Abstract
The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.
Highlights
The post-translational hydroxylation of extracellular proteins such as collagen is well characterized, examples of hydroxylated intracellular proteins are very limited [1]
The notch intracellular domain (ICD) is recruited to target genes by the transcription factor CSL (CBF-1/suppressor of hairless/Lag-1), where, through its ARD, it coordinates the assembly of a nuclear transcriptional activation complex involving Mastermind-like (MAML) proteins and Ski-interacting protein (SKIP) [7, 8]
Given tion to levels comparable with that observed with HIF1␣C-terminal transactivation domain (CAD); mutation of both Asn residues reduced it to control levels
Summary
The post-translational hydroxylation of extracellular proteins such as collagen is well characterized, examples of hydroxylated intracellular proteins are very limited [1]. GST or GST-⌬MAML1 (amino acids 7–254 of human MAML1 containing the Notch binding domain) was incubated with extract from PK-⌬N1 ICD-transfected cells overexpressing (FIH transfection) or underexpressing (siRNA) FIH (see Fig. 1D).
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