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The roles of integrin-linked kinase at the centrosome and its regulation of microtubule dynamics

University of British Columbia

Integrin-linked kinase (ILK) is both a scaffolding protein and a serine/threonine kinase that localizes to the focal adhesions. It interacts with the cytoplasmic domain of the β1-integrin subunit and acts as a hub for the localization of several actin cytoskeletal and signaling proteins resulting in the transduction of signals from cell-matrix interactions and growth factors into the cell interior. These signaling cascades go on to regulate important cellular processes such as cell migration, survival and proliferation. ILK has a second cellular localization at the centrosomes, where it regulates mitotic spindle organization and the interaction between ch-TOG, TACC3 and Aurora A, which is important for their function in regulating microtubule dynamics and spindle organization. However, the specific role of ILK's kinase activity, separate from a possible scaffolding role, in spindle organization is unclear. For this study, I attempted to characterize the spindle defect caused by QLT-0267, a small molecule inhibitor that is highly selective for ILK kinase activity. Treatment of HeLa cells with 10 μM QLT-0267 is known to result in a disorganized mitotic spindle that appeared arrested in a prometaphase-like phenotype. Here, I show that QLT-0267 exposure resulted in an increase in tension across sister centromeres aligned between two poles, suggesting a possible effect on spindle microtubule dynamics. Treatment with QLT-0267 was also associated with slower microtubule regrowth after depolymerization and the presence of a more stable population of microtubules in the mitotic spindle as evidenced by higher levels of acetylated α-tubulin. To further assess the role of ILK in regulating microtubule dynamics, the parameters of microtubule dynamic instability were measured in both QLT-0267-treated HeLa cells and ILK overexpressing HeLa cells. QLT-0267 appeared to dampen microtubule dynamic instability, while ILK overexpression enhanced it. ILK overexpression was also associated with decreased sensitivity to paclitaxel, a chemotherapeutic agent that stabilizes microtubule dynamics. Taken together, the results suggest a role for ILK's kinase activity in regulating microtubule dynamics. Finally, this study reports a novel mechanism of action for the small molecule inhibitor QLT-0267, which dampens microtubule dynamics and should be taken into consideration when designing future uses for the compound.

Text

2012-03-16

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/41516

Interdisciplinary Oncology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/