Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH)

Abstract

Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IB␣ were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARDcontaining proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.NF-B ͉ 2-oxoglutarate-dependent dioxygenase ͉ protein hydroxylation C ells react to variation in oxygen availability with adaptive responses that involve changes in most basic cellular functions. Analysis of the transcriptional component of this response has defined pathways that regulate hypoxia-inducible factor (HIF) by posttranslational hydroxylation of specific residues. HIF is an ␣͞␤ heterodimer that binds hypoxia response elements in a range of hypoxia-inducible genes (for review, see ref. 1). Regulation is mediated by the ␣-subunits and involves dual mechanisms controlling both the abundance and activity of the protein. Thus, hydroxylation of specific prolyl residues promotes interaction with the von Hippel-Lindau E3 ligase and hence proteolysis, whereas hydroxylation of a C-terminal Asn residue blocks recruitment of the coactivators p300͞CBP. The prolyl and asparaginyl hydroxylase enzymes that catalyze these reactions are 2-oxoglutarate (2-OG) and Fe(II)-dependent dioxygenases that couple the oxidative decarboxylation of 2-OG with oxidation of peptidyl substrates. Dioxygen is an obligate cosubstrate, and reductions in the rate of hydroxylation during hypoxia allow HIF-␣ to escape VHLmediated destruction and to activate transcription (for reviews, see refs. 2 and 3).HIF prolyl hydroxylation is catalyzed by three enzymes, PHD1, -2, and -3 (equivalent to EGLN2, -1, and -3 and HPH-3, -2, and -1). HIF Asn hydroxylation is catalyzed by a more distantly related 2-OG-dependent dioxygenase, factor inhibiting HIF (FIH) (for reviews, see refs. 2 and 3). A key question raised by these findings is whether the roles of all four dioxygenases are specific to HIF regulation, or whether one or more have alternative substrates. Several studies have identified proteins that interact to modulate HIF hydroxylase activity (4) or ...

Turn me on: regulating HIF transcriptional activity

“…All these proteins contain ankyrin-repeat domains (ARDs) and include IkBa; p105; Notch1, 2 and 3; and Ankyrin repeat and SOCS Box protein 4 (ASB4). [44][45][46] Presently, the function of hydroxylation on these novel substrates remains unclear, with only subtle downstream effects, if any, being observed. However, it has been suggested that hydroxylation of ARD-containing proteins occurs in competition with HIF hydroxylation, and could potentially regulate HIF-mediated transcription by sequestering FIH-1, particularly in hypoxia.…”

Hypoxia and gastrointestinal disease

“…Indeed, recent studies have indicated that the nuclear factor (NF) κB pathway may also be regulated in a similar manner. Hypoxia activates NF-κB, and this appears at least in part to be mediated through altered hydroxylation of critical components of this pathway [33][34]. It is interesting to note that like conditional HIF-1α-null mice, deletion of the NF-κB pathway in intestinal epithelial cells leads to increased susceptibility to colitis indicating a protective role for epithelial NF-κB in colitis.…”

“…While the role of hydroxylases in the hypoxic sensitivity of the HIF pathway has been clearly demonstrated, recent data raises the intriguing possibility that components of the NF-κB pathway may also be substrates of hydroxylases including PHD1 and FIH [33][34]. It is interesting to note that as well as being hypoxia sensitive, both the HIF and NF-κB pathways are regulated by inflammatory mediators including cytokines and bacterial products such as lippopolysaccharide [35][36] (Fig.…”

The asparaginyl hydroxylase factor-inhibiting HIF is essential for tumor growth through suppression of the p53–p21 axis

“…However, it is tempting to suggest that FIH acts on p53 through the same mechanism as it does on HIF, that is, through inhibition of its interaction with p300/CBP. Cockman et al (2006) proposed a second class of FIH substrates that contained an ankyrin repeat domain (ARD). p105 and IkBa were the first ARD-containing proteins to be characterized, which was then followed by notch1, rabankyrin-5, RNaseL and toankyrase-1 (Cockman et al, 2009).…”