Journal: Journal of cell science

Article Title: Opposing roles for JNK and Aurora A in regulating the association of WDR62 with spindle microtubules.

doi: 10.1242/jcs.157537

Figure Lengend Snippet: Fig. 6. AURKA interacts with WDR62 and regulates its accumulation on spindle microtubules. (A) Myc–AURKA and HA-tagged WDR62 were transiently coexpressed, and their interaction in asynchronous (AS) or mitotically arrested (NZ, 350 nM, 16 h) cells was evaluated by co-immunoprecipitation (IP) analysis. Coexpression with a Myc vector served as a negative control. WB, western blot. (B) Endogenous AURKA co-immunoprecipitation with WDR62 in mitotically arrested cells (NZ, 350 nM, 16 h). Immunoprecipitation with IgG antibodies served as a negative control. (C) In vitro kinase assays to determine the phosphorylation of full-length WDR62 (GST–WDR62 FL) by AURKA or JNK1. (D) Mitotic AD293 cells were immunostained to determine the localization of endogenous WDR62 and AURKA. (E) WDR62–GFP and AURKA–mCherry or JNK1–mCherry were coexpressed in AD293 cells, and their localization assessed in mitotic cells by live-cell imaging. (F) mCherry-tagged AURKA, kinase-dead AURKA mutant (K162R) or JNK1 were coexpressed with GFP-tagged WDR62, and the microtubule association of WDR62 was determined using live-cell microscopy. The asterisk (*) indicates a cell with enhanced microtubule association of WDR62 with AURKA coexpression. Scale bars: 20 mm. (G) Following MLN8237 (0.5 mM) treatment, spindle pole association of endogenous WDR62 was determined by immunofluorescence staining. (H) The spindle-pole localization of endogenous WDR62 in M-phase-arrested (taxol, 10 nM, 16 h) cells was evaluated at the indicated time intervals following MLN8237 (0.5 mM) treatment.

Article Snippet: AURKA cDNA was obtained from Origene.

Techniques: Immunoprecipitation, Plasmid Preparation, Negative Control, Western Blot, In Vitro, Phospho-proteomics, Live Cell Imaging, Mutagenesis, Microscopy, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Opposing roles for JNK and Aurora A in regulating the association of WDR62 with spindle microtubules.

doi: 10.1242/jcs.157537

Figure Lengend Snippet: Fig. 7. AURKA-mediated phosphorylation of residues within the WDR62 N-terminal region regulates microtubule association. (A) GFP-tagged N-terminally truncated (D1– 52) WDR62, full-length wild-type (WT) WDR62, WDR62 S32/S33-AA, WDR62 S49/ T50/S52-AAA or WDR62 S32/S33/S49/T50/ S52-AAAAA mutants were coexpressed with mCherry–AURKA and localization was evaluated. (B) GFP-tagged N-terminally truncated or alanine-substituted WDR62 mutants were coexpressed with H2B– mCherry and taxol treated (10 mM, 30 min), and protein localization was evaluated. (C) GFP-tagged N-terminally truncated or alanine-substituted WDR62 mutants were coexpressed with H2B–mCherry and the spindle pole association of WDR62 proteins was determined in mitotic cells. Relative GFP signal intensities were quantified along line scans bisecting spindle poles (SP). Scale bars: 20 mm. (D) Schematic of WDR62 depicting the position of AURKA and JNK phosphorylation sites and their respective effects on protein association with microtubules. (E) Proposed model of WDR62 spatiotemporal regulation by JNK and AURKA. Centrosome-associated AURKA phosphorylates WDR62 to trigger association with astral microtubules following mitotic entry. This is required for the accumulation of WDR62–JNK signaling complexes at the spindle pole. JNK-mediated phosphorylation of WDR62 promotes dissociation from microtubules for cytoplasmic localization.

Article Snippet: AURKA cDNA was obtained from Origene.

Techniques: Phospho-proteomics

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PDAC cells (n=3, *p < 0.05 versus control shRNA group). (C) Immunoprecipitation (IP) analysis of the levels of RIPK3 binding to RIPK1 and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (D) IP analysis of the levels of RIPK3 binding to RIPK1 and MLKL in control and stable AURKA-knockdown PANC1 and PANC2.03 cells. (E) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (F) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in HEK293 cells after expressed AURKA wild type and D274A mutant. (G) PANC1 cells were treated with CCT137690 (10 μM) or TNF (50 ng/ml) in the absence or presence of anti-TNFR1 antibody (1 mg/ml) for 24 hours and cell death was analyzed (n=3, *p < 0.05).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Control, Knockdown, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PANC1 and PANC2.03 cells (n=3, *p < 0.05 versus control shRNA group). (C, D) PANC1 and PANC2.03 cells were treated with XXVI (C) or AR-A014418 (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with XXVI or AR-A014418 for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F) Knockdown of RIPK3 and MLKL, but not RIPK1, inhibited XXVI- or AR-A014418-induced cell death (n=3, *p < 0.05 versus control shRNA group). (G–I) HEK293 cells were expressed with GSK3β WT and S9A mutant and then treated with CCT137690 (10 μM) for 24 hours. Protein level (G), cell viability (H), caspase-3 activity (H), and complex formation (I) were assayed.

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Control, Knockdown, shRNA, Mutagenesis, Activity Assay