Fig. 8.

JNK-mediated WDR62 phosphorylation is involved in spindle regulation. (A) M-phase-arrested HeLa cells (NZ, 350 nM, 16 h) were treated with JNK inhibitor VIII (20 µM) and samples immunoblotted for WDR62 and α-tubulin. (B) Schematic representation of WDR62 truncation and JNK-binding (JBD) mutants. Asterisks indicate replacement of leucines with alanines in the JBD motif. (C) JNK1-mediated phosphorylation of full-length WDR62 (WDR62-FL) and truncation mutants containing WDR62 N- (WDR62-N) or C-terminal (WDR62-C) regions as measured by in vitro kinase activity assays. Values are means ± s.d. ³²P (pmol) incorporated into substrates from three independent experiments. A representative autoradiograph and Coomassie stain for protein loading are shown. (D) JNK1-mediated phosphorylation of WDR62-C-AXA was determined by in vitro kinase activity assays and compared to the WDR62-C truncation mutant. (E) Myc-tagged WDR62-C and WDR62-C-AXA were transiently expressed with HA–JNK1 in HeLa cells. HA–JNK1 was immunoprecipitated and WDR62 co-immunoprecipitation determined by immunoblotting for the myc tag. Protein expression (input) in total cell lysates was also determined. (F) GFP-labeled WDR62-FL and truncation mutants were expressed in Ad293 cells. Their subcellular localization were determined during metaphase and colocalization with α- or γ-tubulin and DAPI was examined. (G) Ad293 cells were co-transfected with WDR62 siRNA and either GFP–WDR62-FL, GFP–WDR62-FL-AXA or GFP alone. The proportion of GFP-positive cells with normal bipolar, abnormal bipolar or multipolar spindles in metaphase was determined. n values are the total number of cells counted from three independent experiments. Representative images of multipolar, abnormal or normal bipolar spindles in Ad293 cells stained for γ-tubulin and DAPI are shown. (H) Representative images of GFP–WDR62-FL-AXA spindle localization in Ad293 cells treated with WDR62 or Con siRNA and stained for γ-tubulin and DAPI. Images in F and H are single z-sections.