Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity

Abstract

HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.

Turn me on: regulating HIF transcriptional activity

“…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. 53 Finally, in vitro interaction assays demonstrated that phosphorylation alone at this position did not enhance binding to p300, 41 and rather indicated that phosphorylation at Thr796 inhibited hydroxylation by FIH-1, facilitating increased p300 binding and CAD activity.…”

Structure of Human Phytanoyl-CoA 2-Hydroxylase Identifies Molecular Mechanisms of Refsum Disease*

“…The region between residues 223 and 233 appears to extend from a long loop between DSBH ␤-strands IV and V (␤-9 and ␤-10) and probably serves to enclose the active site. The region between residues 303 and 318 is located prior to the C-terminal helix, which for some 2OG oxygenase family members has been shown to play a role in substrate recognition (56); in FIH, the C-terminal helices are involved in dimerization in addition to substrate recognition (57). Assuming the structure of PAHX as observed represents an "open" conformation with its disordered loops, a large groove leading to the active site is clearly visible on its surface.…”

Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

“…Additionally, it has also been reported that Thr-796 in the C-TAD of HIF-1␣ might be phosphorylated because its mutation to alanine strongly reduced the transcriptional activity of HIF-1␣ (22). It is likely that phosphorylation of this residue, by a yet unidentified kinase, inhibits the interaction with factor inhibiting HIF-1, thereby preventing hydroxylation and facilitating binding of C-TAD to CBP (41). Thus, multiple direct phosphorylation events appear to regulate the activity of HIF-1␣ at different steps along its activation pathway.…”