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  • Original Paper
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ILKAP regulates ILK signaling and inhibits anchorage-independent growth

Abstract

ILKAP is a protein phosphatase 2C that selectively associates with integrin linked kinase, ILK, to modulate cell adhesion and growth factor signaling. We investigated the role of endogenous cellular ILKAP in antagonizing ILK signaling of two key targets, PKB and GSK3β. Silencing of endogenous ILKAP by short interfering RNA (siRNA) stimulated GSK3β phosphorylation at S9, with no effect on PKB S473 phosphorylation. In LNCaP prostate carcinoma cells, transient or stable expression of ILKAP suppressed ILK immune complex kinase activity, demonstrating an interaction between ILKAP and ILK. Consistent with the silencing data, ILKAP inhibition of ILK selectively inhibited S9 phosphorylation of GSK3β without affecting S473 phosphorylation of PKB. The ILKAP-mediated inhibition of S9 phosphorylation was rescued by overexpression of ILK, but not by a dominant-negative ILK mutant. The expression level of cyclin D1, a target of ILK-GSK3β signaling, was inversely correlated with ILKAP protein levels, suggesting that antagonism of ILK modulates cell cycle progression. ILKAP expression increased the proportion of LNCaP cells in G1, relative to vector control cells, and siRNA suppression of ILKAP increased entry of cells into the S phase, consistent with ILK antagonism. Anchorage-independent growth of LNCaP cells was inhibited by ILKAP, suggesting a critical role in the suppression of cellular transformation. Taken together, our results indicate that endogenous ILKAP activity inhibits the ILK-GSK3β signaling axis, and suggest that ILKAP activity plays an important role in inhibiting oncogenic transformation.

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Acknowledgements

We thank Dr Herman Yeger (HSC) for providing LNCaP cells, Dr Michael Walsh (University of Calgary) for the gift of purified MLC20, Garrick Fong for technical assistance and Sherry Zhao (Manager, HSC Flow Cytometry Service) for her expert flow cytometric analyses. AK received the Paul Striata Fellowship in Medicine for prostate cancer research. This work was supported by grants (GH) from the Canadian Institutes of Health Research, and the National Cancer Institute of Canada (with funds from the Terry Fox Run). GH was a CIHR Scholar.

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Correspondence to Gregory E Hannigan.

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Kumar, A., Naruszewicz, I., Wang, P. et al. ILKAP regulates ILK signaling and inhibits anchorage-independent growth. Oncogene 23, 3454–3461 (2004). https://doi.org/10.1038/sj.onc.1207473

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