Figure 5.

(See previous page). Phosphorylation of CHO1 at S716^S717 is required for the anchoring and activation of LIM-kinase 1 (LIMK1) on the centrosome. (A) Centrosomal localization of LIMK1-pT508 and γ-tubulin in metaphase HeLa-S3 cells stably expressing vector alone or 6Myc-tagged CHO1-WT, -SA (S717A), or -SD (S717D). The cells were synchronized by a thymidine single block-and-release (10 h). Scale bar, 10 μm. The insets show enlarged images of signals at the centrosome. (B, C) The signal intensities of LIMK1-pT508 (B) and γ-tubulin (C) on centrosomes in metaphase HeLa-S3 cells stably expressing the constructs described in (A). Data represent the mean ± SD of n = 3 independent experiments (30 cells per experiment). (D, E) The signal intensities of Aurora-A-pT288 on centrosomes in G2 phase (D) and metaphase (E) HeLa-S3 cells stably expressing the constructs described in (A). Cells were synchronized by a thymidine single block-and-release (10 h). Data represent the mean ± SD of n = 3 independent experiments (30 cells per experiment). (F) The mitotic spindle in metaphase HeLa-S3 cells stably expressing the constructs described in (A). Cells were synchronized by a thymidine single block-and-release (10 h) and stained with anti-α-tubulin. Scale bar, 10 μm. (G) The percentages of the cells described in (F) with a mitotic spindle during metaphase. The cells were classified according to the α-tubulin signal intensity. Data represent the mean ± SD of n = 3 experiments. (H, I) The centrosomal localization of LIMK1-pT508 in anaphase HeLa-S3 cells stably expressing the constructs described in (A). In (I), the data represent the mean ± SD of n = 3 independent experiments (30 cells per experiment). (J) The level of Cofilin-pS3 in metaphase HeLa-S3 cells stably expressing the constructs described in (A). Cells were synchronized by a thymidine single block-and-release (10 h). Scale bar, 10 μm.