Integrin-linked kinase (ILK) is an ankyrinrepeat containing serine-threonine protein kinase capable of interacting with the cytoplasmic domains of integrin ␤1, ␤2, and ␤3 subunits. Overexpression of ILK in epithelial cells disrupts cell-extracellular matrix as well as cell-cell interactions, suppresses suspension-induced apoptosis (also called Anoikis), and stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of ␤-catenin, where the latter associates with a T cell factor͞ lymphocyte enhancer-binding factor 1 (TCF͞LEF-1) to form an activated transcription factor. We now demonstrate that ILK activity is rapidly, but transiently, stimulated upon attachment of cells to fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore, phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane targetted constitutively active Pi(3)K activates ILK in vivo. We also demonstrate here that ILK is an upstream effector of the Pi(3)K-dependent regulation of both protein kinase B (PKB͞AKT) and glycogen synthase kinase 3 (GSK-3). Specifically, ILK can directly phosphorylate GSK-3 in vitro and when stably, or transiently, overexpressed in cells can inhibit GSK-3 activity, whereas the overexpression of kinasedeficient ILK enhances GSK-3 activity. In addition, kinaseactive ILK can phosphorylate PKB͞AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB͞AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB͞AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB͞AKT, and inhibition of GSK-3.

We show that integrin-linked kinase (ILK) stimulates the expression of VEGF by stimulating HIF-1alpha protein expression in a PKB/Akt- and mTOR/FRAP-dependent manner. In human prostate cancer cells, knockdown of ILK expression with siRNA, or inhibition of ILK activity, results in significant inhibition of HIF-1alpha and VEGF expression. In endothelial cells, VEGF stimulates ILK activity, and inhibition of ILK expression or activity results in the inhibition of VEGF-mediated endothelial cell migration, capillary formation in vitro, and angiogenesis in vivo. Inhibition of ILK activity also inhibits prostate tumor angiogenesis and suppresses tumor growth. These data demonstrate an important and essential role of ILK in two key aspects of tumor angiogenesis: VEGF expression by tumor cells and VEGF-stimulated blood vessel formation.

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