Beschreibung:
<jats:p>The asparaginyl hydroxylase Factor Inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. Besides HIF-α, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD containing substrate to date. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity via interaction with partitioning defective 3 homolog (Par-3). We identified ASPP2 as a new substrate of FIH-1 by mass spectrometry while inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of the enzyme. However, FIH-1 depletion led to impaired binding of Par-3 to ASPP2 while neither interaction between ASPP2 and p53, nor apoptosis or proliferation of the cancer cells were affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in dislocation of ASPP2 from cell cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD containing substrates can be modified by FIH-1 dependent hydroxylation. The large cellular pool of ARD containing proteins may imply effects of FIH-1 on a broad range of cellular functions and signalling pathways, for example in response to severe hypoxia.</jats:p>