Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization

Abstract

Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffold protein with well defined roles in focal adhesions in integrin-mediated cell adhesion, spreading, migration, and signaling. Using mass spectrometry-based proteomic approaches, we identify centrosomal and mitotic spindle proteins as interactors of ILK. alpha- and beta-tubulin, ch-TOG (XMAP215), and RUVBL1 associate with ILK and colocalize with it to mitotic centrosomes. Inhibition of ILK activity or expression induces profound apoptosis-independent defects in the organization of the mitotic spindle and DNA segregation. ILK fails to localize to the centrosomes of abnormal spindles in RUVBL1-depleted cells. Additionally, depletion of ILK expression or inhibition of its activity inhibits Aurora A-TACC3/ch-TOG interactions, which are essential for spindle pole organization and mitosis. These data demonstrate a critical and unexpected function for ILK in the organization of centrosomal protein complexes during mitotic spindle assembly and DNA segregation.

Figure 1.

ILK interacts with tubulin, RUVBL1, and ch-TOG and localizes to centrosomes. (A) FLAG-ILK was immunoprecipitated from the cytoskeleton of HEK293 cells, and the presence of α- and β-tubulin, ch-TOG, and RUVBL1 was determined by Western blot. (B) Detergent-soluble fractions (S1 and S2), isolation buffer–soluble fraction (S3), and purified mitotic spindles (P) from HeLa cells Western blotted for indicated proteins are shown. (C–H) HeLa cells costained for ILK and indicated proteins as well as DNA. Insets are close-ups of small squares (C) or of centrosomes (F). Arrowheads, centrosomes; arrow, focal adhesions. Bars, ∼10 μm.

Figure 2.

RUVBL1 is required for ILK localization to the centrosome and its absence leads to mitotic spindle defects. (A) RUVBL1 protein was depleted by RUVBL1 siRNA as shown by Western blot. (B) RUVBL1 siRNA–treated cells. (C) Histogram showing the percentage of disrupted mitotic cells in control and RUVBL1 siRNA–treated cells. Data are mean ± SD of three independent experiments in which >100 mitotic cells were counted for each condition. (D) Cells stained with ILK and pericentrin show localization of ILK to centrosomes in control but not RUVBL1 siRNA–treated cells. Bars, ∼10 μm.

Figure 3.

Treatment with the ILK inhibitor QLT-0267 causes apoptotic-independent effects on mitotic spindles and an accumulation of cells in mitosis. (A) HEK293 cells were treated with increasing concentrations of QLT-0267 for 7 h and the mitotic index was calculated. Data are mean ± SD of three independent experiments in which >400 cells were counted for each condition. (B and C) Cells were treated with QLT-0267 for 7 h and stained as indicated. (D) Cells were treated with the MEK inhibitor PD098059. (E and F) HeLa cells were treated with QLT-0267 for 1 h (E) or QLT-0267 + Z-VAD-FMK for 7 h (F). Bars, ∼10 μm. (G) Quantification of apoptosis in indicated samples by Cell Death Detection ELISA PLUS. All samples, except QLT-0267 5 μm 1 h, are from the 7-h time point. Data are mean ± SD from two experiments. (H) Indicated cell lysates (all 7-h treatments) were Western blotted for PARP.

Figure 4.

ILK siRNA results in aberrant mitotic spindles, misaligned chromosomes, and the prevention of progress through mitosis. (A) Western blot of HeLa cell extracts after ILK siRNA treatment. The amount of ILK remaining after knockdown is shown as a percentage of ILK in control siRNA and is adjusted according to β-actin levels. (B) HeLa cells treated with control or ILK siRNA show similar aberrant spindle and misaligned chromosome phenotypes to the QLT-0267–treated cells. Bars, ∼10 μm. (C–E) Histograms showing the percentage of mitotic cells with aberrant spindles (C), with misaligned chromosomes (D), and in the various stages of cell division (E) in ILK and control siRNA-treated cells. Data are mean ± SD from three independent experiments in which >50 mitotic cells were analyzed.

Figure 5.

ILK siRNA causes a disruption of Aurora A–TACC3/chTOG interaction. (A) HeLa cells were transfected with control or ILK siRNA and synchronized with nocodazole. Aurora A kinase was immunoprecipitated from cytoskeletal extracts with a monoclonal anti–Aurora A antibody and then Western blotting was performed with polyclonal antibodies against Aurora A, TACC3, and ch-TOG. (B) A similar experiment to A was performed on cells treated with QLT-0267 and a DMSO control. (C–F) Effect of ILK siRNA on localization of ILK-interacting proteins. Hela cells were transfected and stained as indicated. Phospho–Aurora A/B/C staining is presumably phospho–Aurora A rather than Aurora B or C, as it colocalizes with total Aurora A; Aurora B is not found on centrosomes, and Aurora C is only expressed in testis. Bars, ∼10 μm.