The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More

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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Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phoshpatidylinositol (PI) 3-kinasedependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB-serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.Interaction of cells with the extracellular matrix results in the suppression of apoptosis and promotes cell cycle progression (1-4). The molecular basis for this anchorage-dependent cell growth and survival is an intensive area of study, since oncogenically transformed cells very often grow in an anchorage-independent manner. Alterations in anchorage-dependent signaling pathways are also likely to be of importance in tumor progression leading to metastasis. The integrin-linked kinase (ILK) 1 is an intracellular protein kinase that couples integrins and growth factors to downstream signaling pathways involved in the suppression of apoptosis and in promoting cell cycle progression. Cell-extracellular matrix interactions stimulate two major signaling pathways, leading to the regulation of cell cycle progression and cell survival. These are the Ras/Raf-MAP kinase pathway, and the protein kinase B/Akt (PKB/Akt) cell survival pathway (5). ILK regulates both the cell cycle, by stimulating the expression of cyclin D1 (6, 7) and cyclin A (7), and the activity of PKB/Akt, by stimulating the phosphorylation of PKB/Akt on serine 473 in a PI-3 kinase-dependent manner (8, 9), a requirement for full activation of this enzyme. Overexpression of ILK suppresses anoikis by activating PKB/ Akt (10), and ILK activity is constitutively up-regulated in tumor cells lacking expression of the PI(3,4,5)P 3 phosphatase tumor suppress...

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