Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase.

Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine-threonine protein kinase capable of interacting with the cytoplasmic domains of integrin beta1, beta2, and beta3 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 beta-catenin, where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-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 (PKB/AKT) 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 kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB/AKT, and inhibition of GSK-3.

[1]  J. Brugge,et al.  Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation , 1997, Molecular and cellular biology.

[2]  G. Shaw,et al.  The pleckstrin homology domain: An intriguing multifunctional protein module , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[3]  R. Moon,et al.  Signal transduction through beta-catenin and specification of cell fate during embryogenesis. , 1996, Genes & development.

[4]  L. Cantley,et al.  A Comparative Analysis of the Phosphoinositide Binding Specificity of Pleckstrin Homology Domains* , 1997, The Journal of Biological Chemistry.

[5]  E. Querfurth,et al.  Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase. , 1996, The EMBO journal.

[6]  B. Hemmings,et al.  Constitutive activation of protein kinase B and phosphorylation of p47 phox by a membrane-targeted phosphoinositide 3-kinase , 1996, Current Biology.

[7]  Dario R. Alessi,et al.  3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro , 1998, Current Biology.

[8]  Asim Khwaja,et al.  Matrix adhesion and Ras transformation both activate a phosphoinositide 3‐OH kinase and protein kinase B/Akt cellular survival pathway , 1997, The EMBO journal.

[9]  Andrius Kazlauskas,et al.  The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase , 1995, Cell.

[10]  Colin B. Reese,et al.  3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase , 1997, Current Biology.

[11]  J. Holik,et al.  Signaling by Phosphoinositide-3,4,5-Trisphosphate Through Proteins Containing Pleckstrin and Sec7 Homology Domains , 1997, Science.

[12]  E. Ruoslahti,et al.  Association of insulin receptor substrate-1 with integrins. , 1994, Science.

[13]  P. Cohen,et al.  Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B , 1995, Nature.

[14]  J. Cheng,et al.  Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[15]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[16]  J. Downward SIGNAL TRANSDUCTION: Lipid-Regulated Kinases: Some Common Themes at Last , 1998 .

[17]  G. Garcı́a-Cardeña,et al.  The synergistic activity of alphavbeta3 integrin and PDGF receptor increases cell migration. , 1998, Journal of cell science.

[18]  G. Thomas,et al.  TOR signalling and control of cell growth. , 1997, Current opinion in cell biology.

[19]  S. Dedhar,et al.  Cell adhesion and the integrin-linked kinase regulate the LEF-1 and beta-catenin signaling pathways. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J Downward,et al.  PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. , 1997, Trends in biochemical sciences.

[21]  S. Dedhar,et al.  Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase , 1996, Nature.

[22]  B. Seed,et al.  αLβ2 Integrin/LFA-1 Binding to ICAM-1 Induced by Cytohesin-1, a Cytoplasmic Regulatory Molecule , 1996, Cell.

[23]  S. Dedhar,et al.  Integrin cytoplasmic interactions and bidirectional transmembrane signalling. , 1996, Current opinion in cell biology.

[24]  P. Polakis,et al.  Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes , 1996, Molecular and cellular biology.

[25]  J. Woodgett,et al.  Genetic analysis of protein kinase B (AKT) in Drosophila , 1998, Current Biology.

[26]  A. Toker,et al.  Signalling through the lipid products of phosphoinositide-3-OH kinase , 1997, Nature.

[27]  J. Schlessinger,et al.  PH Domains: Diverse Sequences with a Common Fold Recruit Signaling Molecules to the Cell Surface , 1996, Cell.

[28]  S. Dedhar,et al.  Integrin-linked Protein Kinase Regulates Fibronectin Matrix Assembly, E-cadherin Expression, and Tumorigenicity* , 1998, The Journal of Biological Chemistry.

[29]  G. Panayotou,et al.  Phosphoinositide 3-kinases: a conserved family of signal transducers. , 1997, Trends in biochemical sciences.

[30]  K. Yamada,et al.  Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors , 1996, The Journal of cell biology.

[31]  S. Dedhar,et al.  Overexpression of the Integrin-linked Kinase Promotes Anchorage-independent Cell Cycle Progression* , 1997, The Journal of Biological Chemistry.

[32]  M. Schwartz,et al.  Integrins: emerging paradigms of signal transduction. , 1995, Annual review of cell and developmental biology.