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.

Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. Although there is evidence that the two main signaling pathways can modulate each other, the precise mechanism is not completely understood. This report shows that beta-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that beta-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that beta-catenin could upregulate the level of Notch1 and NICD, possibly by competing the common ubiquitin-dependent degradation machinery. In addition, beta-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by beta-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. beta-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either beta-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and beta-catenin are sufficient to induce ALP activation. Furthermore, effects of beta-catenin on Notch1 were dramatically diminished by overexpressed LEF1. Overall, our data suggest that beta-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway.

DJ-1 has been reported as a gene linked to early onset familial Parkinson's disease, and is functionally involved in transcriptional regulation and oxidative stress-induced cell death. To understand the role of DJ-1 in cellular stress, this study investigated DJ-1's effect on stress-activated protein kinase signaling and H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). According to the results, the overexpression of DJ-1 inhibited H(2)O(2)-induced activation of ASK1 as well as the activation of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. The results of both in vivo binding and kinase studies have revealed that ASK1 is the direct target of DJ-1, whereas it has shown no effect on either MKK3 or p38. DJ-1 blocked both the homo-oligomerization of ASK1 and inhibited ASK1 activity. Taken together, our data strongly suggest that DJ-1, by directly inhibiting ASK1, may act as a negative regulator in ASK1 signaling cascades.

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This study suggests the existence of a novel GnRH-II binding protein, in addition to a conventional GnRH-I receptor, in prostate cancer cells. These data may facilitate the development of innovatory therapeutic drugs for the treatment of prostate cancer.