Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC(20)). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K(m) for MnATP is approximately 150-fold less than that for MgATP).Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases

Integrin-linked kinase regulates interphase and mitotic microtubule dynamics

Integrin-linked kinase (ILK) localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells

QLT-0267 treatment of HeLa cells increases sister centromere tension.

<p>(A) Representative immunofluorescence images of mitotic HeLa cells showing α-tubulin (green) and centromere (red). Cells were treated with DMSO, QLT-0267 for 6 hours or 1 µM nocodazole for 16 hours. Images were acquired as z-stacks with 0.25 µm spacing, and individual stacks were browsed through to identify sister centromeres. Small white boxes indicate examples of identifiable sister centromere pairs used for distance measurement between them. Bar = 10 µm. (B) Quantification of distance between sister centromeres in mitotic HeLa cells treated with DMSO, QLT-0267 (aligned chromosomes between both spindle poles and mis-aligned chromosomes on the side of only one pole) and nocodazole (basal control for lack of tension between sister centromeres). Results represent mean ± S.E.M. (DMSO control N = 81, QLT-0267 (aligned) N = 84, QLT-0267 (mis-aligned) N = 84, nocodazole N = 85) from 2 independent experiments.</p

ILK overexpression in HeLa cells is associated with decreased sensitivity to paclitaxel.

<p>(A) Percentage viability of HeLa clones (control: Vector 6 and Vector 8; ILK overexpressing: ILK 7 and ILK 10) after various concentrations of paclitaxel treatment for 48 hours. Results indicate mean ± S.D., and are representative of 3 independent experiments. An asterisk (*) indicates significant difference (P<0.01) between each of the control and overexpressing cell lines. (B) ILK overexpression dampens the paclitaxel-induced increase in mitotic arrest in HeLa cells. The mitotic indices of control Vector 8 and ILK-overexpressing ILK 10 were quantified after treatment with DMSO or 10 nM paclitaxel for 48 hours. Bar graph shows mean ± S.E.M., N>1000 cells.</p

ILK overexpression shortens duration of mitosis in HeLa cells.

<p>(A) Immunoblot analysis of HeLa cell clones stably expressing control vector (clones Vector 6 and Vector 8), and the epitope-tagged Flag-ILK (clones ILK 7 and ILK 10). (B) Representative time-lapse images (taken every 2 minutes) of HeLa clone Vector 8. Images show the four stages of mitosis used for measurement of time required for each phase of mitosis: cell rounding up (start of mitosis), metaphase, onset of anaphase (arrows indicate separating chromosomes), and formation of cleavage furrow (end of mitosis). Bar = 10 µm. (C–E) Scatter plot of time taken by vector control HeLa cells (Vector 8, N = 36) and ILK-overexpressing HeLa cells (ILK 10, N = 42) to complete the various stages of mitosis. Results represent mean ± S.E.M.</p

QLT-0267 treatment affects microtubule dynamics in HeLa cells during mitosis.

<p>(A) QLT-0267 treatment retards the rate of microtubule regrowth during mitosis. Representative immunofluorescence images of mitotic HeLa cells stained for α-tubulin (green), centrosome (red) and chromosomes (blue). HeLa cells were treated with DMSO or QLT-0267 for 5 hours, then chilled at 4°C for 1 hour to depolymerize microtubules, before being returned to 37°C for various time-points followed by methanol fixation. Steady state: un-chilled cells. Bar = 10 µm. (B) Quantification of microtubule length at different time-points of microtubule regrowth. Results represent mean ± S.D., N = 40 (4 longest microtubules per cell, 10 cells per condition). (C) ILK inhibition increases microtubule stability during mitosis. Representative images of HeLa cells treated with DMSO or QLT-0267 were stained for acetylated α-tubulin (green), total α-tubulin (red) and chromosomes (blue). QLT-0267-treated cells show a higher proportion of acetylated α-tubulin relative to the total amount of α-tubulin when compared to normal prometaphase cells. Bar = 10 µm. (D) Quantification of the integrated density of acetylated α-tubulin relative to total α-tubulin. Results represent mean ± S.E.M., N = 15 (DMSO prometaphase), 20 (DMSO metaphase), 20 (QLT-0267).</p

ILK overexpression affects microtubule dynamic instability in living interphase HeLa cells.

<p>(A) Time-lapse images of microtubule ends in living ILK-overexpressing HeLa clone ILK 10. Images were obtained every 3 s to track the movement of microtubule ends. This series of representative images shows a microtubule end (tracked by a blue dot) pausing from 3 s to 6 s, then shortening from 6 s to 12 s. Bar = 2 µm. (B–D) ILK inhibition suppresses, while ILK overexpression increases microtubule dynamic instability. HeLa clones Vector 8 and ILK 10 were stably transfected with tubulin-venus. The rates and parameters of microtubule dynamic instability were compared between Vector control (Vector 8) and ILK overexpression (ILK 10), as well as between DMSO- or QLT-0267-treated Vector 8 cells. Bar graphs show mean ± S.E.M., N = 75 (DMSO), 77 (QLT-0267), 54 (Vector control), 46 (ILK overexpression).</p