| Physiologic inhibition of ILK signalling is poorly understood. ILKAP is a serine/threonine protein phosphatase that directly interacts with and inhibits ILK kinase activity and signalling in response to stimulation by IGF-1 and integrin adhesion. The data presented in this thesis extend previous work and provide novel findings that convincingly demonstrate ILKAP to be a specific physiologic negative regulator of ILK. Expression of ILKAP in LNCaP prostate carcinoma cells, in which ILK function is constitutively elevated, inhibited oncogenic ILK kinase activity, ILK mediated GSK-3β signalling, cyclin D1 protein expression accompanied by G1 cell cycle arrest, and anchorage independent growth of these cells. Reciprocally, ILKAP RNAi in non-tumourigenic HEK293 cells resulted >85 % suppression of endogenous ILKAP protein expression which was accompanied by promotion of ILK-GSK-3β signalling, cyclin D1 protein expression, and S-phase cell cycle transit. These in vitro findings suggested that ILKAP may function as a putative tumour suppressor. In support of this, examination of ILKAP expression in high grade GBM brain tumour samples revealed significant down regulation or absence of ILKAP protein levels in approximately 40% of the cases assessed. Studies stemming from this work were aimed at investigating the mechanism of ILKAP inhibition of ILK. Confocal microscopy and biochemical fractionation revealed that ILKAP was resident primarily in the nucleus of LNCaP and HEK293 cells. Further investigation revealed that ILKAP is a nuclear phosphatase containing a putative bipartite NLS. Site directed mutagenesis of polybasic residues K78T, R79A, K95T, K96T (ΔNLS-ILKAP) resulted in significantly less nuclear and greater cytoplasmic distribution, relative to wild type ILKAP, when expressed in LNCaP cells. Interestingly, perturbation of ILKAP nuclear localization impaired inhibition of ILK-GSK-3β signalling, cyclin D1 protein expression, and suppression of anchorage independent growth when expressed in LNCaP cells, suggesting a novel mechanism of oncogenic ILK activation. These results collectively demonstrate that ILKAP is nuclear phosphatase that functions to negatively regulate ILK and that nuclear localization of ILKAP is critical for this response. |
