Figure 2: Increased K63-linked polyubiquitination of HAUSP at amino acid K443 under hypoxia and its identification by MS/MS spectra.
(a) Hypoxia further increased the deubiquitinase activity of HAUSP when HAUSP (wild type or CS mutant), HIF-1α, HA-ubiquitin were co-expressed in 293T cells under MG-132 treatment to measure the polyubiquitinated HIF-1α levels under normoxia (N) or hypoxia (H). The densitometer reading of polyubiquitinated HIF-1α levels was listed underneath each lane. The first lane was arbitrarily set as 1. (b) Increased K63-linked polyubiquitination of HAUSP under hypoxia in two cell lines (H1299 and PC3) using anti-HAUSP antibodies to pull down HAUSP proteins followed by Western blot analysis using either anti-K63, anti-K48 or anti-ubiquitin antibodies. N, normoxia; H, hypoxia. (c) Increased polyubiquitination in HAUSP wild type, but not the K443R mutant, under hypoxia when HAUSP (wild type or K443R mutant) and HA-ubiquitin were co-expressed in 293T cells followed by precipitation by anti-HA antibodies. (d) The ubiquitination site K443 on QKTDPK (2+, m/z 416.2) was annotated to conjugate with ubiquitin-remnant peptide, Gly-Gly adduct. The peptide NIQKESTL (2+, m/z 524.2) of ubiquitin containing Gly-Gly adduct was identified from digested HAUSP in hypoxia. (e) Inability of the HAUSPK443R mutant to increase the HIF-1α levels when they were co-expressed in 293T cells by Western blot analysis. The HAUSP-CS mutant was used as a negative control. (f) Co-expression of wild type, but not the K443R mutant, HAUSP with HIF-1α further increased the Twist1 promoter-driven reporter activity in transient transfection assays. The condition not-transfected with HIF-1α or HAUSP plasmids under normoxia was used as a control. Data from three independent experiments are expressed as mean±s.d. The asterisk (*) indicated statistical significance (P<0.05) between experimental and control transfections, t-test. (g) In vitro deubiquitination assays showed that the HAUSP wild type (WT), but not the K443R mutant, deubiquitinated the polyubiquitinated HIF-1α. The densitometer reading of polyubiquitinated HIF-1α levels was listed underneath each lane. The first lane was arbitrarily set as 1.
