Differences in hydroxylation and binding of Notch and HIF-1alpha demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1).
FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-alpha. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1-3, p105 and I?B?. 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-1alpha. 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-1alpha, predominantly through binding affinity rather than maximal reaction velocity. The K(m) value of FIH-1 for Notch1, < 0.2 microM, is at least 250-fold lower than that of 50 microM for HIF-1alpha. 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 K(m) of FIH-1 for oxygen at the preferred Site 1 of Notch1-3, 10-19 microM, is an order of magnitude lower than that for Site 2 or HIF-1alpha. 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-1alpha hydroxylation would be reduced.
Wilkins
Sarah E
SE
School of Molecular and Biomedical Science and the Australian Research Council Special Research Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, SA 5005, Australia.
Hyvärinen
Jaana
J
Chicher
Johana
J
Gorman
Jeffrey J
JJ
Peet
Daniel J
DJ
Bilton
Rebecca L
RL
Koivunen
Peppi
P
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010455:Peptides; D051880:Receptors, Notch; D011994:Recombinant Proteins; D012097:Repressor Proteins; D001216:Asparagine; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; D010100:Oxygen