We analysed breast tumors and breast cancer cell lines for the expression of b-parvin (ParvB), an adaptor protein that binds to the integrin-linked kinase (ILK). Quantitative RT-PCR indicated that ParvB mRNA was downregulated, by at least 60%, in four of nine breast tumors, relative to patient-matched normal mammary gland tissue. We also found that ParvB protein levels were reduced by X90% in five of seven advanced tumors, relative to matched normal breast tissue. Conversely, ILK protein and kinase activity levels were elevated in these tumors, suggesting that downregulation of ParvB stimulates ILK signaling. Western blot analyses indicated very low levels of ParvB protein in MDA-MB-231 and MCF7 breast cancer cells, facilitating functional studies of the effects of ParvB on ILK signaling. Expression of ParvB in MDA-MB-231 and MCF7 cells increased cell adhesion to collagen. ParvB inhibited ILK kinase activity, anchorageindependent cell growth and in vitro matrigel invasion by MDA-MB-231 cells. EGF-induced phosphorylation of two ILK targets, PKB (Ser473) and glycogen synthase kinase 3b (Ser9), was also inhibited by ParvB. These results indicated that ParvB inhibits ILK signaling downstream of receptor tyrosine kinases. Our results suggest that loss of ParvB expression is a novel mechanism for upregulating ILK activity in tumors.
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 GSK3b. Silencing of endogenous ILKAP by short interfering RNA (siRNA) stimulated GSK3b 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 GSK3b without affecting S473 phosphorylation of PKB. The ILKAP-mediated inhibition of S9 phosphorylation was rescued by overexpression of ILK, but not by a dominantnegative ILK mutant. The expression level of cyclin D1, a target of ILK-GSK3b 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-GSK3b signaling axis, and suggest that ILKAP activity plays an important role in inhibiting oncogenic transformation.
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