The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase

E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 2 4 3 8– 2 44 6

available at www.sciencedirect.com

www.elsevier.com/locate/yexcr

Research Article

The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase Mi-Yeon Kim, Jane Jung, Jung-Soon Mo, Eun-Jung Ann, Ji-Seon Ahn, Ji-Hye Yoon, Hee-Sae Park⁎ Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, Republic of Korea

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding

Received 10 March 2011

to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and

Revised version received 15 July 2011

releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular

Accepted 16 July 2011

domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1

Available online 29 July 2011

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

Keywords:

degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD

Notch1-IC

functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.

Jagged-1 intracellular domain

© 2011 Elsevier Inc. All rights reserved.

Fbw7 RBP-Jk Mastermind

Introduction The Notch1 receptor performs a crucial role in cell fate decisions, cell growth, differentiation, and proliferation [1–4]. Four members of the Notch family and five ligands have been identified in vertebrates [5,6]. The receptor–ligand interaction by cell–cell contact is accompanied by the proteolytic cleavage of Notch by an ADAM metalloprotease and by a presenilin-dependent gamma-secretase [7–11]. Upon release, the Notch1 intracellular domain (Notch1-IC) translocates to the nucleus, where it interacts with the transcription factors of the CBF1/RBP-Jk/Su(H)/Lag1 (CSL) family [12–14]. In the absence of Notch-IC, RBP-Jk interacts with the SKIP, SMRT, CoR, and HDAC proteins, thereby resulting in the formation of a transcriptional repressor complex [15–17]. Notch-IC dissociates the co-repressors, and Notch-IC interacts with co-activator complexes-including the

Lag-3/mastermind, p300/CBP, and P/CAF/GCN5-to form a transcriptional active complex and activates RBP-Jk-dependent transcription [18–21]. Notch1-IC is degraded in the nucleus by the proteasomedependent pathway via Fbw7, an E3 ligase for the ubiquitination of Notch1-IC [22–26]. However, the regulation of Notch1-IC protein stability via Fbw7 remains poorly understood. We demonstrated previously that ILK and SGK1 downregulate the protein stability of Notch1-IC via the ubiquitin–proteasome pathway by means of Fbw7 [24,27]. Several research groups have demonstrated that the ubiquitin–proteasome degradation pathway performs a role in the regulation of Notch signaling [22–24,28]. Notch ligand Jagged-1 is a type 1 transmembrane protein that shares common structural features including a DSL domain, which is required for Notch binding, and multiple epidermal growth factor-like (EGF) repeats within their extracellular domains

⁎ Corresponding author at: School of Biological Sciences and Technology, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju, 500-757, Republic of Korea. Fax: + 82 62 530 2199. E-mail address: [email protected] (H.-S. Park). 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.07.014

E XP E RI ME N T AL C E L L R E SE A RC H 31 7 ( 20 1 1) 2 4 38 – 2 44 6

[5,29,30]. The Jagged-1 ligand undergoes proteolytic cleavage upon Notch receptor binding, resulting in the production of a free intracellular domain [31,32]. Upon release, the Jagged-1 intracellular domain (JICD) translocates to the nucleus, where it participates in a transcriptional activator complex [31,32]. Notch receptors and ligands are type 1 transmembrane proteins present at the cell surface [33]. Ligand–receptor trans-interaction has the capacity to activate Notch signaling in between neighboring cells. However, many studies have indicated that ligand–receptor interactions can also take place within the same cell in both vertebrates and invertebrates [34–39]. This cis-interaction attenuates the ability of a cell to receive the signal from adjacent cells by a process known as ‘cis-inhibition’ of the receptor by the ligand. Therefore, cis-Inhibitory interactions have revealed as key regulatory mechanism of Notch signaling. Recent our report have shown that Delta-like ligand intracellular domain (DICD) regulates Notch signaling through physical interaction [40]. However, the precise mechanism of cis-inhibition of Notch1 signaling by Jagged1 still remains to be elucidated at the molecular level. In this study, we determined that the JICD inhibits Notch1 signaling via a reduction in the protein stability of Notch1-IC. We have now evaluated the mechanism relevant to the JICD-mediated regulation of Notch signaling. Our data indicate that JICD inhibits the transcriptional activity of Notch1-IC. Interestingly, the level of the Notch1-IC protein was markedly downregulated in the presence of JICD via the proteasomal degradation of Notch1-IC through Fbw7. Collectively, our findings demonstrated that JICD functions as a negative regulator of Notch1-IC through the downregulation of Notch1-IC protein.

Materials and methods Cell culture and transfection HEK293 cells were maintained at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, in a humidified incubator with an atmosphere containing 5% CO2. The cultured cells were transiently transfected via the calcium phosphate method or Lipofectamine (Invitrogen).

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were lysed in chemiluminescent lysis buffer [18.3% of 1 M K2HPO4, 1.7% of 1 M KH2PO4, 1 mM phenylmethyl sulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT)] and assayed for luciferase activity using a luciferase assay kit (Promega). The activity of the luciferase reporter protein in the transfected cells was normalized in reference to the β-galactosidase activity in the same cells [41].

Coimmunoprecipitation assay The cells were lysed in 1 ml of RIPA buffer for 30 min at 4 °C. After 20 min of centrifugation at 12,000 ×g, the supernatants were subjected to immunoprecipitation with appropriate antibodies coupled to protein A-agarose beads. Preparation of the cytoplasmic fraction was conducted as previously described [42]. The resultant immunoprecipitates were washed three times in phosphate-buffered solution (PBS, pH 7.4). Laemmli's sample buffer was subsequently added to the immunoprecipitated pellets; the pellets were heated for 5 min at 95 °C and analyzed via SDS-PAGE. The Western blotting was conducted using the indicated antibodies [43]. The blotted proteins were subsequently probed with anti-Myc antibody (9E10), anti-HA (12CA5) antibody, or anti-FLAG M2 antibody (Sigma), followed by incubation with anti-mouse horseradish-peroxidase-conjugated secondary antibodies (Amersham). The blots were then developed using enhanced chemiluminescence (ECL).

Immunofluorescence staining The assays were conducted as previously described, with cells plated at 1× 105 cells per well onto cover slips (Fisher) [44]. A total of 0.5 μg of appropriate DNA per well was then transfected with Geneporter2 (Genetherapysystems). The transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and then permeabilized with 0.1% Triton X-100 in PBS. Mouse anti-Flag M2 antibody (Sigma) and anti-Myc 9E10 antibody (Novus Biologicals) were employed as the primary antibodies at a dilution of 1:100. Alexa Fluor 594 goat anti-mouse Red secondary antibody (1:100) was added, followed by staining with TOPRO3. The stained cells were then evaluated for localization via confocal microscopy (Leica TCS SPE).

Protein accumulation assay Plasmid constructs The mouse Notch1-IC and rat JICD constructed in the present study were as follows: pCS4-Myc-Notch1-IC (amino acid residues 1744– 2531), pCMV-3xFlag-Notch1-IC (amino acid residues 1744–2531), pCMV-3xFlag-Notch1-IC-N (amino acid residues 1744–2110), pCMV-3xFlag-Notch1-IC-ΔNΔC (amino acid residues 2110–2369), pCMV-3xFlag-Notch1-IC-C (amino acid residues 2369–2531), pEGFP-JICD (amino acid residues 1087–1220) and pCS4-HA-JICD (amino acid residues 1087–1220). The 4xCSL-Luc, Hes1-Luc, and Hes5-Luc promoter reporter and ΔEN1-6xMyc were a kind gift from Raphael Kopan (Washington University, St.Louis). The N-terminal HA and Myc tagged human RBP-Jk constructed via standard PCR, and inserted into XhoI and XbaI sites of pCS4-HA and pCS4-Myc mammalian expression vector.

Cells were treated with the proteasomal inhibitor MG-132 (SigmaAldrich), the lysosomal inhibitor NH4Cl (Sigma-Aldrich), or the translational inhibitor cycloheximide (Sigma-Aldrich). MG-132 was used at 0, 5 and 10 μM for 6 h for the dosage assay of the proteasomal inhibitors. Lactacystin was used at 0, 5 and 10 μM for 6 h for the dosage assay of the proteasomal inhibitors. NH4Cl was employed at 0, 10 and 50 mM for 6 h for the dosage assay of lysosomal inhibitors. Cycloheximide was used at 100 μM for 0, 1, 2, 4 and 6 h for the time-course assay of the translational inhibitors. Protein levels were analyzed via immunoblotting [27].

Results JICD inhibits Notch1 transcriptional activity

Luciferase reporter assay The HEK 293 cells were co-transfected with 4XCSL-Luc and βgalactosidase together with the indicated vector constructs. The cells

In an effort to determine whether JICD is involved in the regulation of the transcriptional activation of Notch1 target genes, a reporter assay was conducted in HEK293 cells using luciferase reporter genes. In

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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 4XCSL promoter (4XCSL-Luc). We assessed the effects of JICD on Notch1-IC-mediated transcriptional activity. With the cotransfection of increasing amounts of JICD expression vector, we detected a dose-dependent inhibition of Notch1-IC-mediated transcriptional activity (Fig. 1A). Fig. 1A similar result was observed with the Hes1-Luc and Hes5-Luc reporter systems (Fig. 1B and C). These results demonstrated that JICD inhibits Notch1-IC-mediated transcriptional activity in intact cells.

complex was prevented, in comparison with the association observed in the absence of JICD (Fig. 2B). We investigated the subcellular location of Notch1-IC and RBP-Jk within the HEK 293 cells in the presence of JICD. As shown in Fig. 2C, Notch1-IC and RBP-Jk were localized principally in the nuclei. The subcellular localization of Notch1-IC and RBP-Jk was unchanged by coexpression of JICD. This result indicates that JICD exerts no significant effects on Notch1-IC and RBP-Jk cellular localization, and may exist within the same compartment (Fig. 2C).

JICD interacts with Notch1-IC in intact cells

JICD disrupts the formation of the Notch1-IC-RBP-Jk-Mastermind complex

In order to demonstrate that JICD prevents the binding of Notch1-IC to RBP-Jk, we conducted coimmunoprecipitation experiments. To confirm the interaction between Notch1-IC and JICD in a mammalian cell line, coimmunoprecipitation was conducted in the HEK293 cells via the cotransfection of a vector encoding for Myc-Notch1-IC or GFPJICD. GFP-JICD was detected only in anti-Myc immune complexes from cells expressing Myc-Notch1-IC. The results revealed that JICD interacts with Notch1-IC (Fig. 3A). Also, to confirm the interaction between membranes tethered Notch1 receptor and JICD, we transfected HEK293 cells with ΔEN1-Myc and GFP-JICD. This finding showed that JICD interacts with ΔEN1-Myc (Fig. 3B). Next, to determine which domains of Notch1-IC and JICD mediated their interaction, we carried out coimmunoprecipitation assays using a

In order to determine whether JICD is involved in the regulation of the interactions between Notch1-IC and RBP-Jk, we conducted a coimmunoprecipitation assay using the nuclear extracts of HEK 293 cells expressing Notch1-IC-Myc, Flag-RBP-Jk, and GFP-JICD. In the presence of JICD, the association occurring between Notch1-IC and RBP-Jk was severely disrupted, in comparison with the association observed in the absence of JICD (Fig. 2A). In order to determine the role of JICD in the negative regulation of Notch1-IC-RBP-Jk-Mastermind-mediated signaling, we evaluated the formation of coactivator complexes. In the presence of JICD, the formation of the Notch1-IC-RBP-Jk-Mastermind

A

4

*

4xCSL Luc 4xCSL-Luc

R.L.U (fold)

3

2

1

0 Notch1-IC Notch1 IC : JICD :

+ -

-

+ +

+ ++

+ +++

C

B *

5

+

+++

++

5

Hes1-Luc

Hes5-Luc

* 4

R.L.U (fold))

R.L.U (fold)

4 3 2

2

1

1 0 Notch1-IC : JICD :

3

-

+ -

+ +

+ ++

+ +++

+

++

+++

0 Notch1-IC : JICD :

-

+ -

+ +

+ ++

+ +++

+

++

+++

Fig. 1 – JICD suppresses the Notch1 signaling pathway. HEK293 cells were transfected with expression vectors for (A) 100 ng of 4XCSL-Luc, (B) 100 ng of Hes1-Luc, (C) 100 ng of Hes5-Luc and 100 ng of β-galactosidase, along with 100 ng of Notch1-IC and 100 ng (+), 200 ng (++) and 300 ng (+++) of JICD, as indicated. After 48 h of transfection, the cells were lysed, and the luciferase activity was determined. The data was normalized with β-galactosidase. These results represent the means± standard deviation of three independent experiments. R.L.U means relative luciferase units. *ANOVA, P < 0.001.

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E XP E RI ME N T AL C E L L R E SE A RC H 31 7 ( 20 1 1) 2 4 38 – 2 44 6

B

A Myc-Notch1-IC : HA-RBP-Jk : GFP-JICD : IB : Myc IB : HA

-

+ -

+ -

+ + -

+ + +

95

Myc-Notch1-IC

72 72

HA-RBP-Jk

Flag-Notch1-IC : Myc-RBP-Jk : HA-MAML : HA-JICD :

IB : Myc

-

+ -

+ -

+ -

+ + + -

+ + + + Myc-RBP-Jk

72 55 170

IB : HA

HA-MAML 130

IP : HA

Cell Lysates

IB : Flag

95

95 72

Myc-Notch1-IC

72

72

HA-RBP-Jk

95

IP : Flag 72

50

GFP-JICD

kDa

72

Myc-Notch1-IC Myc-RBP-Jk

Cell Lysates 130

C

Flag-Notch1-IC

GFP-JICD

Topro3

Merge

26

HA-MAML JICD HA HA-JICD

kDa Flag-Notch1-IC

Myc-RBP-Jk

Fig. 2 – JICD disrupts the formation of the Notch1-IC-RBP-Jk-Mastermind complex. (A) HEK293 cells were transfected for 48 h with expression vectors encoding for the indicated combinations of 1 μg of Myc-Notch1-IC, 1 μg of HA-RBP-Jk, and 1 μg of GFP-JICD. The cell lysates were subjected to immunoprecipitation with anti-HA antibody and the immunoprecipitates were immunoblotted with anti-Myc (9E10) or anti-HA antibody. The cell lysates were also subjected to immunoblot analysis with anti-Myc (9E10), anti-HA, and anti-GFP antibodies, respectively. (B) HEK293 cells were transfected for 48 h with expression vectors encoding for the indicated combinations of 1 μg of Flag-Notch1-IC, 1 μg of Myc-RBP-Jk, 1 μg of HA-MAML and 1 μg of HA-JICD. The cell lysates were subjected to immunoprecipitation with anti-Flag antibody and the immunoprecipitates were immunoblotted with anti-Myc (9E10), anti-Flag or anti-HA antibody. The cell lysates were also subjected to immunoblot analysis with anti-Myc (9E10), anti-Flag M2 and anti-HA antibodies, respectively. (C) HEK293 cells were transfected with expression vectors for 100 ng of Flag-Notch1-IC, 100 ng of Myc-RBP-Jk, and 100 ng of GFP-JICD, as indicated. The cells were stained for Notch1-IC (Red) and RBP-Jk (Red). The nuclei were stained with TOPRO3. The cells were visualized via confocal microscopy (Leica TCS SPE). Scale bar, 25 μm.

variety of Flag-Notch1-IC deletion mutants including Notch1-IC-N, Notch1-IC-ΔNΔC, and Notch-IC. As shown in Fig. 3C, the N-terminus harboring the RAM and ANK domain of Notch1-IC interacts with JICD (Fig. 3C). These results demonstrated that the N-terminal regions of Notch1-IC are required for its interaction with JICD.

JICD facilitates the degradation of Notch1-IC via a proteasomedependent pathway Next, we conducted immunoblot analysis on HEK293 cells to determine whether JICD performs a function in the regulation of Notch1-IC protein levels. The cells were cotransfected with MycNotch1-IC and GFP-JICD (1:3 ratio), and the steady-state level of the Notch1-IC protein was evaluated after various periods of cycloheximide treatment with or without JICD. After cycloheximide treatment, the level of Notch1-IC protein declined gradually, with approximately half of the protein being degraded after 2 h without

JICD (Fig. 4A). Upon cycloheximide treatment, the reduced level of Notch1-IC protein declined rapidly, with approximately half of the protein being degraded after 1 h in the presence of JICD (Fig. 4A). This result showed that Notch1-IC is rapidly turned over in the presence of JICD. In order to determine whether the Notch1-IC protein is degraded by the proteasome, the proteasome inhibitor Lactacystin was used to treat Myc-Notch1-IC- and GFP-JICD-transfected HEK293 cells. The Notch1-IC protein level was reduced in the presence of JICD, but was restored to a significant degree by treatment with Lactacystin (Fig. 4B). We subsequently attempted to investigate whether the Notch1-IC protein is degraded by the lysosome; the lysosome inhibitor NH4C1 was used to treat Myc-Notch1-IC- and GFP-JICD-transfected HEK293 cells. Notch1-IC protein levels were reduced in the presence of JICD, but were not restored significantly by NH4C1 treatment (Fig. 4C). We conducted immunoblot analysis on HEK293 cells to determine whether JICD performs a function in the regulation of RBP-Jk protein

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A

E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 2 4 3 8– 2 44 6

Myc-Notch1-IC : GFP-JICD : IB : GFP

50

IB : Myc

95 72

-

+ -

+ +

+

C Notch1-IC N t h1 IC N Notch1-IC-N

2110

Myc-Notch1-IC Notch1-IC-ΔNΔC

IP : Myc

Cell Lysates

2531

1744

GFP-JICD

2369

Notch1-IC-C

95 72

2531

Myc-Notch1-IC

50 GFP-JICD kDa

GFP-JICD : Flag- Notch1-IC-N : Flag- Notch1-IC-ΔNΔC : Flag- Notch1-IC-C : IB : GFP

+ -

+ + -

+ + -

50 GFP-JICD 43 34

Flag-Notch1-IC-N Flag-Notch1-IC-ΔNΔC

26

Flag-Notch1-IC-C

IB : Flag

B

MG132 Δ EN1-Myc : GFP-JICD :

-

+ -

+ +

+ +

+

IP : Flag 50

IB : Myc

Cell L t Lysates

IP : GFP

Cell Lysates

GFP-JICD

ΔEN1-Myc

95

95

ΔEN1-Myc

50

GFP-JICD

43 34

Flag-Notch1-IC-N Flag Notch1 IC N Flag-Notch1-IC-ΔNΔC

26

Flag-Notch1-IC-C

kDa

kDa a

Fig. 3 – Physical interaction of JICD with Notch1-IC in intact cells. (A) HEK293 cells were transfected with expression vectors encoding for 1 μg of Myc-Notch1-IC and 1 μg of GFP-JICD as indicated. After 48 h of transfection, the cells were lysed, and the cell lysates were subjected to immunoprecipitation with anti-Myc antibody. The immunoprecipitates were then immunoblotted with anti-GFP or anti-Myc antibody. The cell lysates were also immunoblotted with anti-Myc and anti-GFP antibody. (B) HEK293 cells were transfected with expression vectors encoding for 1 μg of ΔEN1-Myc and 1 μg of GFP-JICD as indicated. After 42 h of transfection, the cells were treated for 6 h with 10 μM of MG132 and the cell lysates were subjected to immunoprecipitation with anti-GFP antibody. The immunoprecipitates were then immunoblotted with anti-Myc (9E10) antibody. The cell lysates were also immunoblotted with anti-Myc and anti-GFP antibody. (C) HEK293 cells were transfected with expression vectors encoding for 1 μg of Flag-Notch1-IC, 1 μg of Flag-Notch1-IC-N, 1 μg of Flag-Notch1-IC-ΔNΔC, 1 μg of Flag-Notch1-IC-C, and 1 μg of GFP-JICD as indicated. After 48 h of transfection, the cells were lysed, and the cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The immunoprecipitates were then immunoblotted with anti-GFP or anti-Flag antibody. Cell lysates were also immunoblotted with anti-Flag and anti-GFP antibody.

levels. The cells were cotransfected with Myc-RBP-Jk and GFP-JICD, and steady-state level of the RBP-Jk protein did not changed in the presence of JICD (Fig. 4D). The steady-state level of the JICD protein did not change in the presence of MG132 (Fig. 4E). These data demonstrate that the degradation of the Notch1-IC protein is regulated by JICD via the proteasome pathway.

JICD facilitates the polyubiquitination of Notch1-IC by E3 ligase Fbw7 Generally, the ubiquitination of proteins results in the rapid degradation of proteins, and Notch1-IC was ubiquitinated by the F-box protein, Fbw7 [25,26]. Therefore, we assessed the involvement

of Fbw7 by using the F-box deleted and hence dominant-negative mutant form of Fbw7 (Fbw7ΔF). Notch1-IC transcriptional activity was reduced in the presence of JICD, but was restored significantly via cotransfection with Fbw7ΔF (Fig. 5A). The results of Western blot analysis showed that the levels of the Notch1-IC protein were reduced by JICD, and were restored markedly by the cotransfection of Fbw7ΔF (Fig. 5B). These results demonstrate that Fbw7ΔF can recover and enhance the transcriptional activity and protein level of Notch1-IC in the presence of JICD. Accordingly, we suggest that JICD negatively regulates Notch1-IC through Fbw7. Next, we evaluated the involvement of JICD in the physical association between Fbw7 and Notch1-IC via coimmunoprecipitation. As shown in Fig. 5C, JICD facilitates the physical association between

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E XP E RI ME N T AL C E L L R E SE A RC H 31 7 ( 20 1 1) 2 4 38 – 2 44 6

A

0

CHX : Mock

95 72

+ JICD

95 72

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2

6

4

C

(hr)

Myc-Notch1-IC

NH4Cl (mM)

Myc-Notch1-IC : GFP-JICD :

+ +

10

50

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+ +

95 IB : Myc

50

50

IB : GFP β-actin

55

Myc-Notch1-IC

72

GFP-JICD

IB : GFP IB : β-actin

+ -

-

GFP-JICD

kDa

kDa

D

120 Mock JICD

Notch1-IC (%)

100

Myc-RBP-Jk: GFP-JICD:

-

+ -

+ +

+ ++

+ +++

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IB : Myc

72

Myc-RBP-Jk

IB : GFP

50

GFP JICD GFP-JICD

40 20 0

kDa 0

1

2

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6 (hr)

(Time)

B

E

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Myc-Notch1-IC : GFP-JICD :

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10

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+ +

MG132

GFP-JICD: IB : Myc

95 72 50

+

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20 (μM)

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Myc-Notch1-IC IB : GFP

IB : GFP

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GFP-JICD

GFP-JICD kDa

kDa

Fig. 4 – JICD facilitates the degradation of Notch1-IC via a proteasome-dependent pathway. (A) HEK293 cells were transfected for 48 h with the indicated combinations of expression vectors encoding for 0.5 μg of Myc-Notch1-IC and 1.5 μg of GFP-JICD. The cells were then treated with 100 μM of cycloheximide for the indicated periods of time. Quantification of each band intensity using densitometer represents as relative percent for Notch1-IC protein level. (B) HEK293 cells were transfected for 42 h with the indicated combinations of expression vectors encoding for Myc-Notch1-IC and GFP-JICD. The cells were then treated with the indicated amount of Lactacystin for 6 h. (C) HEK293 cells were transfected for 42 h with the indicated combinations of expression vectors encoding for 0.5 μg of Myc-Notch1-IC and 1.5 μg of GFP-JICD. The cells were then treated with the indicated amounts of NH4Cl for 6 h. (D) HEK293 cells were transfected for 48 h with expression vectors encoding for 1 μg of Myc-RBP-Jk and 1 μg (+), 1.5 μg (++) and 2 μg (+++) of GFP-JICD in the indicated combinations. (E) HEK293 cells were transfected for 42 h with the expression vector encoding for 1 μg of GFP-JICD. The cells were then treated for 6 h with the indicated amount of MG132. (A-E) The cell lysates were also subjected to immunoblotting analysis with the indicated antibodies.

Fbw7 and Notch1-IC in the cells. These results demonstrated that the downregulation of the Notch1-IC protein by JICD occurred via an Fbw7-dependent pathway. We evaluated the formation of a trimeric complex between JICD and Notch1-IC or Fbw7, in an effort to define more precisely the role of JICD in the negative regulation of Notch1 signaling. We detected binding between Notch1-IC and JICD, but not between JICD and Fbw7, although the trimeric complex was detected in this case (Fig. 5D). Therefore, the results demonstrated that JICD interacts with Fbw7 in the presence of Notch1-IC, thereby forming a trimeric complex. We also attempted to determine whether JICD could regulate the level of Notch1-IC ubiquitination through Fbw7. As shown in Fig. 5E, the levels of ubiquitinated Notch1-IC were increased

with the coexpression of JICD. These results demonstrate that the downregulation of the Notch1-IC protein by JICD occurs via an Fbw7dependent pathway.

Discussion Notch receptors in a sending cell are activated by cell surface ligands in adjacent cells but can also be inhibited by the ligands present in the same cell. This regulatory process is known as cis-inhibition of Notch. Recent report suggests that this cis-inhibition between Notch and their ligand is symmetric: Notch inhibits their ligands, and the

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A

C *

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Myc-Notch1-IC : Flag-Fbw7 Flag Fbw7 : GFP-JICD :

4XCSL-Luc

+ -

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MG132

E

MG132

HA-Ub : Myc-Notch1-IC : Flag-Fbw7: Flag Fbw7: GFP-JICD :

+ + +

IB : Flag

R.L.U (fold)

IB : Myc

3

95 72

+ + + +

+ + +

170

Myc-Notch1-IC IP : Myc y IB : HA

IP : Myc

Cell Lysates

1 0

Myc-Notch1-IC

72 130

50 + + -

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+

Notch1- IC-(Ub)n

130 95

95

2

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Flag-Fbw7

4

-

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130

*

Notch1-IC : JICD : Fbw7Δ ΔF :

+ -

Flag-Fbw7

95

GFP-JICD

72 130

Cell Lysates

kDa

Myc-Notch1- IC Flag-Fbw7

50

GFP-JICD

kDa

B

MG132

D Myc-Notch1-IC : GFP-JICD : Flag-Fbw7Δ F :

-

+ -

+ + -

+ + +

+ +

+ -

+

Myc-Notch1-IC : Flag-Fbw7: GFP-JICD :

95 IB : Myc IB : GFP

72 50

Myc-Notch1- IC

IB : Myc

GFP-JICD

IB : Flag

+ -

+ -

+

+ +

95 72

+ +

+ + + Myc-Notch1-IC

130 Flag-Fbw7

130

IP : GFP

Flag-Fbw7Δ F

IB : Flag

-

95

kDa

Myc-Notch1-IC

72 Cell Lysates

130

50

Flag-Fbw7

GFP-JICD

kDa

Fig. 5 – JICD facilitates the polyubiquitination of Notch1-IC by E3 ligase Fbw7. (A) HEK293 cells were transfected with expression vectors for 100 ng of 4XCSL-Luc, and 100 ng of β-galactosidase, along with 100 ng of Notch1-IC, 300 ng of JICD and 100 ng of Fbw7ΔF as indicated. After 48 h of transfection, the cells were lysed and the luciferase activity was determined. The data were normalized with β-galactosidase. The results are expressed as the means ± SEM of three independent experiments. R.L.U means relative luciferase units. *ANOVA, P < 0.001 (B) HEK293 cells were transfected with expression vectors encoding for 0.5 μg of Myc-Notch1-IC, 1.5 μg of GFP-JICD, and 1.5 μg of Flag-Fbw7ΔF in the combinations indicated. After 48 h of transfection, the cell lysates were subjected to immunoblotting analysis with the indicated antibodies. (C) HEK293 cells were transfected with expression vectors encoding for 0.5 μg of Myc-Notch1-IC, 0.5 μg of Flag-Fbw7, and 1.5 μg of GFP-JICD in the combinations indicated. After 42 h of transfection, the cells were treated with 10 μM of MG132 for 6 h and the cell lysates were immunoprecipitated with anti-Myc antibody, after which the immunoprecipitates were immunoblotted with anti-Flag or anti-Myc antibody. (D) HEK293 cells were transfected with expression vectors encoding for Myc-Notch1-IC, Flag-Fbw7, and GFP-JICD in the indicated combinations. After 42 h of transfection, the cells were treated for 6 h with 10 μM of MG132 and the cell lysates were immunoprecipitated with anti-GFP antibody and the immunoprecipitates were immunoblotted with anti-Myc or anti-Flag antibody. Cell lysates were also subjected to immunoblotting analysis with the indicated antibodies. (E) HEK293 cells were transfected with expression vectors for 0.5 μg of Myc-Notch1-IC, 0.5 μg of Flag-Fbw7, 1.5 μg of GFP-JICD, and 1 μg of HA-Ub, as indicated. After 42 h of transfection, the cells were treated with 10 μM of MG132 for 6 h and the cell lysates were immunoprecipitated with anti-Myc antibody; the immunoprecipitates were immunoblotted with anti-HA antibody.

ligand inhibits Notch [45,46]. Furthermore, we recently reported that the possible regulation of Notch signaling by Delta-like ligand 1 intracellular domain via gamma-secretase independent manner [40]. The molecular mechanism of cis-inhibition between Notch and their ligand is still unclear. It is also unclear whether or not it occurs at the cell nucleus. Jagged-1 is a type I transmembrane protein which has the capacity to activate Notch in adjacent cells (trans-Activation) and

to inhibit Notch present within the same cell (cis-inhibition) [38,46,47]. Notch receptor and its ligand coexist in the same cell. It has been previously reported that Notch and its ligands are processed by the same molecular machinery and proposed that the regulated intramembrane proteolysis of Notch receptor and ligand may play important, potentially roles in cell signaling [32]. The Jagged-1 undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain and

E XP E RI ME N T AL C E L L R E SE A RC H 31 7 ( 20 1 1) 2 4 38 – 2 44 6

translocates to the nucleus [32]. Our report are wholly consistent with the observation of several groups that ligand overexpression inhibits Notch function and the finding ligand and Notch coexpression block the Notch-induced inhibition of neurite outgrowth in a cellular model of Notch function [48]. We and others recently reported evidence for a strong cis-inhibition interaction that occurs between Notch and their ligand in the same cell. In this study, we determined that Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). JICD inhibits Notch1 transcriptional activity via the disruption of the Notch1-IC-RBP-Jk complex. Additionally, the formation of the Notch1-IC-RBP-Jk-Mastermind complex is prevented in the presence of JICD. JICD interacts directly with Notch1-IC in intact cells. JICD accelerates the protein degradation of Notch1-IC via an Fbw7dependent proteasomal pathway. This study suggests a novel role of JICD in reducing Notch signaling. JICD overexpression increases the interaction between Fbw7 and Notch1-IC and enhances Notch1-IC ubiquitination and degradation. Thus, the enhancement of the interaction between Notch1-IC and Fbw7 may be a possible mechanism for the JICD-mediated proteasomal degradation of Notch1-IC. Recent studies have emphasized the role of ubiquitination in the regulation of Notch signaling [22]. Several research groups have demonstrated that the F-box containing protein Sel10/Fbw7 mediates Notch ubiquitination in the nucleus, and targets it for proteasome-dependent degradation [25,28,49,50]. In one of our recent reports, we demonstrated that ILK and SGK1 downregulate the protein stability of Notch1-IC via the ubiquitin–proteasome pathway by means of Fbw7 [24,27]. Our results demonstrate that the inhibitory mechanism functions via the acceleration of protein degradation of Notch1-IC via an Fbw7-dependent proteasomal pathway. In this study, we determined that JICD stimulated the proteasomal degradation of Notch1-IC. The results of previous studies have demonstrated that Numb interacts with Itch, and that together Numb and Itch cooperate to increase the ubiquitination of Notch1 in the nucleus [51–53]. Numb functions as an adaptor for the recruitment of the E3 ligase Itch and components of the ubiquitination machinery to the Notch1 receptor, thereby promoting Notch1 ubiquitination [53]. We have demonstrated that JICD interacts with Notch1-IC, and thereby increases the binding between Fbw7 and Notch1-IC. Thus, enhancement of the interaction between Notch1-IC and Fbw7 may be a possible mechanism for the JICD-mediated proteasomal degradation of Notch1-IC. Our data indicate that JICD may play a novel role as an adaptor for the recruitment of Notch1-IC to the E3 ligase Fbw7. In summary, our results demonstrate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC protein degradation. Henceforth, the findings of this study may begin to shed some light onto what may be a signal cross-talk mechanism of Notch1 and Jagged-1 after cleavage by gamma-secretase.

Acknowledgments We would like to thank Raphael Kopan (Washington University Medical School, St. Louis, USA) for the Notch-related constructs and B. E. Clurman (Fred Hutchinson Cancer Research Center) for the Fbw7 construct. This study was supported by a grant from the

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Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A090106).

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