Regulation of Notch1 signaling by the APP intracellular domain ...

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In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates ...... Activation of the cell death program by inhibition of proteasome.
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Research Article

Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk Mi-Yeon Kim1,*, Jung-Soon Mo1,*, Eun-Jung Ann1,*, Ji-Hye Yoon1, Jane Jung1, Yun-Hee Choi1, Su-Man Kim1, Hwa-Young Kim1, Ji-Seon Ahn1, Hangun Kim2, Kwonseop Kim2, Hyang-Sook Hoe3 and Hee-Sae Park1,‡ 1

Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea The College of Pharmacy and Research Institute for Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Neuroscience and Neurology, Department of Neurology, Georgetown University Medical Center, Washington, DC 20007, USA

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*These authors equally contributed to this work ‡ Author for correspondence ([email protected])

Journal of Cell Science

Accepted 24 January 2011 Journal of Cell Science 124, 1831-1843 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.076117

Summary The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC–RBP-Jk complex after processing by -secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation. Key words: APP, Notch1, Protein degradation

Introduction Notch is a highly conserved transmembrane receptor that performs a key role in the determination of cell fate, differentiation, adult cell self-renewal, cancer, neurodegenerative disease, wound healing, and inflammation (Artavanis-Tsakonas et al., 1995; Egan et al., 1998; Lai, 2004; Weinmaster, 1998). The Notch1 receptor plays the role of a membrane-bound transcription factor. Notch1 is processed by furin in the endoplasmic reticular Golgi complex (S1 cleavage) during transport to the cell surface, where it is expressed in heterodimeric form (Lieber et al., 2002; Pan and Rubin, 1997). Upon binding to the specific ligands, Jagged and Delta, the transmembrane C-terminal fragment of Notch is generated via proteolytic cleavage (S2 cleavage) (Brou et al., 2000; Mumm and Kopan, 2000). Cleavage of this fragment by -secretase (S3 cleavage) induces the release of the Notch intracellular domain (Notch-IC) from the membrane, and induces the nuclear translocation of Notch-IC, thus resulting in the formation of a complex with the CSL transcription factor family [CBF1/RBPJk/KBF2 in mammals, Su(H) in Drosophila and Xenopus, and Lag2 in Caenorhabditis elegans] (Capell et al., 2000; De Strooper et al., 1999; Mumm and Kopan, 2000; Ray et al., 1999; Steiner et al., 1999; Weinmaster, 1997). In the absence of Notch-IC, CSL interacts with the SKIP, SMRT, CoR and HDAC proteins, resulting in the formation of a transcriptional repressor complex (Espinosa et al., 2002; Kao et al., 1998; Zhou et al., 2000; Zhou and Hayward, 2001). Notch-IC dissociates the co-repressors, and Notch-IC interacts with co-activator complexes, including the Lag3/mastermind, p300/CBP and P/CAF/GCN5, to form a

transcriptional active complex and activates CSL-dependent transcription (Kurooka and Honjo, 2000; Oswald et al., 2001; Petcherski and Kimble, 2000; Schuldt and Brand, 1999; Wallberg et al., 2002). The RAM domain of Notch1, which mediates the interaction of RBP-Jk/Su(H) with the Notch1-IC, induces the activation of target gene transcription (Tamura et al., 1995; Tani et al., 2001). In addition to the enhancer of split [E(spl)] complex genes, and the mammalian homologues of the Hairy and E(spl) genes, Hes1, Hes5, Hes7, Hey1, Hey2 and Heyl are the downstream target genes of Notch signaling (Abu-Issa and Cavicchi, 1996; Bessho et al., 2001; de Celis et al., 1996; Fischer et al., 2004; Jennings et al., 1994; Jouve et al., 2000; Leimeister et al., 2000; Ligoxygakis et al., 1998; Maier and Gessler, 2000; Ohtsuka et al., 1999). Following the transcriptional regulation of the target genes, Notch1-IC undergoes proteasomal degradation in the nucleus via the ubiquitin-proteasome system, including Fbw7, an E3 ligase relevant to the ubiquitylation of Notch1-IC (Gupta-Rossi et al., 2001; Lai, 2002; Minella and Clurman, 2005; Mo et al., 2007; Oberg et al., 2001; Wu et al., 2001). Several E3 ubiquitin ligases have been implicated in the half-life of Notch1-IC, including Fbw7, which promotes PEST-dependent Notch1-IC degradation in the nucleus, and Itch, which regulates the PEST-independent degradation of cytoplasmic Notch protein (Lai, 2002). We demonstrated previously that ILK downregulates the protein stability of Notch1-IC via the ubiquitin-proteasome pathway by means of Fbw7 (Mo et al., 2007).

Journal of Cell Science

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Journal of Cell Science 124 (11)

The amyloid- precursor protein (APP) is a type 1 integral transmembrane protein composed of a large extracellular sequence, a single transmembrane region and a short intracellular fragment, which is a cytotoxic 39–43 residue peptide that performs a crucial function in the pathogenesis of Alzheimer’s disease (Beyreuther and Masters, 1991; Tang, 2005; Younkin, 1991). Under physiological conditions, APP is cleaved proteolytically by secretase activity. APP is cleaved in a fashion similar to that of Notch, which undergoes regulated intramembranous proteolysis induced by secretase to release the APP intracellular domain (AICD), which modulates transcription (Tomita et al., 1998; Zhang et al., 2000). Indeed, AICD is capable of inducing transcriptional activation by interacting with the adaptor protein Fe65 and the acetyltransferase Tip60 (Cao and Sudhof, 2001). AICD was initially identified in the brains of patients with AD and was demonstrated to either sensitize or induce cells to undergo apoptosis. We demonstrated previously that Notch1-IC downregulates the AICD transcriptional activity through physical binding with AICD, Fe65 and Tip60 (Kim et al., 2007b). We have also demonstrated that Notch1-IC is a novel substrate for Tip60 and acetylation is one of the key factors in the regulation of the Notch1 signaling pathway (Kim et al., 2007a). D’Adamio’s group has demonstrated that AICD binds to the cytosolic Notch inhibitors Numb and Numb-like, both of which can repress Notch activity (Roncarati et al., 2002). Despite the fact that AICD regulates Notch1 signaling, the precise mechanism underlying this control remains to be clarified. In this study, we demonstrate that signal crosstalk occurs between AICD and Notch1 signaling after their processing by -secretase.

We have now evaluated the mechanism relevant to the AICDmediated regulation of Notch signaling. Our data indicate that AICD inhibits the transcriptional activity of Notch1-IC by an induced reduction in the protein stability of Notch1-IC and RBPJk. Interestingly, the level of the Notch1-IC protein was downregulated markedly in the presence of AICD by the proteasomal degradation of Notch1-IC through Fbw7. Additionally, the level of RBP-Jk protein was downregulated markedly in the presence of AICD by the lysosomal degradation of RBP-Jk. Collectively, our findings demonstrate that AICD functions as a negative regulator of the protein turnover of Notch1-IC and RBPJk. Results AICD inhibits Notch1 transcriptional activity

To evaluate the possible function of AICD in Notch1 signaling, a reporter assay was conducted with HEK293 cells, using luciferase reporter genes. HEK293 cells were transfected with 4⫻CSL-Luc, and either the active Notch1 mutant EN1 or an empty vector. As anticipated, EN1-mediated transcription activity was found to have increased in these samples. We determined that AICD attenuated the ability of EN1 to stimulate transcription (Fig. 1A). The basic helix-loop-helix (bHLH) proteins, Hes1 and Hes5, both of which harbor several RBP-Jk-binding sequences on their promoters, were identified as essential targets of Notch in epithelial cells (Kageyama and Ohtsuka, 1999). Therefore, we confirmed the effects of AICD on the Notch1 signaling pathway, using the Hes1 reporter system. The expression of the active form of Notch1

Fig. 1. AICD inhibits Notch transcriptional activity. (A,B)HEK293 cells were transfected with expression vectors for 4⫻CSL-Luc (A), Hes-1-Luc (B) and galactosidase, along with EN1, as indicated. (C,D)HEK293 cells were transfected with expression vectors for 4⫻CSL-Luc (C), Hes-1-Luc (D) and galactosidase, along with Notch1-IC, as indicated. (E)HEK293 cells were transfected with expression vectors for GAL4–Luc, and -galactosidase, along with APP–GAL4, as indicated. After 42 hours of transfection, the cells were pretreated with 1M DAPT and exposed to 1–3M PMA for 6 hours, as indicated. (F)HEK293 cells were transfected with expression vectors for 4⫻CSL-Luc, APP and -galactosidase, along with Notch1-IC, as indicated. After 42 hours of transfection, the cells were pretreated with 1M DAPT and then exposed to 1M PMA for 6 hours. (G)HEK293 cells were transfected for 48 hours with expression vectors for 4⫻CSL-Luc, APP, siAPP and -galactosidase, along with Notch1-IC. After 42 hours of transfection, the cells were treated with 1M PMA for 6 hours. In A–G, the cells were lysed and the luciferase activity was determined. The data were normalized to -galactosidase. These results are expressed as the mean ± s.d. of three independent experiments. RLU, relative luciferase unit. The data were evaluated for significant differences by Student’s t-test; *P