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

Abstract

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.

Figure 1

Identification of phosphoinositide lipid-binding motif in ILK. Amino acid sequence alignment of ILK residues 180–212 with amino acid sequences found in the PH domains of indicated proteins. Amino acids highlighted indicate residues found to be critical for the binding of phosphatidylinositol lipids (30).

Figure 2

(A) PtdIns(3, 4, 5)P3 specifically stimulates the activity of recombinant wild-type ILK in vitro. The indicated phospholipids were prepared as lipid vesicles as described in Materials and Methods and incubated with ILK. ILK activity was then determined by using γ³²P-ATP and MBP as an exogenous substrate. Phosphorylated MBP was detected by SDS/PAGE and autoradiography. (B) Stimulation of ILK activity by insulin. IEC-18 cells were serum-starved overnight and then exposed to insulin (100 nM) for the indicated time periods. ILK was immunoprecipitated from cell extracts and ILK activity determined by using MBP as described in Materials and Methods. Pi(3)K activity was inhibited by preincubating the cells with Wortmannin (200 nM) for 20 min. Bottom shows equivalent amount of ILK in each extract as determined by Western blotting with an anti-ILK antibody. (C) Stimulation of ILK activity by cell adhesion on fibronectin. IEC-18 cells were serum-starved overnight, resuspended in serum-free medium, and allowed to adhere to fibronectin- (10 μg/ml) coated nontissue culture plastic plates for the indicated time periods. Nonadherent cells were removed, and adherent cells were lysed. ILK kinase activity was determined as described for B. Pi(3)K activity was inhibited by preincubating the cells with Ly294002 (50 μM) for 20 min. Bottom shows equivalent levels of ILK in each extract as determined by Western blotting with anti-ILK antibody. Quantification of the extent of phosphorylation was determined by phosphorimage analysis. Data shown in A, B, and C are representative of three independent experiments.

Figure 3

Stable overexpression of HA-tagged constitutively active P110 Pi(3)K-CAAX in NIH 3T3 cells results in increased ILK activity and constitutive phosphorylation of PKB/AKT on serine-473. NIH 3T3 cell clones overexpressing constitutively active membrane-targeted p110 subunit of Pi(3)K, or kinase-deficient P110 (15), were serum-starved for 18 hr and then analyzed for ILK activity by using MBP, or for PKB/AKT serine-473 phosphorylation. Pi(3)K activity was inhibited by preincubating the cells with Ly294002 (50 μM) for 15 min. P110 expression was detected in the transfectants by Western blotting with anti-HA antibody (data not shown).

Figure 4

ILK inhibits GSK-3 activity. (A) ILK and GSK-3 activities were measured in cell clones stably overexpressing wild-type ILK (ILK-13), kinase-deficient (KD) ILK (ILK-KD), or antisense ILK (ILK-14) (–19) as described in Materials and Methods. Overexpression of wild-type ILK results in high ILK activity and drastic inhibition of GSK-3 activity. The level of expression of GSK-3 protein is similar in cell clones, as determined by Western blot analysis by using anti-GSK-3 antibody. (Inset) Quantification of GSK-3 activity in different IEC-18 clones. Data shown are the means of two separate experiments. (B) Transient transfection of ILK and HA-tagged GSK-3 in HEK-293 cells. Coexpression of wild-type ILK results in inhibition of GSK-3 activity whereas coexpression of kinase-deficient ILK (ILK-KD) results in enhanced GSK-3 activity relative to cells not transfected with ILK. Transfection efficiency was determined by monitoring GSK–HA expression by Western blot analysis by using anti-HA antibody. (C) ILK can phosphorylate GSK-3 in vitro. Recombinant kinase-deficient GST–GSK-3 coupled to glutathione-Sepharose beads was incubated with increasing concentrations of GST-ILK (7.5 ng/μl) in the presence of ³²P-orthophosphate. After 2-hr incubation at 30°C, phosphorylated GSK was detected by SDS/PAGE and autoradiography. ILK did not phosphorylate GST.

Figure 5

Kinase-deficient ILK inhibits phosphorylation of PKB/AKT on serine-473, and wild-type ILK directly phosphorylates PKB/AKT on serine-473 in vitro. (A) The status of phosphorylation of PKB/AKT on serine-473 was determined in IEC-18 cells and in wild-type ILK overexpressing (ILK-13) cells or IEC-18 cells expressing kinase-deficient ILK (ILK-KD, GH31R) (18, 19). Cells growing in serum (5 μg/ml) containing tissue culture medium were lysed and extracts were analyzed by Western blotting by using either anti-PKB/AKT antibody or serine-473 phospho-specific antibody. PKB/AKT is expressed at equivalent levels in all cell lines but the extent of serine-473 phosphorylated PKB/AKT is dramatically inhibited in cells expressing kinase-deficient ILK. (B) Transient transfection of kinase-deficient ILK with HA-tagged PKB/AKT inhibits serine-473 phosphorylation of PKB/AKT. Cotransfection of PKB/AKT with wild-type ILK results in enhanced phosphorylation on serine-473, whereas cotransfection with kinase-deficient ILK results in inhibition of serine-473 phosphorylation of PKB/AKT. Transfection efficiency was monitored by analyzing PKB/AKT-HA expression by using Western blot analysis with anti-HA antibody. (C) Direct phosphorylation of PKB/AKT on serine-473 by ILK in vitro. Recombinant GST-PKB/AKT coupled to glutathione-Sepharose beads was incubated with or without recombinant GST-ILK (75 ng) in kinase reaction buffer. After 2 hr at 30°C, the GST-PKB/AKT beads were precipitated and analyzed by Western blot analysis. The amount of PKB/AKT detected is equivalent in the presence or absence of ILK, whereas serine-473 phosphorylated PKB/AKT is detectable only in the presence of ILK.