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ABSTRACT: Notch1 genes encode receptors for a signaling pathway that regulates various aspects of cell growth and differentiation; however, the role of Notch1 signaling in p38 mitogen-activated protein kinase (MAPK) signaling pathway is still not well defined. In this study, we found that Notch1 intracellular domain (Notch1-IC) prevents oxidative stress-induced cell death through the suppression of the Apoptosis signal-regulating kinase (ASK) 1 signaling pathway. Notch1-IC inhibited H2O2-induced activation of ASK1 and the activation of downstream kinases in the p38 MAPK signaling cascade. The results of both in vivo binding and kinase studies have revealed that ASK1 is the direct target of Notch1-IC, whereas it produced no effect on either MAP kinase kinase (MKK) 3 or p38 MAPK. Notch1-IC blocked both the homooligomerization of ASK1 and inhibited ASK1 activity. Furthermore, Notch1-IC facilitated the translocation of activated ASK1 toward the nucleus. Notch1 knockdown was determined to be highly susceptible to oxidative stress-induced activation of ASK1-MKK3/MKK6-p38 MAPK signaling cascade and cell death. Taken together, our findings suggest that Notch1-IC may act as a negative regulator in ASK1 signaling cascades.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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ABSTRACT: The Notch signaling pathway plays a crucial role in the regulation of cell fate decision, and is also a key regulator of cell differentiation, including bone homeostasis, in a variety of contexts. However, the role of Notch1 signaling in osteoclast differentiation is still controversial. In this study, we show that Receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation is promoted by the Notch1 intracellular domain (Notch1-IC) and Ca(2+)/Calmodulin dependent protein kinase IV (CaMKIV) signaling. Notch1-IC protein level was augmented by CaMKIV through escape from ubiquitin dependent protein degradation. In addition, CaMKIV remarkably increased Notch1-IC stability, and the kinase activity of CaMKIV was essential for facilitating Notch1 signaling. CaMKIV directly interacted with Notch1-IC and phosphorylates Notch1-IC, thereby decreasing proteasomal protein degradation through F-box and WD repeat domain-containing 7 (Fbw7). We also found that Notch1-IC prevented inhibition of osteoclast differentiation by KN-93 but not the phosphorylation deficient form of Notch1-IC. These results suggest that phosphorylated Notch1-IC by CaMKIV increases Notch1-IC stability, which enhances osteoclast differentiation.
Biochimica et Biophysica Acta 10/2012; · 4.66 Impact Factor
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ABSTRACT: Serine-threonine Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the key component in non-canonical Wnt5a signaling and has been shown to regulate its signaling. In this study, we found that CaMKII induced by Wnt5a remarkably reduced the protein stability of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), a co-repressor of Notch signaling, through proteasomal degradation. Wnt5a was found to enhance Notch1 intracellular domain (Notch1-IC) transcription activity, which could be inhibited by treatment with KN93, a CaMKII inhibitor. The kinase activity of CaMKII was essential for the activation of Notch signaling. We also determined that CaMKII could enhance the association between Notch1-IC and RBP-Jk. Furthermore, the physical association between RBP-Jk and SMRT was substantially suppressed by CaMKII. We demonstrated that CaMKII directly bound and phosphorylated SMRT at Ser1407, thereby facilitating SMRT translocation from the nucleus to the cytoplasm and proteasome dependent degradation. These results suggest that CaMKII down-regulated the protein stability of SMRT through proteasomal degradation.
Journal of Biological Chemistry 08/2012; · 4.77 Impact Factor
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ABSTRACT: Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.
Journal of Cell Science 02/2012; 125(Pt 5):1296-308. · 6.11 Impact Factor
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Jung-Soon Mo,
Ji-Hye Yoon,
Ji-Ae Hong,
Mi-Yeon Kim, Eun-Jung Ann,
Ji-Seon Ahn,
Su-Man Kim,
Hyeong-Jin Baek,
Florian Lang,
Eui-Ju Choi,
Hee-Sae Park
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ABSTRACT: The gamma-secretase complex is involved in the intramembranous proteolysis of a variety of substrates, including the amyloid precursor protein and the Notch receptor. Nicastrin (NCT) is an essential component of the gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we determined that serum- and glucocorticoid-induced protein kinase 1 (SGK1) markedly reduced the protein stability of NCT. The SGK1 kinase activity was decisive for NCT degradation and endogenous SGK1 inhibited gamma-secretase activity. SGK1 downregulates NCT protein levels via proteasomal and lysosomal pathways. Furthermore, SGK1 directly bound to and phosphorylated NCT on Ser437, thereby promoting protein degradation. Collectively, our findings indicate that SGK1 is a gamma-secretase regulator presumably effective through phosphorylation and degradation of NCT.
PLoS ONE 01/2012; 7(5):e37111. · 4.09 Impact Factor
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ABSTRACT: Notch1 receptor functions as a critical controller of cell fate decisions and also as a key regulator of cell growth, differentiation, and proliferation in invertebrates and vertebrates. In this study, we have demonstrated that the adaptor protein Fe65 attenuates Notch1 signaling via the accelerated degradation of the membrane-tethered Notch1 in the cytoplasm. Fe65 also suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-recombining binding protein suppressor of hairless (RBP)-Jk complex within the nucleus. Fe65 is capable of forming a trimeric complex with Itch and membrane-tethered Notch1, and Fe65 enhances the protein degradation of membrane-tethered Notch1 via an Itch-dependent proteasomal pathway. Collectively, our results demonstrate that Fe65 carries out different functions depending on its location in the regulation of Notch1 signaling.
Journal of Biological Chemistry 12/2011; 287(7):4690-701. · 4.77 Impact Factor
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ABSTRACT: Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC-RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.
Experimental Cell Research 07/2011; 317(17):2438-46. · 3.58 Impact Factor
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ABSTRACT: Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. The Delta-like ligand 1 also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. In this study, we have demonstrated that the Delta-like 1 intracellular domain (Dll1-IC) specifically binds to Notch1-IC in the nucleus, thereby disrupting the association of the Notch1-IC-RBP-Jk-MAM transcription activator complex. Additionally, the Notch1-mediated blockage of the induction of MyoD is abolished by the co-expression of Dll1-IC. Collectively, our results show that Dll1-IC functions as a negative regulator in Notch signaling via the disruption of the Notch1-IC-RBP-Jk complex.
Molecules and Cells 06/2011; 32(2):161-5. · 2.18 Impact Factor
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Mi-Yeon Kim,
Jung-Soon Mo, Eun-Jung Ann,
Ji-Hye Yoon,
Jane Jung,
Yun-Hee Choi,
Su-Man Kim,
Hwa-Young Kim,
Ji-Seon Ahn,
Hangun Kim,
Kwonseop Kim,
Hyang-Sook Hoe,
Hee-Sae Park
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ABSTRACT: 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 γ-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.
Journal of Cell Science 06/2011; 124(Pt 11):1831-43. · 6.11 Impact Factor
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Eun-Jung Ann,
Hwa-Young Kim,
Yun-Hee Choi,
Mi-Yeon Kim,
Jung-Soon Mo,
Jane Jung,
Ji-Hye Yoon,
Su-Man Kim,
Jeong-Sik Moon,
Mi-Sun Seo,
Ji-Ae Hong,
Won-Gu Jang,
Paul Shore,
Toshihisa Komori,
Jeong-Tae Koh,
Hee-Sae Park
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ABSTRACT: Notch1 genes encode receptors for a signaling pathway that regulates cell growth and differentiation in various contexts, but the role of Notch1 signaling in osteogenesis is not well defined. Notch1 controls osteoblast differentiation by affecting Runx2, but the question arises whether normal osteoblastic differentiation can occur regardless of the presence of Notch1. In this study, we observed the downregulation of Notch1 signaling during osteoblastic differentiation. BMPR-IB/Alk6-induced Runx2 proteins reduced Notch1 activity to a marked degree. Accumulated Runx2 suppressed Notch1 transcriptional activity by dissociating the Notch1-IC-RBP-Jk complex. Using deletion mutants, we also determined that the N-terminal domain of Runx2 was crucial to the binding and inhibition of the N-terminus of the Notch1 intracellular domain. Notably, upregulation of the Runx2 protein level paralleled reduced expression of Hes1, which is a downstream target of Notch1, during osteoblast differentiation. Collectively, our data suggest that Runx2 is an inhibitor of the Notch1 signaling pathway during normal osteoblast differentiation.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 02/2011; 26(2):317-30. · 6.04 Impact Factor
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Jung-Soon Mo, Eun-Jung Ann,
Ji-Hye Yoon,
Jane Jung,
Yun-Hee Choi,
Hwa-Young Kim,
Ji-Seon Ahn,
Su-Man Kim,
Mi-Yeon Kim,
Ji-Ae Hong,
Mi-Sun Seo,
Florian Lang,
Eui-Ju Choi,
Hee-Sae Park
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ABSTRACT: Notch is a transmembrane protein that acts as a transcriptional factor in the Notch signaling pathway for cell survival, cell death and cell differentiation. Notch1 and Fbw7 mutations both lead the activation of the Notch1 pathway and are found in the majority of patients with the leukemia T-ALL. However, little is known about the mechanisms and regulators that are responsible for attenuating the Notch signaling pathway through Fbw7. Here, we report that the serum- and glucocorticoid-inducible protein kinase SGK1 remarkably reduced the protein stability of the active form of Notch1 through Fbw7. The protein level and transcriptional activity of the Notch1 intracellular domain (Notch1-IC) were higher in SGK1-deficient cells than in SGK1 wild-type cells. Notch1-IC was able to form a trimeric complex with Fbw7 and SGK1, thereby SGK1 enhanced the protein degradation of Notch1-IC via a Fbw7-dependent proteasomal pathway. Furthermore, activated SGK1 phosphorylated Fbw7 at serine 227, an effect inducing Notch1-IC protein degradation and ubiquitylation. Moreover, accumulated dexamethasone-induced SGK1 facilitated the degradation of Notch1-IC through phosphorylation of Fbw7. Together our results suggest that SGK1 inhibits the Notch1 signaling pathway via phosphorylation of Fbw7.
Journal of Cell Science 01/2011; 124(Pt 1):100-12. · 6.11 Impact Factor
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ABSTRACT: 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.
Journal of Cellular Biochemistry 03/2010; 110(1):229-37. · 2.87 Impact Factor
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S Sakura Minami,
You Me Sung,
Sonya B Dumanis,
Seong Hwan Chi,
Mark P Burns, Eun-Jung Ann,
Toshiharu Suzuki,
R Scott Turner,
Hee-Sae Park,
Daniel T S Pak,
G William Rebeck,
Hyang-Sook Hoe
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ABSTRACT: The goal of this study was to determine the effect of X11alpha on ApoE receptor 2 (ApoEr2) trafficking and the functional significance of this interaction on cell movement in MCF 10A epithelial cells. We found that X11alpha increased surface levels of ApoEr2 by 64% compared to vector control, as determined by surface protein biotinylation. To examine the functional significance of this effect, we tested whether ApoEr2 played a novel role in cell movement in a wound-healing assay. We found that overexpression of ApoEr2 in MCF 10A cells increased cell migration velocity by 87% (P<0.01, n=4) compared to GFP control. Cotransfection of X11alpha had an additive effect on average velocity compared to ApoEr2 alone (13%; P<0.05, n=4). In addition, we tested whether ApoEr2 ligands altered the effect of ApoEr2 on cell movement. We found that treatment with concentrated medium containing the extracellular matrix protein Reelin, but not control medium, further increased the velocity of ApoEr2- but not APP-transfected cells (20%; P<0.001, n=4). Similarly, Reelin treatment increased cell velocity in the presence of ApoEr2 and X11alpha (10%; P<0.05, n=4). In the present study, we are the first to demonstrate that ApoEr2 regulates cell movement, and both X11alpha and Reelin enhance this effect.
The FASEB Journal 08/2009; 24(1):58-69. · 5.71 Impact Factor
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ABSTRACT: Notch proteins perform a critical function in cell-fate decisions and in differentiation. In this study, we determined that indirubin-3'-monoxime reduced Notch1 signaling to a remarkable extent. Indirubin-3'-monoxime has been shown to inhibit both constitutive active mutants of Notch1 and Notch1-IC-mediated transactivation activity. However, in such cases, neither the Notch cleavage pattern nor the protein stability of Notch1-IC was determined to have been significantly altered. Indirubin-3'-monoxime suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-RBP-Jk complex. Notably, the transcriptional activity of Notch1-IC was not suppressed significantly in the GSK-3beta null cells by indirubin-3'-monoxime as compared to what was observed with GSK-3beta wild-type cells. In the previous study, we synthesized a series of indirubin derivatives. Interestingly, some of these indirubin derivatives were characterized as potent inhibitors of Notch1 signaling. Taken together, the results of this study indicate that indirubin-3'-monoxime downregulated Notch1 signaling in a GSK-3beta-dependent and proteosomal degradation-independent manner.
Cancer Letters 08/2008; 265(2):215-25. · 4.24 Impact Factor
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Mi-Yeon Kim,
Ji-Hye Park,
Jung-Soon Mo, Eun-Jung Ann,
Seung-Ok Han,
Sang-Hyun Baek,
Kyoung-Jin Kim,
Suhn-Young Im,
Jeen-Woo Park,
Eui-Ju Choi,
Hee-Sae Park
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ABSTRACT: The Notch signaling pathway appears to perform an important function in inflammation. Here, we present evidence to suggest that lipopolysaccharide (LPS) suppresses Notch signaling via the direct modification of Notch by the nitration of tyrosine residues in macrophages. In the RAW264.7 macrophage cell line and in rat primary alveolar macrophages, LPS was found to inhibit Notch1 intracellular domain (Notch1-IC) transcription activity, which could then be rescued by treatment with N(G)-nitro-l-arginine, a nitric oxide synthase (NOS) inhibitor. Nitric oxide (NO), which was produced in cells that stably express endothelial NOS (eNOS) and brain NOS (bNOS), also induced the inhibition of Notch1 signaling. The NO-induced inhibition of Notch1 signaling remained unchanged after treatment with 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylyl-cyclase inhibitor, and was not found to be mimicked by 8-bromo-cyclic GMP in the primary alveolar macrophages. With regards to the control of Notch signaling, NO appears to have a significant negative influence, via the nitration of Notch1-IC, on the binding that occurs between Notch1-IC and RBP-Jk, both in vitro and in vivo. By intrinsic fluorescence, we also determined that nitration could mediate conformational changes of Notch1-IC. The substitution of phenylalanine for tyrosine at residue 1905 in Notch1-IC abolished the nitration of Notch1-IC by LPS. Overall, our data suggest that an important relationship exists between LPS-mediated inflammation and the Notch1 signaling pathway, and that this relationship intimately involves the nitration of Notch1-IC tyrosine residues.
Journal of Cell Science 06/2008; 121(Pt 9):1466-76. · 6.11 Impact Factor
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ABSTRACT: The Notch signaling pathway appears to perform an important function in a wide variety of organisms and cell types. In our present study, we provide evidence that UV irradiation-induced Tip60 proteins reduced Notch1 activity to a marked degree. Accumulated UV irradiation-induced Tip60 suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-CSL complex. The binding between endogenous Tip60 and Notch1-IC in UV radiation-exposed cells was verified in this study by coimmunoprecipitation. Interestingly, the physical interaction of Tip60 with Notch1-IC occurs to a more profound degree in the presence of CSL but does not exist in a trimeric complex. Using Notch1-IC and Tip60 deletion mutants, we also determined that the N terminus, which harbors the RAM domain and seven ankyrin repeats of Notch1-IC, interacts with the zinc finger and acetyl coenzyme A domains of Tip60. Furthermore, here we report that Notch1-IC is a direct target of the acetyltransferase activity of Tip60. Collectively, our data suggest that Tip60 is an inhibitor of the Notch1 signaling pathway and that Tip60-dependent acetylation of Notch1-IC may be relevant to the mechanism by which Tip60 suppresses Notch1 signaling.
Molecular and Cellular Biology 10/2007; 27(18):6506-19. · 5.53 Impact Factor
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ABSTRACT: The Notch signaling pathway appears to perform an important function in the determination of cell fate and in differentiation, in a wide variety of organisms and cell types. In this study, we provide evidence that the inactivation of Notch signaling by zinc is achieved via a PI3K-Akt-dependent, cytoplasmic retention of Notch1-IC and RBP-Jk. Extracellular zinc has been determined to inhibit constitutive active mutants of both Notch1 (DeltaEN1) and Notch1-IC-mediated transcription. However, in such cases, neither the cleavage pattern of Notch nor the protein stability of Notch1-IC and RBP-Jk was found to have significantly changed. With regard to the modulation of Notch signaling, zinc appears to exert a significant negative influence on the binding occurring between Notch1 and RBP-Jk, both in vivo and in vitro. The zinc-induced inhibition of Notch signaling can be rescued via pretreatment with wortmannin or LY294002, both of which are specific PI3K signaling pathway inhibitors. Furthermore, we ascertained that zinc triggers the cytoplasmic retention of Notch1-IC and RBP-Jk, and that cytoplasmic retention could be rescued via treatment with wortmannin. Overall, we have determined that an important relationship exists between zinc and the Notch1 signaling pathway, and that this relationship is intimately involved with the cytoplasmic retention of Notch and RBP-Jk.
Cancer Letters 10/2007; 255(1):117-26. · 4.24 Impact Factor
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Jung-Soon Mo,
Mi-Yeon Kim,
Seung-Ok Han,
In-Sook Kim, Eun-Jung Ann,
Kyu Shik Lee,
Mi-Sun Seo,
Jin-Young Kim,
Seung-Chul Lee,
Jeen-Woo Park,
Eui-Ju Choi,
Jae Young Seong,
Cheol O Joe,
Reinhard Faessler,
Hee-Sae Park
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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.
Molecular and Cellular Biology 09/2007; 27(15):5565-74. · 5.53 Impact Factor
