt2zu