Integrin-Linked Kinase Controls Notch1 Signaling by Down-Regulation of Protein Stability through Fbw7 Ubiquitin Ligase

Abstract

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.

Notch-dependent cell cycle arrest and apoptosis in mouse embryonic fibroblasts lacking Fbxw7

“…A phosphodegron of Notch has been identified and found to differ slightly from the 'typical' degrons of c-Myc and cyclin E. The serine or threonine residue at position þ 4 relative to the central threonine is thus replaced with glutamic acid in the phosphodegron of Notch (O'Neil et al, 2007;Thompson et al, 2007). The turnover of NICD1 is thought to be regulated by the cyclin C-CDK8 complex and integrin-linked kinase (Fryer et al, 2004;Mo et al, 2007). Together, these observations suggest that the efficiency of substrate recognition by Fbxw7 is dependent on the balance of various kinase activities that are regulated in a cell type-or developmental stage-specific manner.…”

Inhibition of Notch1 signaling by Runx2 during osteoblast differentiation

“…(45) The luciferase reporter plasmids were under the control of 4XCSL-Luc (a four-time repeating section of the RBP-Jk target sequence, CGTGGGAA, with the luciferase gene) and Hes1-Luc (-467 to þ46 of the Hes1 promoter with the luciferase gene). In brief, Myc-Notch1-IC and the Notch1 transcription reporter system (4XCSL-Luc or Hes1-Luc) were transfected along with bgalactosidase in cells (12-well plates).…”

“…The luciferase reporter activity in each sample was normalized according to the b-galactosidase activity, which had been measured in the same sample. (45) Knockdown of Runx2 in cells…”

“…The precipitates were separated via SDS-PAGE, and the pull-down proteins were detected via immunoblotting with specific antibodies. (45) Alkaline phosphatase (ALP) staining MC3T3-E1 and MC3T3-E1-Notch1-IC stable cells were plated in 24-well plates (1.2 Â 10 4 cells/well) and cultured in complete medium (10% FBS/a-MEM with 10 mM b-glycerol-phosphate and 50 mg/mL of ascorbic acid for several days). The cultured cells were washed with PBS twice, fixed with 70% EtOH at room temperature for 1 hour, and stained for alkaline phosphatase production using a kit (Sigma, St Louis, MO, USA).…”

Wnt5a Controls Notch1 Signaling through CaMKII-mediated Degradation of the SMRT Corepressor Protein

“…In Vitro Binding Assay-The recombinant GST-SMRT proteins were expressed in Escherichia coli strain BL21, using the pGEX system as indicated (66). The GST-SMRT proteins were then purified using glutathione-agarose beads (Sigma), in accordance with the manufacturer's instructions.…”