MOLECULAR AND CELLULAR BIOLOGY, Aug. 2007, p. 5565–5574 0270-7306/07/$08.00⫹0 doi:10.1128/MCB.02372-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 15
Integrin-Linked Kinase Controls Notch1 Signaling by Down-Regulation of Protein Stability through Fbw7 Ubiquitin Ligase䌤 Jung-Soon Mo,1† Mi-Yeon Kim,1† Seung-Ok Han,1 In-Sook Kim,1 Eun-Jung Ann,1 Kyu Shik Lee,1 Mi-Sun Seo,1 Jin-Young Kim,1 Seung-Chul Lee,2 Jeen-Woo Park,3 Eui-Ju Choi,4 Jae Young Seong,5 Cheol O. Joe,6 Reinhard Faessler,7 and Hee-Sae Park1* Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757,1 Department of Dermatology, Chonnam National University, Gwangju 501-757,2 Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701,3 National Creative Research Initiative Center for Cell Death, School of Life Science and Biotechnology, Korea University, Seoul 136-701,4 Lab of G Protein Coupled Receptors, Graduate School of Medicine, Korea University College of Medicine, Seoul 136-705,5 and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701,6 Republic of Korea, and Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried D-82152, Germany7 Received 20 December 2006/Returned for modification 1 February 2007/Accepted 26 April 2007
Integrin-linked kinase (ILK) is a scaffold and protein kinase that acts as a pivotal effector in integrin signaling for various cellular functions. In this study, we found that ILK remarkably reduced the protein stability of Notch1 through Fbw7. The kinase activity of ILK was essential for the inhibition of Notch1 signaling. Notably, the protein level and transcriptional activity of the endogenous Notch1 intracellular domain (Notch1-IC) were higher in ILK-null cells than in ILK wild-type cells, and the level of endogenous Notch1-IC was increased by the blocking of the proteasome, suggesting that ILK enhances the proteasomal degradation of Notch1-IC. ILK directly bound and phosphorylated Notch1-IC, thereby facilitating proteasomal protein degradation through Fbw7. Furthermore, we found down-regulation of Notch1-IC and up-regulation of ILK in basal cell carcinoma and melanoma patients but not in squamous cell carcinoma patients. These results suggest that ILK down-regulated the protein stability of Notch1-IC through the ubiquitin-proteasome pathway by means of Fbw7.
ovarian, breast, and prostate carcinoma and skin cancer (23). High expression of Notch1 and Jagged1 is associated with poor prognosis in breast cancer and with metastasis in prostate cancer (23). However, the molecular basis for the oncogenic activity of Notch1-IC remains unclear. Integrin-linked kinase (ILK) is an integrin receptor-proximal cytoplasmic scaffold protein with a kinase domain that acts as a pivotal effector for various cellular functions, such as cell migration and invasion, cell proliferation, cell differentiation, cell metabolism, and cell survival (6, 7, 16, 37, 43, 44). Increased expression of ILK has been reported in malignant melanomas relative to benign lesions and melanocytes (5). High levels of ILK expression have also been detected in basal cell carcinoma (BCC) and wounded regions of skin (40). In contrast to ILK, a recent report has shown decreased expression of Notch1 in BCC (40). Moreover, Notch1 emerged as a tumor suppressor in BCC-like skin cancer (25). ILK is activated in a phosphoinositide 3-kinase-dependent manner; activated ILK phosphorylates and directly activates Akt (protein kinase B) on Ser473 (10, 30). ILK also phosphorylates and inhibits the activity of glycogen synthase kinase-3 (GSK-3), resulting in the activation of the transcription factors -catenin/Lef-1 and AP-1 (36, 41). GSK-3 modulates Notch1 signaling through direct phosphorylation of Notch1-IC, and the active GSK-3 protects or facilitates proteasomal degradation of Notch1-IC (12). For that reason, it was expected that ILK, the upstream kinase of GSK-3, could be a possible regulator for Notch1 signaling through GSK-3. Phosphorylation of
Notch1, a highly conserved transmembrane protein, has an essential role in the regulation of cell fate determination, cell differentiation, cell survival, and cell death in vertebrates and invertebrates (1). Notch1 is processed by furin in the endoplasmic reticulum Golgi apparatus (S1 cleavage), TACE after ligand binding (S2 cleavage), and ␥-secretase complex (S3 cleavage) in the plasma membrane, thereby generating the Notch1 intracellular domain (Notch1-IC), after which Notch1-IC is finally released from the membrane (2, 3, 13). Notch1-IC is translocated into the nucleus and functions as a transcriptional activator by binding to a transcription factor, CSL (CBF1/ RBP-Jk in vertebrates, Suppressor of Hairless [SuH] in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans) (33, 35, 38). After the transcriptional regulation of the target genes, Notch1-IC is degraded in the nucleus by the ubiquitin-proteasome system with the aid of Fbw7, an E3 ligase for the ubiquitination of Notch1-IC (19, 20, 24, 26, 39, 42, 46). At present, the regulator of Notch1-IC protein stability via Fbw7 is not well defined. Deregulated expression of Notch receptors, ligands, and targets is observed in solid tumors, including cervical, head and neck, endometrial, renal, lung, pancreatic,
* Corresponding author. Mailing address: School of Biological Sciences and Technology, Chonnam National University, Yongbongdong, Buk-ku, Gwangju 500-757, Republic of Korea. Phone: 82-62530-0021. Fax: 82-62-530-2199. E-mail:
[email protected]. † J.-S.M. and M.-Y.K. contributed equally to this work. 䌤 Published ahead of print on 25 May 2007. 5565
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Notch1-IC by GSK-3, CDK8, and possibly other kinases regulates its half-life (12, 14). However, little is known of any other protein kinase(s) that may contribute to the turnover of Notch1-IC. Therefore, in this study, we evaluated the signal cross talk occurring between ILK and Notch1 signaling. Through observation of a reduction in the protein stability of Notch1-IC, we determined that the transcriptional activity of Notch1-IC was inhibited by ILK. Interestingly, the level of the Notch1-IC protein was markedly down-regulated in the presence of ILK via the enhancement by ILK of the phosphorylation and proteasomal degradation of Notch1-IC through Fbw7. Furthermore, we also determined that the up-regulation of ILK and the down-regulation of Notch1-IC occurred in melanoma and BCC but not in squamous cell carcinoma (SCC). Collectively, our findings indicate that ILK functions as a negative regulator of the protein turnover of Notch1-IC through Fbw7. MATERIALS AND METHODS Cell culture and transfection. NIH 3T3 and human embryonic kidney (HEK) 293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum, penicillin (100 U/ml), and streptomycin (100 g/ml). Along with the NIH 3T3 and HEK293 cells, ILK wild-type and ILK-null mouse embryonic fibroblasts were cultured in DMEM supplemented with 10% bovine calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. GSK-3 wild-type and GSK-3-null mouse embryonic fibroblasts were routinely maintained in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml). For plasmid DNA transfection, cells were plated at a density of 2 ⫻ 106/100-mm-diameter dish, grown overnight, and transfected with appropriate expression vectors in the presence of the indicated combinations of plasmid DNAs by using the calcium phosphate method (29). Cloning and preparation of recombinant proteins. A mouse Notch1-IC gene and deletion mutants were constructed via standard PCR and inserted into the bacterial expression vector pGEX4T-3 (Amersham Pharmacia). The Notch1-IC deletion mutants constructed in the present study were as follows: Notch1-IC-N (amino acid residues 1744 to 2283), Notch1-IC-N1 (amino acid residues 1744 to 2076), Notch1-IC-N2 (amino acid residues 1744 to 2014), Notch1-IC-N3 (amino acid residues 1744 to 1939), Notch1-IC-N4 (amino acid residues 1744 to 1872), and Notch1-IC-N5 (amino acid residues 1744 to 1808). Expression of the recombinant glutathione S-transferase (GST)–Notch1-IC proteins within the transformed bacteria was induced using 1 mM isopropyl--D-thiogalactopyranoside (Sigma). GST–Notch1-IC and its mutant proteins were purified with glutathione (GSH)-agarose (Sigma) in accordance with the manufacturer’s instructions. Sitedirected mutagenesis of Notch1-IC cDNA was performed with a QuikChange kit (Stratagene). The mutations were verified by automatic DNA sequencing. Reporter assay. The cells were lysed in chemiluminescent lysis buffer (18.3% 1 M K2HPO4, 1.7% 1 M KH2PO4, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1 mM dithiothreitol [DTT]) and assayed for luciferase activity with a luciferase assay kit (Promega). The activity of the luciferase reporter protein in the transfected cells was normalized in reference to the -galactosidase activity in the same cells (28). Coimmunoprecipitation assays. The cells were lysed in 1 ml of radioimmunoprecipitation assay buffer for 30 min at 4°C. After centrifugation at 12,000 ⫻ g for 20 min, supernatants were subjected to immunoprecipitation with appropriate antibodies coupled to protein A-agarose beads. The resulting immunoprecipitates were washed three times with phosphate-buffered saline (PBS; pH 7.4). Laemmli sample buffer was then added to the immunoprecipitated pellets; the pellets were heated at 95°C for 5 min and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed with the indicated antibodies (27). Immunocomplex kinase assay. To analyze the kinase activity, confluent cells were harvested and lysed in lysis buffer. Cell lysates were then subjected to 10 min of centrifugation at 12,000 ⫻ g and 4°C. The soluble fraction was incubated for 1 h with appropriate antibodies against the indicated protein kinases at 4°C. The immunocomplexes were then coupled to protein G-agarose during an additional 1 h of incubation at 4°C, after which they were pelleted by centrifugation. The immunopellets were rinsed three times with buffer A and then twice with 20 mM HEPES, at a pH of 7.4. Immunocomplex kinase assays were conducted by
MOL. CELL. BIOL. incubation of the immunopellets for 30 min at 30°C with 2 g of substrate proteins in 20 l reaction buffer containing 0.2 mM sodium orthovanadate, 10 mM MgCl2, 2 Ci [␥32P]ATP, 20 mM HEPES (pH 7.4). Phosphorylated substrates were then visualized by SDS-PAGE and quantified using a Fuji BAS 2500 phosphorimager (27). Skin cancer samples. Human skin was obtained from mammary reduction operations at the plastic surgery department of the Chonnam National University hospital, South Korea. All patients from the Chonnam National University hospital gave informed consent for biopsy specimens to be taken. All had stable psoriatic plaques; none of them had been treated with systemic drugs or phototherapy in the month preceding sampling. When harvested, biopsy specimens were frozen in liquid nitrogen and stored at ⫺80°C until use. Immunofluorescence staining. Assays were conducted as previously described with HEK293 cells plated at 1 ⫻ 105 per well onto coverslips (Fisher). A total of 0.5 g of appropriate DNA per well was then transfected using Geneporter2 (Gene Therapy Systems). The transfected cells were fixed with 4% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X-100 in PBS. Mouse antihemagglutinin (anti-HA) (Sigma) and anti-Myc (Novus Biologicals) monoclonal antibodies were used as primary antibodies at a dilution of 1:100. A Rhodamine Red- or fluorescein-conjugated anti-mouse secondary antibody (1: 100) was added, and then the cells were stained with 4⬘,6⬘-diamidino-2-phenylindole dihydrochloride (DAPI). The stained cells were evaluated for localization by confocal microscopy (Leica TCS SP5). Preparation of cytosolic and nuclear fractions. The cells were rinsed with ice-cold PBS and then harvested by 5 min of centrifugation at 3,000 rpm and 4°C. The dispersed cells were then homogenized with buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.5 mM EDTA, 1 mM DTT, and 0.5 mM PMSF). After 15 min on ice, 10% NP-40 was added, and the mixture was vortexed vigorously for 10 s. The resultant supernatant was then used as a cytosolic fraction, via 1 to 2 min of centrifugation at 13,000 rpm and 4°C. The pellet was then homogenized with buffer B (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF). After 10 min of vigorous vortexing, the homogenates were centrifuged for 10 min at 13,000 rpm and 4°C. The resultant supernatants were then used as nuclear fractions. The nuclear and cytosolic fractions were quantified using the Bradford method, and 20 g of each fraction was analyzed by SDS-PAGE.
RESULTS ILK suppresses the transcriptional activity of Notch1. To determine whether ILK is involved in regulating the transcriptional activation of Notch1 target genes, a reporter assay was performed with NIH 3T3 cells using luciferase reporter genes. In this study, three types of luciferase reporter genes were evaluated under the control of the Hes1 promoter (Hes1-Luc), the Hes5 promoter (Hes5-Luc), and the artificial 4⫻CSL promoter (4⫻CSL-Luc) (33, 35, 38). We investigated the effect of ILK on Notch1-IC transcriptional activity. The Notch1-ICinduced 4⫻CSL luciferase reporter activity was also inhibited by the cotransfection of ILK using 4⫻CSL-Luc reporter genes (Fig. 1A). We also found similar results using the Hes1-Luc and Hes5-Luc reporter systems (data not shown). To investigate whether the kinase activity of ILK is necessary for the down-regulation of the transcriptional activation of Notch1 target genes, we used a dominant-negative, kinasedeficient ILK mutant (ILK-E359K, called ILK-KD) to block the kinase activity of ILK (17, 45). In the luciferase reporter gene assay with NIH 3T3 cells, ILK-KD was transfected instead of ILK, and the effect of this transfection on the transcriptional activation of Notch1-IC target genes was then assessed using 4⫻CSL-Luc. The transcriptional activity of Notch1-IC was inhibited by ILK but was not inhibited by the cotransfection of Notch1-IC and ILK-KD instead of ILK (Fig. 1B). To determine the role of endogenous ILK in Notch1 signaling, we performed a transcription reporter assay using ILK wild-type (ILK⫹/⫹) and ILK-deficient (ILK⫺/⫺) fibroblasts (32). The transcriptional activity of Notch1-IC in ILK-
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FIG. 1. ILK suppresses the transcriptional activity of Notch1 target genes. (A) NIH 3T3 cells were transfected with expression vectors encoding Notch1-IC and ILK with 4⫻CSL-Luc. (B) NIH 3T3 cells were transfected with expression vectors encoding Notch1-IC, ILK, and ILK-KD with 4⫻CSL-Luc as indicated. (C) ILK⫹/⫹ and ILK⫺/⫺ fibroblast cells were transfected with expression vectors encoding Notch1-IC with 4⫻CSL-Luc. (D) ILK⫺/⫺ fibroblast cells were transfected with expression vectors encoding Notch1-IC and ILK with 4⫻CSL-Luc. After 48 h of transfection, cells were lysed and the luciferase activity was assayed. Data were normalized to -galactosidase activity. Results are means ⫾ average deviations from three independent experiments.
deficient fibroblasts was threefold higher than that in ILK wild-type cells (Fig. 1C). We also found that Notch1-IC transcriptional activity was suppressed by overexpressed ILK in ILK-deficient cells (Fig. 1D). These results indicated that ILK suppresses the transcriptional activity of Notch1 in intact cells. ILK down-regulates the level of Notch1-IC protein. To determine whether ILK is involved in regulating the interactions between Notch1-IC and RBP-Jk, coimmunoprecipitation was performed for ILK wild-type and ILK-deficient fibroblasts. Endogenous binding between Notch1-IC and RBP-Jk in ILKdeficient fibroblasts was much higher than that in ILK wildtype cells (Fig. 2A). To observe the effects of ILK on the molecular interactions between Notch1-IC and RBP-Jk, coimmunoprecipitation was performed in HEK293 cells by cotransfection of Myc-tagged Notch1-IC, Flag-tagged RBP-Jk, and HA-tagged ILK. Notch1-IC and RBP-Jk were coimmunoprecipitated, but when they were cotransfected with ILK, the band of Notch1-IC that interacted with RBP-Jk disappeared (Fig. 2B). Immunoprecipitation was performed on cell lysates with an anti-Myc antibody, and immunoblotting was performed with the anti-Flag antibody; the results reconfirmed the disruption of the Notch1-IC–RBP-Jk complex in the presence of ILK (data not shown). Surprisingly, on the cell lysate immunoblot, the level of Notch1-IC protein was down-regulated upon cotransfection of ILK (Fig. 2B), which shows that ILK may regulate the level of Notch1-IC protein.
Next, we used Western blot analysis on HEK293 cells to determine whether ILK plays a role in the regulation of the Notch1-IC protein level. Cells were cotransfected with Myctagged Notch1-IC and HA-tagged ILK. We found that the Notch1-IC protein level was reduced upon cotransfection of ILK but was not reduced upon cotransfection of ILK-KD (Fig. 2C). This result showed that the kinase activity of ILK is essential for the regulation of the Notch1-IC protein level. ILK has been shown to phosphorylate GSK-3 on Ser9, leading to inactivation of GSK-3 kinase activity (8). Also, it has already been reported that GSK-3 is a positive regulator of Notch1 (12). Therefore, we attempted, by using GSK3(S9A), to determine whether ILK down-regulates the transcriptional activity of Notch1 through GSK-3. To test the involvement of GSK-3 in the down-regulation of Notch1-IC protein by ILK, HEK293 cells were transfected with GSK3(S9A) and ILK. The results showed that the down-regulation of Notch1-IC protein by ILK was independent of GSK-3 (Fig. 2D). Thus, the down-regulation of the Notch1-IC protein level by ILK was dependent on the intact kinase activity of ILK but independent of the downstream kinase GSK-3. ILK negatively regulates Notch1 signaling via an E3 ligase, Fbw7. We tested whether transiently expressed Notch1-IC could be subjected to proteasome-mediated proteolysis as reported previously (19, 20, 24, 26, 39, 42, 46). Notch1-IC was stabilized by treatment with MG132, a proteasome inhibitor, in
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FIG. 2. ILK down-regulates the level of Notch1-IC protein. (A) ILK⫹/⫹ and ILK⫺/⫺ fibroblast cells were lysed and subjected to immunoprecipitation (IP) with immunoglobulin G (IgG) and anti-Notch1-IC antibodies as indicated. Immunoprecipitates were immunoblotted (IB) with an anti-RBP-Jk antibody. Cell lysates were immunoblotted with anti-ILK as a control. (B) HEK293 cells were transfected for 48 h with expression vectors encoding the indicated combinations of Myc–Notch1-IC, Flag-RBP-Jk, and HA-ILK. Cell lysates were subjected to immunoprecipitation with the anti-Flag antibody M2, and the immunoprecipitates were immunoblotted with an anti-Myc antibody (9E10). The cell lysates were also subjected to immunoblot analysis with anti-Myc (9E10), anti-Flag (M2), and anti-HA antibodies, respectively. (C and D) HEK293 cells were transfected for 48 h with expression vectors encoding Myc–Notch1-IC, V5-ILK, HA-ILK, V5-ILK-KD, and HA-GSK-3(S9A) in the indicated combinations. The cell lysates were immunoblotted with anti-Myc (9E10), anti-HA, and anti-V5 antibodies.
a dose-dependent manner (data not shown). Therefore, we confirmed that Notch1-IC was degraded in a proteasome-dependent pathway. Next, we evaluated the involvement of ILK in the Notch1-IC proteasome-dependent degradation pathway by performing luciferase reporter gene assays and Western blot analysis. The transcriptional activity of Notch1-IC was inhibited by ILK but recovered in the presence of MG132 (Fig. 3A). The Notch1-IC protein level was decreased in the presence of ILK but was significantly restored by treatment with MG132 (Fig. 3A). Moreover, the endogenous Notch1-IC protein level was lower in ILK wild-type cells than in ILK-null cells (Fig. 3B, first and third lanes), and treatment with MG132 enhanced the endogenous Notch1-IC protein level by inhibiting proteasomal degradation (Fig. 3B, second and fourth lanes). 3B). These results revealed that the stability of the Notch1-IC protein was down-regulated by ILK through the proteasome-dependent pathway. Generally, the ubiquitination of proteins leads to their rapid degradation, and Notch1-IC is degraded by the ubiquitin-proteasome system in the nucleus. Notch1-IC is ubiquitinated by the F-box protein Fbw7/Sel-10/hCdc4/Ago, which was first isolated from a process of genetic screening for negative regulators of Notch in C. elegans (26, 46). Fbw7 consists of the subdomain of the F-box and seven WD40 repeats. The F-box domain mediates the interaction with the core ubiquitination complexes, and the WD40 repeats mediate interactions with
substrates (15, 18). We expected that Fbw7 might act as a mediator for the negative regulation of Notch1-IC by ILK. Therefore, we tested the involvement of Fbw7 by using the F-box-deleted and hence dominant-negative mutant form of Fbw7 (Fbw7⌬F). When Fbw7⌬F was cotransfected with Notch1-IC and ILK, the transcriptional activation of Notch1 was increased (Fig. 3C). In agreement with previous reports, coexpression of the dominant-negative Fbw7⌬F construct with Notch1-IC increased reporter expression approximately twofold, suggesting that ILK-independent Notch1IC degradation also contributed to the down-regulation of Notch1-IC reporter activity (Fig. 3C) (46). By Western blot analysis, the Notch1-IC protein level was found to be decreased by ILK and was remarkably restored by the cotransfection of Fbw7⌬F (Fig. 3C). These results indicated that Fbw7⌬F could recover and enhance the transcriptional activity and protein level of Notch1-IC in the presence of ILK. Accordingly, we suggest that ILK negatively regulates Notch1-IC through Fbw7. Next, we evaluated the involvement of ILK in the physical association between Fbw7 and Notch1-IC by coimmunoprecipitation. HEK293 cells were cotransfected with vectors encoding V5-tagged wild-type ILK, dominant-negative ILK, green fluorescent protein (GFP)-tagged Fbw7, and Myc-tagged Notch1IC and were then subjected to coimmunoprecipitation analysis (Fig. 3D). Immunoblot analysis of Myc immunoprecipitates
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FIG. 3. ILK negatively regulates Notch1 signaling via an E3 ligase, Fbw7. (A) NIH 3T3 cells were transfected with expression vectors encoding Myc–Notch1-IC and HA-ILK with the 4⫻CSL-Luc reporter in the indicated combinations. After 42 h of transfection, the cells were treated with MG132 (3 M) for 6 h. The cells were then lysed, and the luciferase activity was assayed. Data were normalized to -galactosidase activity. These results are means ⫾ average deviations from three independent experiments. The cell lysates were immunoblotted (IB) with anti-Myc (9E10) and anti-HA antibodies, respectively. (B) ILK wild-type and null cells were treated with MG132 (3 M) for 6 h, and cell lysates were immunoblotted with anti-Notch1-IC and anti-ILK antibodies. We quantified the intensity of each band using a densitometer and plotted relative intensities, taking the results for wild-type cells without MG132 treatment as 1. Data are expressed as means ⫾ standard deviations from three independent experiments. *, P ⬍ 0.001 by ANOVA. (C) NIH 3T3 cells were transfected with expression vectors encoding Myc–Notch1-IC, V5-ILK, and HA-Fbw7⌬F with the 4⫻CSL-Luc reporter in the indicated combinations. After 48 h of transfection, cells were lysed and the luciferase activity was assayed. Data were normalized to -galactosidase activity. These results are means ⫾ average deviations from three independent experiments. The cell lysates were immunoblotted with an anti-Myc antibody (9E10) and anti-V5 antibodies. (D) HEK293 cells were transfected with expression vectors encoding Myc–Notch1-IC, V5-ILK, V5-ILK-KD, and GFP-Fbw7 as indicated. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with anti-Myc. The immunoprecipitates were then immunoblotted with an anti-Myc antibody. Cell lysates were also immunoblotted with an anti-Myc, anti-V5, or anti-His antibody.
from the transfected cells using an anti-GFP antibody revealed that ILK facilitated the physical association between Fbw7 and Notch1-IC in the cells (Fig. 3D). These results indicated that the down-regulation of the Notch1-IC protein by ILK occurred via an Fbw7-dependent pathway. Physical interaction of ILK with Notch1-IC in intact cells. Given that our results suggest that Notch1 is a target of ILK, we next investigated whether these two proteins interact physically in intact cells. In the in vitro binding studies, purified GST and GST–Notch1-IC proteins were immobilized on GSHagarose. Cell lysates expressing V5-ILK were incubated either with immobilized GST or with GST–Notch1-IC on GSH-agarose. The interaction between GST–Notch1-IC and ILK was detected on bead complexes (Fig. 4A). HEK293 cells were cotransfected with vectors encoding V5-tagged wild-type ILK and Myc-tagged Notch1-IC and were then subjected to coimmunoprecipitation analysis (Fig. 4B). Immunoblot analysis of Myc immunoprecipitates from the transfected cells with an anti-V5 antibody revealed that V5-ILK physically associated
with Myc–Notch1-IC in the cells. Conversely, immunoblot analysis of the V5 immunoprecipitates with an anti-Myc antibody also showed the interaction between the two proteins (Fig. 4B). We also examined whether endogenous ILK and Notch1-IC could interact in intact cells. Using ILK⫹/⫹ and ILK⫺/⫺ fibroblast cells, immunoblot analysis of the Notch1-IC immunoprecipitates with an anti-ILK antibody indicated the physical association of endogenous ILK and Notch1-IC in ILK⫹/⫹ cells (Fig. 4C). Notch1-IC has a CDC domain including six ankyrin repeats, an OPA domain, and a PEST domain in its structure. We investigated which of the domains might be involved in the interaction of Notch1-IC and ILK. We used various Flagtagged Notch1 deletion mutants: Notch1-IC-N (CDC domain), Notch1-IC-⌬N⌬C (OPA domain), or Notch1-IC-C (PEST domain). We performed coimmunoprecipitation using three Notch1-IC deletion mutants and V5-tagged ILK (Fig. 4D). Our results show that ILK bound to Notch1-IC-N but not to Notch1-IC-⌬N⌬C or Notch1-IC-C.
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FIG. 4. Physical interaction of ILK with Notch1-IC in intact cells. (A) Recombinant GST or GST–Notch1-IC proteins were immobilized onto GSH-agarose. HEK293 cells were transfected with an expression vector encoding V5-ILK or an empty vector. After 48 h of transfection, the cell lysates were subjected to GST pulldown experiments with immobilized GST or GST–Notch1-IC. Proteins bound to GST or GST–Notch1-IC were analyzed via immunoblotting with an anti-V5 antibody. (B) HEK293 cells were transfected with expression vectors encoding Myc–Notch1-IC and V5-ILK as indicated. After 42 h of transfection, the cells were treated with MG132 (3 M) for 6 h. The cell lysates were subjected to immunoprecipitation (IP) with an anti-Myc or anti-V5 antibody. The immunoprecipitates were then immunoblotted (IB) with an anti-V5 or anti-Myc antibody. Cell lysates were also immunoblotted with anti-Myc and anti-V5 antibodies. Asterisk indicates a nonspecific band. (C) ILK⫹/⫹ and ILK⫺/⫺ fibroblast cells were lysed and subjected to immunoprecipitation with immunoglobulin G (IgG) and anti-Notch1-IC antibodies as indicated. Immunoprecipitates were immunoblotted with an anti-ILK antibody. Cell lysates were immunoblotted with anti-ILK as a control. (D) HEK293 cells were transfected with expression vectors encoding Flag–Notch1-IC, Flag–Notch1-IC-N, Flag–Notch1-IC-⌬N⌬C, Flag–Notch1IC-C, and V5-ILK. After 42 h of transfection, the cells were treated with MG132 (3 M) for 6 h. The cell lysates were then subjected to immunoprecipitation with an anti-ILK antibody. The immunoprecipitates were then immunoblotted with an anti-Flag antibody. Cell lysates were also immunoblotted with an anti-Flag or anti-V5 antibody.
ILK phosphorylates Notch1 on Ser2173. We next conducted an in vitro kinase assay with V5-ILK and purified GST– Notch1-IC. The V5-ILK immunocomplexes prepared from HEK293 cells catalyzed the phosphorylation of purified recombinant GST–Notch1-IC (Fig. 5A). Serial deletion mutants of Notch1-IC were employed in the ILK phosphorylation reaction in order to determine the possible phosphorylation site of Notch1-IC (Fig. 5B). ILK phosphorylated GST–Notch1-IC-N but did not phosphorylate the other six deletion mutants (Fig. 5B). According to these results, we may surmise that the possible phosphorylation sites are located between residues 2076 and 2283 of Notch1-IC, a region that harbors two conserved serine residues: Ser2152 and Ser2173. Furthermore, via site-directed mutagenesis, we determined that the replacement of Ser2173 of Notch1-IC with alanine effected a reduction in the in vitro phosphorylation of the recombinant protein by the V5-ILK immunoprecipitates (Fig. 5C). Moreover, we ascertained that Notch1-IC(S2173A) is resistant to ILK-induced degradation, which implies that the ILK-induced phosphorylation of Notch1-IC is crucial for the
degradation of the Notch1 protein (Fig. 5D). We then attempted to characterize the involvement of phosphorylation in the physical association between Fbw7 and Notch1-IC via coimmunoprecipitation. HEK293 cells were cotransfected with vectors coding for HA-tagged ILK, His-tagged Fbw7, Myctagged Notch1-IC, and Myc-tagged Notch1-IC(S2173A) and were then subjected to coimmunoprecipitation analysis. Immunoblot analysis of the His immunoprecipitates from the transfected cells with an anti-Myc antibody showed that ILK does not facilitate the physical association between Fbw7 and Notch1-IC(S2173A) in the cells (Fig. 5E). Furthermore, we also determined that ILK is present in the same complex with Notch1-IC and Fbw7 and forms a trimeric complex in a phosphorylation-dependent manner (Fig. 5E). These results showed that the phosphorylation of Notch1-IC by ILK is crucial to its ability to bind with Fbw7. Up-regulation of ILK and down-regulation of Notch1-IC in melanoma and BCC but not in SCC. We then attempted to determine whether ILK activity is crucial to Notch1 signaling in three different skin cancer types: melanoma, BCC, and SCC.
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FIG. 5. ILK phosphorylates Notch1 on Ser2173. (A) HEK293 cells were transfected with expression vectors encoding V5-ILK as indicated. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, and the resulting precipitates were examined for ILK kinase activity by an immune complex kinase assay using GST–Notch1-IC. (B) HEK293 cells were transfected with expression vectors encoding V5-ILK or an empty vector as indicated. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, and the resulting precipitates were examined for ILK kinase activity by an immune complex kinase assay using purified recombinant GST–Notch1-IC-N, GST–Notch1-IC-N1, GST–Notch1-IC-N2, GST–Notch1-IC-N3, GST–Notch1-IC-N4, GST–Notch1-IC-N5, and GST–Notch1-IC-C proteins. (C) HEK293 cells were transfected with expression vectors encoding V5-ILK as indicated. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, and the resulting precipitates were examined for ILK kinase activity by an immune complex kinase assay using GST–Notch1-IC (wild type [WT]), GST–Notch1-IC(S2152A), and GST–Notch1-IC(S2173A). (D) HEK293 cells were transfected with expression vectors encoding Myc–Notch1-IC (WT), Myc–Notch1-IC(S2152A), Myc–Notch1-IC(S2173A), and V5-ILK as indicated. After 48 h of transfection, the cell lysates were subjected to immunoblotting (IB) with an anti-Myc or anti-V5 antibody. (E) HEK293 cells were transfected with expression vectors encoding Myc–Notch1-IC, Myc–Notch1-IC(S2173A), V5-ILK, and His-Fbw7 as indicated. After 48 h of transfection, the cell lysates were subjected to immunoprecipitation with anti-His. The immunoprecipitates were then immunoblotted with an anti-Myc or anti-V5 antibody. Cell lysates were also immunoblotted with an anti-Myc, anti-V5, or anti-His antibody.
In HaCaT cells, which are normal keratinocytes, ILK expression and Notch1-IC expression were comparable (Fig. 6A). However, in A375P (low-metastasis human melanoma) cells, high ILK expression levels were observed but Notch1-IC levels were significantly down-regulated (Fig. 6A). We also verified the expression levels of ILK and Notch1-IC using another melanoma cell line, A375SM (p53 mutated, pRb positive). These cells evidenced moderate ILK expression levels, but
Notch1-IC levels were significantly down-regulated (Fig. 6A). Interestingly, we observed high levels of Notch1-IC expression and low levels of ILK expression in A431 (human epidermoid carcinoma [SCC]) cells (Fig. 6A). We then determined the levels of ILK and Notch1-IC expression in samples taken from skin cancer patients. In melanoma cells, the expression of ILK was slightly higher than that in control cells, whereas the expression of Notch1-IC was shown to be severely down-regu-
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FIG. 6. Expression pattern of ILK and Notch1-IC in melanoma, BCC, and SCC. (A) HaCaT, A375P (low-metastasis human melanoma), A375SM (p53-mutated, pRb-positive melanoma), and A431 (human epidermoid carcinoma [SCC]) cells were lysed and subjected to immunoblotting with anti-ILK and anti-Notch1-IC antibodies. (B) Melanoma, BCC, and SCC patients’ normal and cancer tissues were lysed and subjected to immunoblotting with anti-ILK and antiNotch1-IC antibodies. Con, control.
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lated (Fig. 6B). In the case of BCC, we observed a robust increase in the level of ILK expression, but Notch1-IC expression was severely attenuated (Fig. 6B). However, only minimal ILK proteins were detected in the SCC samples, whereas Notch1-IC expression was dramatically increased (Fig. 6B). Notch1-IC enhances the nuclear accumulation of ILK. The nucleus may constitute the primary site of Notch1-IC degradation by Fbw7. Notch1-IC is located predominantly within the nucleus. However, ILK is located predominantly within the focal adhesions of the cytoplasm. Therefore, in this study, we conducted immunofluorescence staining analyses in order to determine whether ILK was colocalized with Notch1-IC or not. In order to identify the cellular compartment, HA-ILK or Myc–Notch1-IC was expressed in HEK293 cells. As had been expected, ILK and Notch1-IC were localized predominantly in the cytoplasm (80% of stained cells) and nucleus (90% of stained cells), respectively (Fig. 7A). We then attempted to assess the subcellular localization of ILK within HEK293 cells in the presence of Notch1-IC. When the cells were coexpressed with Notch1-IC, ILK was detected within both the nucleus and the cytoplasm (Fig. 7A). Both the nuclear and the cytoplasmic localization of ILK in the presence of Notch1-IC were corrob-
FIG. 7. Notch1-IC enhances the nuclear accumulation of ILK. (A) HEK293 cells were transfected with HA-ILK or Myc–Notch1-IC as indicated. After 48 h, ILK and Notch1-IC were stained with rhodamine (red) and fluorescein (green), respectively. (B) HEK293 cells were transfected with HA-ILK or Myc–Notch1-IC as indicated. After 48 h, the cells were fractionated into cytosolic (C), and nuclear (N) fractions. Lamin B and -actin were used as nuclear and cytoplasmic fraction markers, respectively. The cell lysates were also subjected to immunoblot (IB) analysis with antibodies against HA and Myc. (C) HaCaT, A375P, and A375SM cells were stained with antibodies against endogenous ILK (red) and Notch1 (green). (D) HaCaT, A375P, and A375SM cells were fractionated into cytosolic (C) and nuclear (N) fractions. The cell lysates were also subjected to immunoblot analysis with antibodies against ILK and Notch1-IC. Lamin B and -actin were used as nuclear and cytoplasmic fraction markers, respectively.
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orated by immunoblotting of extracts of the nuclear and cytoplasmic fractions (Fig. 7B). Previous studies have indicated that a large proportion of cellular ILK was present in the nuclei of skin cancer cells (9). We therefore attempted to evaluate the localization of endogenous levels of ILK and Notch1 in A375P, A375SM, and HaCaT cells. ILK and Notch1 were detected in both the nucleus and the cytoplasm (Fig. 7C). We also assessed the cellular distribution of endogenous ILK and Notch1-IC via the subcellular fractionation of the nuclear and cytoplasmic fractions. Endogenous ILK was detected in both the nucleus and the cytoplasm, whereas endogenously processed Notch1-IC was determined to be localized principally within the nucleus (Fig. 7D). Collectively, our data indicate that Notch1-IC and ILK may exist within the same compartment. DISCUSSION A recent study has suggested that GSK-3, a downstream kinase of ILK, phosphorylates and positively or negatively regulates the Notch1 signaling pathway both at the transcriptional level and in terms of protein stability (11, 12). For that reason, it was expected that ILK, the upstream kinase of GSK-3, could be a possible regulator for Notch1 signaling through GSK-3. However, no direct relationship between ILK and Notch1 signaling has been reported yet. In this study, we found that ILK inhibited the transcriptional activity of Notch1-IC and that the kinase activity of ILK was essential in order for this function to occur. Our findings also support the idea that the suppression of the transactivation of the Notch1-IC target genes by ILK was independent of the downstream effector GSK-3 (data not shown). Several groups have shown that Sel-10/Fbw7, via its WD40 domains, binds to phosphorylated Notch1-IC and mediates its ubiquitination and subsequent rapid degradation (15, 18, 26, 46). Our results showed that the inhibitory mechanism functioned through the suppression of the interaction of Notch1-IC and RBP-Jk due to the down-regulation of Notch1-IC protein stability; it was also dependent on the kinase activity of ILK and independent of GSK-3. In this study, we found that ILK stimulated the proteasomal degradation of ectopically expressed Notch1-IC, and the reduction of endogenous Notch1-IC levels by ILK was also observed as a proteasome-dependent regulation in ILK-null cells. Collectively, our findings show that the kinase activity of ILK plays a crucial role in the proteasomal degradation of Notch1-IC. Phosphorylation of Notch1-IC by GSK3, CDK8, and possibly other kinases regulates its half-life in a positive or negative manner (14). However, little is known of any other protein kinase(s) that may contribute to the turnover of Notch1-IC. The negative regulation of Notch1-IC by ILK is further supported by our observation that endogenous ILK, when activated, physically interacts with endogenous Notch1-IC in intact cells. Furthermore, in this study we demonstrate that ILK-mediated Notch1-IC phosphorylation on Ser2173 results in a decrease in the degradation of Notch1-IC protein. Moreover, we found that ILK negatively regulates the transcriptional activation of the Notch1-IC target genes and the stability of Notch1-IC protein in an Fbw7- and proteasomedependent manner. ILK, Notch1-IC, and Fbw7 form a trimeric complex, and the formation of this complex is likely controlled
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by phosphorylation of Notch1-IC at the Ser2173 residue. Thus, enhancement of the interaction between Notch1-IC and Fbw7 may be a possible mechanism for the ILK-mediated phosphorylation and proteasomal degradation of Notch1-IC. When the cells overexpressed Notch1-IC or ILK, Notch1-IC was located predominantly within the nucleus and ILK was located mainly in focal adhesions of the cytoplasm. However, when the cells coexpressed ILK with Notch1-IC, ILK was found in both the nucleus and the cytoplasm, suggesting that Notch1-IC facilitates nuclear accumulation of ILK under certain conditions. Previous reports have shown that the nuclear distribution of ILK may be attributable to an impairment of its association with either PINCH or caveolin (4, 22). Further studies will therefore be necessary in order to obtain deeper insight into the overall function of Notch1-IC in ILK subcellular localization control. In addition to its prominent focal adhesion localization, ILK was also observed in the nuclear and cytoplasmic compartments. The nuclear distribution of ILK is somewhat surprising but has been demonstrated previously in Cos-1 and skin cancer cells (4, 9). Chun et al. suggest that nuclear localization sequences are present in front of the caveolin-1-binding motif. ILK seems to be localized to the nucleus through the putative nuclear localization signal (4). We also observed the colocalization of ILK and Notch1-IC in both the nucleus and the cytoplasm in HaCaT and melanoma cell lines. ILK is involved in tumor growth and angiogenesis through the generation of vascular endothelial growth factor (37) and usually transduces signals through the downstream effectors Akt and GSK-3, but the negative regulation of Notch1 signaling is mediated by ILK itself, in a manner that is independent of GSK-3, according to our observations. The results also coincided in terms of the regulation of the transcriptional activation of Notch1 target genes and the stability of Notch1-IC protein. Recently, several reports concentrated on the function of Notch1 as a tumor suppressor in various tumors (21, 31, 34). Our data now demonstrate the expression patterns of ILK and Notch1-IC in skin cancers including melanoma, BCC, and SCC. Analysis of ILK expression in human melanoma, BCC, and SCC biopsy samples demonstrated that ILK expression levels increased. The expression pattern of Notch1-IC is the reverse of the expression pattern of ILK in melanoma and BCC. Therefore, the mechanism of down-regulation of Notch1 signaling by ILK holds promise in controlling developmental programs or reducing the tumor suppressor functions of Notch1 proteins. For this purpose, the precise mechanism of the function of ILK in the regulation of Notch1 signaling must be intensively investigated. ACKNOWLEDGMENTS We thank R. Kopan (Washington University Medical School, St. Louis, MO), S. Dedhar (British Columbia Cancer Agency, Canada), K. A Jones (Salk Institute), and B. E. Clurman (Fred Hutchinson Cancer Research Center) for providing Notch, ILK, GST–Notch1-IC, and Fbw7 clones. GSK-3-null cells were kindly provided by J. R. Woodgett (Ontario Cancer Institute, Canada). This work was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program, funded by the Ministry of Science and Technology (to H.-S. Park), and by a Korea Research Foundation grant (MOEHRD, Basic Research Promotion Fund) (KRF-2005-070-C00105) (to H.-S. Park) from the Republic of Korea.
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