Fig 1: Abrogation of the interaction between centrosomal protein of 192 kDa (CEP192) conserved helix (Helix-1) and Aurora kinase A (AURKA) leads to the mitotic defects.(A) Examples of immunofluorescence confocal images of the HeLa cells transfected with pEGFP-C1-empty vector (control), pEGFP-C1-CEP192506–530WT, or pEGFP-C1-CEP192506–530F508S constructs. The cells were stained with anti-AURKA (orange), anti-CEP192 (red) antibodies, and Hoechst 33342 (blue). Scale bar, 10 μm. Data shown are representative of three biological replicate experiments. (B) Quantification of mitotic phenotypes shown in (A). Bars represent mean values ± SEM of n = 3 biological replicate experiments. Approximately 100 cells were quantified for each condition. (C) Intercentrosomal distances between centrosomes marked with anti-CEP192 antibody in cells with a bipolar spindle. The statistical significance of the measurements was determined using the Kruskal-Wallis test, followed by a Dunn’s multiple comparisons test; n = 37 (control), n = 46 [wild type (WT)], and n = 45 (F508S) cells. Bars indicate means ± SEM; ns, not significant; ****P ≤ 0.0001.
Fig 2: The conserved Helix-1 within AURKA-binding domain (AURKA-BD) of centrosomal protein of 192 kDa (CEP192) binds the kinase domain of AURKA.(A) Alignment of CEP192 sequences from different species [Homo sapiens (UniProt ID: Q8TEP8), Mus musculus (UniProt ID: E9Q4Y4), Bos taurus (UniProt ID: A0A3Q1LJH6), Cavia procellus (UniProt ID: H0V7I4), Gallus gallus (UniProt ID: A0A1D5PSP1), Danio rerio (UniProt ID: A0A0R4IEX1), and Xenopus tropicalis (UniProt ID: A0A6I8S9H5)] around Helix-1 within AURKA-binding domain (AURKA-BD) (top) and domain diagram showing the constructs used in the Isothermal titration calorimetry (ITC) experiments (bottom). (B to G) ITC titrations of maltose binding protein (MBP)–CEP192 constructs (as indicated) into AURKA123–403. Listed with each titration are the concentrations of the protein in the syringe and in the cell, as well as the parameters of the fit (stoichiometry N, dissociation constant Kd). Errors correspond to the SD of the fits.
Fig 3: Crystal structure of centrosomal protein of 192 kDa (CEP192)–Aurora kinase A (AURKA)chimera.(A) Size exclusion chromatography–multiangle light scattering analysis of CEP192-AURKAchimera and AURKA123–403. The molar mass (right y axis) and the UV280nm (left y axis) are plotted. The theoretical masses are given in parentheses. (B) Left: Ribbon representation of the structure of CEP192-AURKAchimera (colored as blue, pink, and orange for chains A, B, and C, respectively). Right: Schematic representation showing trimeric assembly of the protein. (C) Ribbon representation of CEP192-AURKAchimera structure, from the boxed region in (B). CEP192 region is colored in blue, and the side chains are shown using a stick representation that is colored by atom type. AURKA region is colored in pink, and the surface is shown. Targeting protein for Xklp2 (TPX2)–complexed AURKA structure (PDB code: 1OL5) is superimposed, and TPX2 is shown in yellow. (D) Close-up view of the CEP192 binding site, showing the 2Fo-Fc electron density map (blue mesh) at 2.7-Å resolution contoured at 1 σ around an all-atom representation of CEP192 region. (E) Close-up view of the short loop (E506–G511) of the CEP192 binding site from the boxed region in (C), which overlaps the TACC3 binding site. Left: The TACC3-complexed AURKA structure (PDB code: 5ODT) is superimposed, and TACC3 is shown in yellow. Right: Residues involved in the interaction between CEP192 and AURKA. TACC3 residues F525 and E523 are also shown in yellow sticks. The hydrogen bonding interactions are shown as dotted lines. (F) Close-up view of the conserved helix (Helix-1) binding site, from the boxed region in (C). Hydrogen bonding interactions are shown (dotted lines). (G) Fluorescence polarization (FP) measurements of the indicated AURKA mutants binding into FITC-CEP192506–536. Errors correspond to the SD from triplicate measurements. (H) Isothermal titration calorimetry (ITC) titration of the MBP-CEP192506–536 into AURKA123–403Y148A mutant. (I) ITC titration of the MBP-CEP192506–536I518D mutant into AURKA123–403.
Fig 4: Centrosomal protein of 192 kDa (CEP192) binds to a distinct region of Aurora kinase A (AURKA), and the binding by itself cannot activate AURKA.(A) Alignment of CEP192 sequences around the predicted conserved helix (Helix-1) and targeting protein for Xklp2 (TPX2) sequences from different species (UniProt accession codes are listed with the name of each sequence). (B) Binding affinities of CEP192506–536 (blue) and TPX21–43 (orange) to AURKA123–403 determined with fluorescence polarization (FP) assays. The errors correspond to the SD from triplicate measurements. (C and D) Competitive binding inhibition assay. FITC-labeled CEP192506–536 (C) or FITC-labeled TPX21–43 (D) in complex with AURKA123–403 (1 μM) is displaced by nonlabeled CEP192506–536. The errors correspond to the SD from triplicate measurements. (E) In vitro kinase assay (ADP-glo assay) showing kinase activities of phosphorylated AURKA123–403wild type (WT) (red), phosphorylated AURKA123–403C290,393A (blue), and dephosphorylated AURKA123–403C290,393A (gray) at indicated concentrations (3, 30, and 300 nM). (F to H) In vitro kinase assay [adenosine diphosphate (ADP)–glo assay] showing stimulation of kinase activity of (F) 30 nM phosphorylated AURKA123–403WT, (G) 30 nM phosphorylated AURKA123–403C290,393A, and (H) 30 nM dephosphorylated AURKA123–403C290,393A by TPX21–43 (orange), CEP192506–536 (blue), or CEP1921–995 (green). The errors correspond to the SD from triplicate measurements. N.D., not determined; RLU, relative light unit.
Fig 5: Quantitative analysis of interactions between centrosomal protein of 192 kDa (CEP192) and Aurora kinase A (AURKA).(A) Domain organization of Homo sapiens (H.s.) CEP192 and AURKA. (B to D) Isothermal titration calorimetry (ITC) titrations of CEP192 and AURKA constructs as indicated. Listed with each titration are the concentrations of the protein in the syringe and in the cell, as well as the parameters of the fit (stoichiometry N, dissociation constant Kd). Errors correspond to the SD of the fits.
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