Differences in hydroxylation and binding of Notch and HIF-1α demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1)

Sarah E. Wilkins, Jaana Hyvärinen, Johana Chicher, Jeffrey J. Gorman, Daniel J. Peet, Rebecca L. Bilton, Peppi Koivunen

Research output: Contribution to journalArticlepeer-review

49 Citations (Scopus)

Abstract

FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-α. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1-3, p105 and IκBα. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1-4 and compared them with those for HIF-1α. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time. Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1α, predominantly through binding affinity rather than maximal reaction velocity. The Km value of FIH-1 for Notch1, <0.2 μM, is at least 250-fold lower than that of 50 μM for HIF-1α. Site 1 of Notch1-3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1-3, but no hydroxylation was detected. Importantly, we demonstrate that the Km of FIH-1 for oxygen at the preferred Site 1 of Notch1-3, 10-19 μM, is an order of magnitude lower than that for Site 2 or HIF-1α. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1α hydroxylation would be reduced.

Original languageEnglish
Pages (from-to)1563-1571
Number of pages9
JournalInternational Journal of Biochemistry and Cell Biology
Volume41
Issue number7
DOIs
Publication statusPublished - Jul 2009
Externally publishedYes

Keywords

  • FIH-1
  • HIF
  • Hydroxylation
  • Notch
  • Oxygen

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