Fig 1: VHL R167Q mutant fails to ubiquitinate and degrade AURKA. (a) Schematic of VHL24 (long isoform of VHL, VHLL) showing the ß-domain (amino acid 63–204) and a-domain (amino acid 155–193) and the R167Q mutation. (b) Lysates from 786-0 (parental) to isogenic 786-0 cells stably re-expressing WT (VHL24), or R167Q VHL mutant probed with the indicated antibodies. Arrows indicate the expected bands for VHL. (c) Densitometric quantitation showing an average ratio of AURKA expression to tubulin. Parental-black bar, WT-gray bar, R167Q-black bar with white dots. *P<0.0001. (d) In vivo ubiquitination assay using lysates from 786-0 to isogenic 786-0 VHL24 (WT), and R167Q cell lines treated with MG132. Ubiquitinated AURKA probed using anti-ubiquitin P4D1 antibody. Arrowhead indicates the expected molecular weight of monoubiquitinated AURKA. Input lysates probed with AURKA, VHL and tubulin. Arrows indicate the expected bands for VHL. (e) Graphical representation of densitometric quantitation (measured from 50 to 200 kD) showing an averaged ratio of ubiquitinated AURKA to the pull down efficiency. Black bar (parental 786-0), gray bar (WT), black bar with white dots (R167Q). *P<0.01 All error bars denote s.e.m.
Fig 2: AURKA and HDAC6 inhibition rescues ciliogenesis in VHL-deficient cells. (a) Representative images from hTERT RPE-1 cells transiently transfected with siControl (siC) or siVHL, treated with vehicle (DMSO), alisertib (MLN8237) or rocilinostat (ACY1215) at the time of serum withdrawal for 48 h. Ciliation monitored by immunofluorescent staining using acetylated a-tubulin (cilia marker) and pericentrin (basal body marker). Nuclei counterstained using DAPI. Highlighted boxes show magnified cilia. Scale bar, 3 µM. (b, d) Percentage of cells that fail to ciliate (unciliated) normalized to 1, from siC or siVHL transfected cells treated with (b) alisertib or (d) rocilinostat. * and # denote statistical significance. (c, e) Cilia length (µM) from siC or siVHL transfected cells treated with (c) alisertib or (e) rocilinostat. At least 100 cells were counted from each replicate. * and # denote statistical significance. All error bars indicate s.e.m. (f) Model depicting a role for VHL’s ubiquitin ligase function in conditions of normoxia targeting HIFa and hypoxia targeting AURKA.
Fig 3: VHL modulates AURKA protein levels. (a) Lysates from 786-0 to isogenic 786-0 cells overexpressing VHL (VHL24) cultured at sub-confluent (cycling) or confluent and serum starved conditions (48 h) probed as indicated. (b) Densitometric quantitation of the average ratio of AURKA to tubulin expression from 786-0 (black bars) to 786-0 VHL24 (gray bars) cells. *P<0.01. (c) Lysates harvested from 786-0 to 786-0 VHL24 cells cultured to confluence and serum starved for 24 h before treatment with CHX for the indicated time points probed as shown. (d) Densitometric quantitation showing an average ratio of AURKA to tubulin in 786-0 (black bars) and 786-0 VHL24 (gray bars) cells treated with CHX. *P<0.01. # Denotes significant differences exclusively in the 786-0 VHL24 cell line at the each of the indicated CHX treatment time points compared with the 0- h time point (P<0.05). Error bars denote s.e.m.
Fig 4: VHL promotes AURKA degradation as cells enter quiescence. (a) In vivo ubiquitination assay using hTERT RPE-1 cells overexpressing EGFP-AURKA and HA-Ub with and without overexpressed VHL24 in the presence of MG132, harvested following serum withdrawal at the indicated time points. Ubiquitinated AURKA immunoprecipitated using an anti-HA antibody and probed with an anti-AURKA antibody. Input lysates immunoblotted for the indicated antibodies. (b) Lysates from hTERT RPE-1 cells overexpressing EGFP-AURKA and HA-Ub with and without overexpressed VHL24 in the absence of MG132 probed for the indicated antibodies. (c) Densitometric quantitation showing an average ratio of EGFP-AURKA to tubulin in cells without (black bars) and with (gray bars) overexpressed VHL24 at the indicated time points. *P<0.01. (d, e) Densitometric quantitation of EGFP-AURKA to tubulin expression averaged from three independent experiments at each of the indicated time points normalized to the 0-h time point in the absence of MG132 treatment (d) and the presence of MG132 treatment (e). *P<0.01 (compared with the 0-h time point). All error bars denote s.e.m.
Fig 5: VHL E3 ligase directly ubiquitinates AURKA. (a) 786-0 and isogenic 786-0 cells overexpressing VHL (VHL24) treated with DMSO (vehicle), proteasome inhibitors (MG132 and Bort (Bortezomib)) or lysosome inhibitor (Baf (Bafilomycin A)). Immunoblots probed for the indicated antibodies. (b) Densitometric quantitation from 786-0 (black bars) to 786-0 VHL24 (gray bars) cells showing an averaged ratio of AURKA to tubulin in the absence and presence of MG132. *P<0.00001. (c) In vivo ubiquitination assay using lysates from 786-0 to 786-0 VHL24 cell lines in the absence or presence of MG132. Immunoprecipitated AURKA probed using an anti-ubiquitin (P4D1) antibody, and pull down efficiency measured with an anti-AURKA antibody. Arrowhead indicates the expected molecular weight of monoubiquitinated AURKA. Input shows AURKA, VHL and tubulin. (d) Graphical representation of densitometric quantitation (measured from 50 to 200 kD) showing an average ratio of ubiquitinated AURKA (probed using the P4D1 anti-ubiquitin antibody) to the pull down efficiency (using an anti-AURKA antibody). Black and gray bars represent the ratio in 786-0 and 786-0 VHL24 cells, respectively. *P<0.01. (e) In vivo ubiquitination assay performed using hTERT RPE-1 cells overexpressing Dendra2C-AURKA and HA-Ub with and without overexpressed VHL24 in the absence and presence of MG132. Ubiquitinated AURKA immunoprecipitated under denaturing conditions using an anti-HA antibody and probed with an anti-AURKA antibody. Input lysates immunoblotted for the indicated antibodies. (f) In vitro ubiquitination assay. All error bars denote s.e.m.
Supplier Page from DNASU for AURKA (Homo sapiens) in pLP-EGFP-C1 (EGFP-tagged mammalian expression vector)