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Fe65 negatively regulates Jagged1 signaling by down-

regulation of protein stability through E3 ligase Neurl1.

Eun-Hye Jo, Mi-Yeon Kim, So-I Noh, Hyung-Ju Lee and Hee-Sae Park *

School of Biological Sciences and Technology, Chonnam National University, Yongbong-dong, Buk-ku, Gwangju, 500-757, Republic of Korea

*Corresponding author : Hee-Sae Park, e-mail : proteome@jnu.ac.kr

Abstract Figure 1. Figure 2.

Fe65 suppresses Jagged1 signaling Fe65 destabilizes Jagged1 not JICD by both

Fe65 is a highly conserved adaptor protein that interacts with proteasomal and lysosomal degradation pathway

several binding partners. Fe65 binds to the protein and mediates

various cellular processes, but the interacting partner is largely

unknown as well as the regulatory mechanism controlled with

Fe65. In this study, we found that Fe65 interacts with C-terminus

of Jagged1. Furthermore, Fe65 negatively regulates AP1-

mediated Jagged1 intercellular domain transactivation in a Tip60-

independent manner. We found that Fe65 triggers the

degradation of Jagged1, but not JICD through proteasomal and

lysosomal pathway. We also show that Fe65 promotes the

recruitment of E3 ligase Neuralized-like 1 (Neurl1) to membrane-

tethered Jagged1 and mono-ubiquitination of Jagged1. These

three proteins form stable trimeric complex and thereby down-

regulates Jagged1 targeted by ubiquitin-mediated degradation.

Consequently, Jagged1 is a novel binding partner of Fe65, and

Fe65 may act novel effector on Jagged1 signaling.

Introduction (A) HEK293 cells were transiently transfected with HA-Jagged1

Jagged1 is a single-pass transmembrane protein and one of (A) HEK293 cells were transiently transfected with a luciferase and Fe65-Myc. (B) HEK293 cells were transiently transfected with

ligands for the Notch1 receptor. Jagged1 is overexpressed in reporter plasmid AP1-Luc, Jagged1 and Fe65 along with β- GFP- JICD and Fe65-Myc. (C) HEK293 cells were transiently
transfected with shFe65 in a dose-dependent manner. (D) HEK293

breast cancer, ovarian cancer and, it promotes the oncogenic galactosidase as indicated. (B) HEK293 cells were transiently cells were transiently transfected with HA-Jagged1 and Fe65-Myc.

function such as metastasis and proliferation by enhancing the transfected with a luciferase reporter plasmid AP1 luciferase, The cells were treated with MG132 for 6 h before harvested as

Notch signaling pathway. Also, Jagged1 regulates hematopoiesis, JICD and Fe65 along with β-galactosidase as indicated. (C) indicated concentration. (E) HEK293 cells were transiently

NIH3T3 cells were transiently transfected with a luciferase transfected with HA-Jagged1 and Fe65-Myc. The cells were
and induces embryonic lethality, and inhibits vascular reporter plasmid AP1 luciferase, Jagged1 and Fe65 along with β-

development in Notch-dependent manner. Jagged1 is galactosidase as indicated. (D) NIH3T3 cells were transiently treated with MG132 (5 μM), ALLN (25 μM) and Epoxomicin (100
μM) respectively for 6 h before harvested. (F) HEK293 cells were
sequentially cleaved by alpha-secretase and gamma-secretase transfected with a luciferase reporter plasmid AP1 luciferase, transiently transfected with HA-Jagged1 and Fe65-Myc. The cells

resulting in releases of Jagged1 intracellular domain (JICD) in a JICD and Fe65 along with β-galactosidase as indicated. (A-D) were treated with Chloroquine for 6 h before harvested as

after 48 h transfection, the cells were lysed, and the luciferase
ligand-expressing cell. Jagged1 has PDZ-domain binding site at indicated concentration. (A-F) After 48 h transfection, the cell
activity was determined. The data were normalized with β- lysates were also subjected to immunoblotting analysis with the
the C-terminus. The PDZ-domain of Jagged1 is necessary in galactosidase activity. These results represent the means

transforming activity but has no effect on the Notch signaling average deviation of three independent experiments. indicated antibodies.

pathway. JICD positively regulates transcriptional activity

through the transcription factor Activator Protein 1 (AP1). Notch1

intracellular domain (Notch1-IC) was known to down-regulate the Figure 4.

transcriptional activity induced by transcription factor AP1. Also,

we previously suggested that JICD induces the degradation of Figure 3. Fe65 suppresses Jagged1 by increasing

Notch1-IC by enhancing ubiquitination of Notch1-IC through Fe65 physically interacts with Jagged1 and recruitment of E3 ligase Neurl 1 and forming stable

promoting interaction with E3 ligase Fbw7. Since Jagged1 is JICD. association with a tertiary complex

membrane-tethered, the endocytosis and posttranslational

modification is important to stabilize the protein level of Jagged1

and its activity. Neurl1 regulates protein stability of Jagged1, and

prevents Jagged1-induced Notch signaling pathway. Neurl1

inhibits the expression of Notch target genes to prevent the

development of medulloblastoma. However, to date, the

regulation mechanisms of Jagged1 pathway were not well

demonstrated.

Fe65 is an evolutionally conserved adaptor protein and is

consisted with a WW domain and two consecutive phosphor-

tyrosine interaction domains which were involved in protein-

protein interaction. Fe65 has important role in neuronal

development and brain disease, Alzheimer disease and

Huntington. In previous study, Fe65 regulates APP processing

and the Notch signaling pathway. Fe65 enhances the production

of amyloid beta by promoting the activity of gamma-secretase

and Fe65 inhibits the protein stability of Notch1. Fe65 binds to (A) HEK293 cells were transiently transfected with HA-Jagged1

Notch1-IC, and inhibits binding to transcription factor RBP-Jκ to and Fe65-Myc. After 48 h transfection, the cell lysates were
subjected to immunoprecipitation with anti-Myc antibody. The (A) HEK293 cells were transiently transfected with HA-Jagged1,
suppress the transactivation. Moreover, Fe65 accelerated immunoprecipitates were immunoblotted with anti-HA antibody. His-ub and Fe65-Myc. After 48 h transfection, the cell lysates

degradation of the membrane-tethered Notch1 in the cytoplasm. (B) HEK293 cells were transiently transfected with shControl and were subjected to immunoprecipitation with Nickel-agarose and

However, the study of Fe65 to regulate the Jagged1 signaling is shFe65. After 48 h transfection, the cell lysates were to immunoblotting analysis with anti-HA antibody. (B) HEK293

largely unknown. immunoprecipitated with anti-Fe65 or IgG antibody, and the cells were transiently transfected with HA-Jagged1, His-ub, Fe65-

Here, we show that Fe65 down-regulates the AP1-mediated immunoprecipitates were immunoblotted with anti-Jagged1 Myc, Neurl1-Flag and NeurlRm-Flag. After 48 h transfection, the

antibody. (C) Recombinant GST or GST- JICD proteins were cells were treated with MG132 for 6 h before harvested and lysed.
transactivation of Jagged1 or JICD in a Tip60-independent
immobilized onto GSH-agarose. HEK293 cells were transiently The cell lysates were subjected to immunoprecipitation with

manner. Whereas Fe65 decreases the protein stability of Jagged1 transfected with Fe65-Myc. After 48 h of transfection, the cell Nickel-agarose and to immunoblotting analysis with anti-HA

in both proteasomal and lysosomal degradation pathway, Fe65 lysates were subjected to GST pull-down experiments with antibody. (C) HEK293 cells were transiently transfected with HA-

does not reduce the JICD protein level but suppresses the AP1- immobilized GST or GST- JICD. Proteins bound to GST or GST- Jagged1, Neurl1-Flag and Fe65-Myc. After 48 h transfection, the

mediated JICD transactivation. Also, we demonstrate that JICD JICD was analyzed via immunoblotting with an anti-Myc antibody. cell lysates were subjected to immunoprecipitation with anti-Flag
The input represents 1% of the cell lysate prior to in vitro binding antibody and to immunoblotting analysis with anti-HA antibody.
interacts with Fe65 in vitro and in vivo. We show that Neurl1 is
assay. (D) HEK293 cells were transiently transfected with GFP- (D) HEK293 cells were transiently transfected with HA-Jagged1,

involved in Fe65-mediated inhibition of Jagged1. Fe65 increases JICD and Fe65-Myc. After 48 h transfection, the cell lysates were Neurl1-Flag and Fe65-Myc. After 48 h transfection, the cells were

the interaction between Jagged1 and Neurl1, and mono- subjected to immunoprecipitation with anti-Myc antibody, and the treated with MG132 for 6 h before harvested and lysed. The cell

ubiquitination of Jagged1 thereby facilitates degradation of immunoprecipitates were immunoblotted with anti-GFP antibody. lysates were subjected to immunoprecipitation with anti-Flag

Jagged1. This regulation was promoted by forming the stable Cell lysates were also subjected to immunoblotting analysis with antibody and to immunoblotting analysis as indicated antibodies.

the indicated antibodies.
association as trimeric complex among Jagged1, Neurl1 and

Fe65. These results suggested that Fe65 negatively regulates

Jagged1 signaling pathway.
Conclusion

In conclusion, Jagged1 is a novel binding partner of Fe65, and

Reference Fe65 acts as negative regulator in Jagged1 signaling by

triggering the degradation of membrane-tethered Jagged1. Fe65

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biological chemistry, 287 (2012) 4690-4701. promise about research on the signaling transduction in a

ligand-expressing cell.