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Phosphoinositide-3-OH Kinase-Dependent Regulation of Glycogen Synthase Kinase 3 and Protein Kinase B͞AKT by the Integrin-Linked Kinase

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Phosphoinositide-3-OH Kinase-Dependent Regulation of Glycogen Synthase Kinase 3 and Protein Kinase B͞AKT by the Integrin-Linked Kinase Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11211–11216, September 1998 Cell Biology Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase ByAKT by the integrin-linked kinase MARC DELCOMMENNE*, CLARA TAN*†,VIRGINIA GRAY*, LAURENT RUE*‡,JAMES WOODGETT‡, AND SHOUKAT DEDHAR*†§ *British Columbia Cancer Agency, Jack Bell Research Centre, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6 Canada; †Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3 Canada; and ‡Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 810 University Avenue, Toronto, Ontario M5G 2M9 Canada Communicated by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, July 23, 1998 (received for review July 6, 1998) ABSTRACT Integrin-linked kinase (ILK) is an ankyrin- is negatively regulated in a Pi(3)K-dependent manner upon repeat containing serine–threonine protein kinase capable of insulin stimulation (8) and is also a critical component of the interacting with the cytoplasmic domains of integrin b1, b2, Wnt-signaling pathway (9). GSK-3 can phosphorylate highly and b3 subunits. Overexpression of ILK in epithelial cells conserved serine residues in the protein b-catenin in the disrupts cell–extracellular matrix as well as cell–cell inter- presence of adematous polyposis coli and axin (9). GSK-3 also actions, suppresses suspension-induced apoptosis (also called can regulate protein translation by phosphorylating and inac- Anoikis), and stimulates anchorage-independent cell cycle tivating eIF2B, the guanine nucleotide exchange factor for the progression. In addition, ILK induces nuclear translocation of translation initiation factor eIF2 (3). b-catenin, where the latter associates with a T cell factory Pi(3)K-dependent signals therefore regulate critical cellular lymphocyte enhancer-binding factor 1 (TCFyLEF-1) to form functions such as cell survival, cell fate determination, and an activated transcription factor. We now demonstrate that protein translation via the key molecular components PKBy ILK activity is rapidly, but transiently, stimulated upon AKT, GSK-3, and p70S6K. Recent work (1, 11, 12) has led to attachment of cells to fibronectin, as well as by insulin, in a a better understanding of how these components are regulated phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. by Pi(3)K. The activation of PKByAKT by Pi(3)K requires Furthermore, phosphatidylinositol(3,4,5)trisphosphate spe- both the phosphoinositide products of Pi(3)K as well as protein cifically stimulates the activity of ILK in vitro, and in addition, phosphorylation of threonine-308 and serine-473 of PKBy membrane targetted constitutively active Pi(3)K activates ILK AKT. It is now generally recognized that PtdIns(3,4,5)P3 can in vivo. We also demonstrate here that ILK is an upstream bind to the pleckstrin homology (PH) domain of PKTyAKT, effector of the Pi(3)K-dependent regulation of both protein resulting in its targeting to the plasma membrane and exposure kinase B (PKByAKT) and glycogen synthase kinase 3 (GSK- of threonine-308. A recently identified, constitutively active 3). Specifically, ILK can directly phosphorylate GSK-3 in vitro kinase, PDK-1 (13, 14), then phosphorylates PKB on threo- and when stably, or transiently, overexpressed in cells can nine-308. However, this phosphorylation alone is not sufficient inhibit GSK-3 activity, whereas the overexpression of kinase- to fully activate PKByAKT, which also needs to be phosphor- deficient ILK enhances GSK-3 activity. In addition, kinase- ylated on serine-473 in a PtdIns(3,4,5)P3-dependent manner. y active ILK can phosphorylate PKB AKT on serine-473, PDK-1 also can directly phosphorylate p70S6K (14). Although whereas kinase-deficient ILK severely inhibits endogenous GSK-3 can be directly phosphorylated and inactivated by y phosphorylation of PKB AKT on serine-473, demonstrating PKByAKT, GSK-3 activity also can be clearly regulated inde- that ILK is involved in agonist stimulated, Pi(3)K-dependent, pendently of PKByAKT, for instance via activation of the Wnt y PKB AKT activation. ILK is thus a receptor-proximal effec- pathway (9), which does not require PKByAKT (15). We have tor for the Pi(3)K-dependent, extracellular matrix and growth recently identified a serine–threonine protein kinase called y factor mediated, activation of PKB AKT, and inhibition of integrin-linked kinase (ILK-1), which can interact with the GSK-3. cytoplasmic domain of the b1, b2, and b3 integrin subunits (ref. 16 and G. Downie, unpublished data). Overexpression of Phosphatidylinositol 3-kinase a(Pi(3)K) is a receptor proximal ILK in epithelial cells results in anchorage-independent cell intracellular effector activated in response to a wide variety of survival and cell cycle progression (17), as well as tumorigen- extracellular stimuli, which include growth factors and cyto- esis in nude mice (18). Furthermore, ILK can induce the kines, as well as adhesion to extracellular matrices (1, 2). The translocation of b-catenin to the nucleus, resulting in the phosphatidylinositol lipid product of Pi(3)K, phospha- formation of LEF-1 transcription factoryb-catenin complex tidylinositol(3,4,5)trisphosphate [PtdIns(3,4,5)P3] is a second and the enhancement of LEF-1 transcriptional activity (19). messenger that acts on pathways that control cell proliferation, This latter finding implicates ILK in the activation of compo- cell survival, protein translation, and metabolic changes, nents of the Wnt-signaling pathway. largely through the activation of protein kinase B (PKB) (also In this paper, we now report that the activity of ILK can be known as AKT) and p70S6 kinase (p70S6K), or the inhibition rapidly, but transiently, stimulated by both cell–fibronectin of glycogen synthase kinase 3 (GSK-3) (1, 3). PKByAKT interactions, as well as by insulin, in a Pi(3)K-dependent promotes cell survival by inhibiting apoptosis, is a proto- oncogene (4, 5), and has been shown to be activated in a Abbreviations: ILK, integrin-linked kinase; GSK-3, glycogen synthase Pi(3)K-dependent manner (2, 6, 7). GSK-3, on the other hand, kinase 3; PKB, protein kinase B; PH, pleckstrin homology; HA, haemagglutanin; GST, glutathione S-transferase; MBP, myelin basic The publication costs of this article were defrayed in part by page charge protein; Pi(3)K, phosphoinositide-3 kinase; PtdIns(3,4,5)P, P2, or P3; phosphatidylinositol(3,4,5)monphosphate, bisphosphate, or trisphos- payment. This article must therefore be hereby marked ‘‘advertisement’’ in phate; LEF-1, lymphocyte enhancer-binding factor 1; HEK, human accordance with 18 U.S.C. §1734 solely to indicate this fact. embryonal kidney. © 1998 by The National Academy of Sciences 0027-8424y98y9511211-6$2.00y0 §To whom reprint requests should be addressed. e-mail: sdedhar@ PNAS is available online at www.pnas.org. interchange.ubc.ca. 11211 Downloaded by guest on October 2, 2021 11212 Cell Biology: Delcommenne et al. Proc. Natl. Acad. Sci. USA 95 (1998) manner, likely via the binding of PtdIns(3,4,5)P3 with a For assessing the effect of insulin on ILK activity, IEC-18 cells PH-like domain of ILK. In addition, we demonstrate that ILK were serum starved for 18 hr and then incubated with insulin not only directly phosphorylates GSK-3 and negatively regu- (100 nM) for the indicated time periods (0–30 min). To assess lates its activity but also can directly phosphorylate PKByAKT the role of Pi(3)K on ILK activity, the cells were preincubated on serine-473 in vitro, whereas a kinase-deficient form of ILK for 20 min with Pi(3)K inhibitors, Wortmannin (200 nM) severely inhibits PKByAKT serine-473 phosphorylation in (Sigma), or Ly294002 (50 mM) (Calbiochem) before plating vivo. These data suggest that ILK is a receptor proximal cells on fibronectin or the addition of insulin. Quantification of effector of Pi(3)K signals regulating the kinase activities of the extent of phosphorylation was determined by phosphor PKByAKT and GSK-3. image analysis. Effect of Phosphoinositides on ILK Activity in Vitro. MATERIALS AND METHODS PtdIns(3)P, PtdIns(3,4)P2, or PtdIns(3,4,5)P3 (Matreya, Pleas- ant Gap, PA) were dried under nitrogen and resuspended at Stably Transfected Cells Lines. Clones of IEC-18 cells a concentration of 0.1 mM in Hepes (10 mM), pH 7.0 in the [American Type Culture Collection (ATTC)] expressing either presence of L-a-phosphatidyl-L-serine and L-a-phosphatidyl- wild-type ILK cDNA in the sense orientation (ILK-13), or choline, b-arachidonoyl-g-stearoyl (1 mM each). The lipid anti-sense orientation (ILK-14), or a kinase-deficient ILK suspensions were then vortexed and sonicated for 20 min to cDNA (GH31R), were established as described by us (16, 19). generate unilamellar vesicles. ILK-5-glutathione S-transferase All clones and the parental cell line were maintained in a-ME (GST) (16) was combined with the phosphoinositide lipids (10 medium containing 5% fetal calf serum, insulin (10 mgyliter), mM) and kinase reaction buffer containing MBP (5 mg) and and glucose (3.6 gyliter). The transfected clones also were [g-32P]ATP (5 mCi). The reaction was allowed to proceed for maintained in 40 mgyml G418 to provide selection pressure. 30 min or 2 hr at 30°C and was stopped by adding 2X sample NIH 3T3 cells (ATCC) were transfected, by using electropo- buffer. Reaction products were analyzed by SDSyPAGE fol- ration, with cDNAs-encoding haemagglutanin (HA)-tagged lowed by autoradiography. P110 subunits of Pi(3)K in pcDNA3 expression vectors kindly provided by M. Thelen (University of Bern, Switzerland). After selection in G418 (0.8 mgyml), the transfected cells were RESULTS cloned by limited dilution, and clones expressing HA-tagged ILK Activity Is Stimulated by Fibronectin and Insulin in a p110 Pi(3)K were expanded and maintained in DMEM con- Pi(3)K-Dependent Manner.
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