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Alpha-synuclein negatively regulates Notch1
Abstract
Notch signaling pathway is well known that it is
involved in regulating cell fate, proliferation and
homeostasis. In this study, we show a novel function
of alpha-synuclein (SNCA) to promote degradation of
Notch1 intracellular domain (Notch1-IC) through
Fbw7, ubiquitin E3 ligase. We identified that SNCA
inhibits Notch1 transcription activity and diminishes
the interaction between Notch1-IC and RBP-Jk. We
also found decrease of Notch1-IC protein stability by
exogenous and endogenous SNCA through proteasomal
pathway, not through lysosomal pathway. And, we
found that SNCA promotes interaction between
Notch1-IC and Fbw7. Furthermore, SNCA directly
interacts with Fbw7. SNCA increases ubiquitination of
Notch-IC by Fbw7 through interaction with Fbw7.
Together, these results suggest that SNCA is a novel
regulator of Notch1-IC transcriptional activity with
acting as an enhancer of the interaction of Notch1-IC
and Fbw7 with increasing degradation of Notch1-IC.
Introduction
The Notch signaling pathway, which is evolutionally well
con-versed, is well known that it has important roles to
regulate cell apoptosis, proliferation and homeostasis.
Among several Notch receptors (Notch1–4), we
concentrated on Notch1 signaling path-way [1]. Right
after activation by interaction of extracellular domain
of Notch1 with ligands from neighbor cells,
intracellular domain ofNotch1 is cleaved by gamma-
secretase complex, which results in release of
intracellular domain of Notch1 (Notch1-IC) along with
cleavage and release of extracellular domain of Notch1
[3,5,22]. Then, released Notch1-IC translocate to the
nucleus, and acts as a transcriptional factor by
interacting with recombining
Suppressor of Hairless (RBP-Jk), which binds to the
promoter site of target genes of Notch1 to regulate
transcription by complex with transcriptional co-
repressors or co-activators [11,18].When Notch1-IC
interacts with RBP-Jk, associated transcriptional
repressors to RBP-Jk are detached, on the other hand,
transcriptional co-activators are associated with RBP-
Jk [23]. Overall, not along Notch1-IC; when Notch1
signaling pathway is not activated, transcription of
target genes are prevented [21,23,24]. Through
control of transcriptional factors, Notch1-IC activates
transcription of Notch1 target genes, as an example of
them, Hes (Hairy enhancer of split) family [12,14].
Although Notch1 has both pro-survival and pro-
apoptotic functions according to environment of each
tissues,Notch1 is an important activator of cell
proliferation and differentiation in neuronal cells
[4,20]. Recent studies have demonstrated that SNCA
inhibits neurogenesis of mouse stem cells and
hippocampus of SNCA
neurogenesis of adult rat hippocampus neural progeni-
tors as well [7,8]. Crews et al. found that SNCA
reduces neurogenesis which is caused by increased
apoptosis and reduced proliferation of neuronal
progenitor cells by hindering Notch1 signaling; down-
regulation of mRNA level of Hes-5, and protein
stability offull-length Notch1 and Notch1-IC [7].
Moreover, Desplats et al. found that the inhibition of
Notch1 signaling is derived from capacity of SNCA to
bind to the promoter site of Notch1 with p53 to hinder
transcription of Notch1 [8].In the present study, we
focused on that attenuation of Notch1signaling by
SNCA is through regulation of Notch1 protein sta-
bility. First, we confirmed decrease of Notch1-IC
transcriptional activity by SNCA, and it is derived
from reduction of physical inter-action between
Notch1-IC and RBP-Jk by SNCA. Moreover, we found
that protein stability of Notch1-IC is reduced by
SNCA through proteasomal pathway. Importantly, we
identified Fbw7 as an E3ligase required for SNCA-
induced Notch1-IC protein degradation. We identified
that SNCA facilitates the interaction of Fbw7
withNotch1-IC and thereby facilitates ubiquitination
of Notch1-IC. From this observation, we implicated
that down-regulation of Notch1signaling by SNCA is
resulted from Notch1-IC protein destabilization
through Fbw7-mediated ubiquitination.
intracellular domain protein stability through
promoting interaction with Fbw7
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, Gwangju 500-757, Republic of Korea
Results
Fig. 1. SNCA reduces Notch1-IC transcriptional Fig. 3. SNCA reduces Notch1-IC protein
activity. stability through Fbw7 dependent manner.
(A–B) HEK293 cells were transfected with expression vectors encoding (A) HEK293 cells were transiently transfected with expression vectors
encoding for shCon or shFbw7, Myc-Notch1-IC and Myc-SNCA. 48 h
for 4xCSL-Luc and -galatosidase, (A) along with Notch1-IC and 100 ng
(+), 200 ng (+ +), 300 ng (+ + +)of SNCA, (B) and control shRNA, or after transfection, the cells werelysates and lysates were immunoblotted
Notch1 shRNA, as indicated. (C) HEK293 cells were transiently with anti-Myc antibody. The expressions of Myc-SNCA or Fbw7 were
transfected with shCon or shSNCA as indicated. 48 h after transfection, analyzed via immunoblotting using anti-Myc and anti-Fbw7antibody,
thecells were lysed, and lysates were subjected to immunoprecipitation respectively. (B) HEK293 cells were transfected with expression vectors
using IgG or anti-Notch1 antibodies, as indicated. The encoding for Myc-Notch1-IC, Myc-SNCA and Flag-Fbw7 or Fbw7 F. 48
immunoprecipitates were immunoblotted withanti-RBP-Jk antibody. The h after transfection,the cells were lysates and lysates were
immunoblotted with anti-Myc antibody. The expressions of Myc-SNCA or
expressions of Notch1-IC, RBP-Jk or SNCA were analyzed via
immunoblotting using anti-Notch1, anti- RBP-Jk and anti-SNCA antibody, Flag-Fbw7 or Fbw7 F were analyzed via immunoblottingusing anti-Myc
respectively.(D) HEK293 cells were transfected with expression vectors and anti-Flag antibody, respectively. (C) HEK293 cells were transfected
with expression vectors encoding for Myc-SNCA and Flag-Fbw7. 48 h
encoding for Myc-Notch1-IC, Flag-RBP-Jk and Myc-SNCA as indicated.
48 h after transfection, the cells lysates weresubjected to after transfection,the cells lysates were subjected to
immunoprecipitation analysis with anti-Flag. The immunoprecipitates were immunoprecipitation analysis with anti-Flag. The immunoprecipitates were
then immunoblotted with anti-Myc. The expressions of Myc-Notch1-IC, then immunoblotted with anti-Myc. The expressions of Myc-SNCA or
Flag-Fbw7 were analyzed via immunoblotting using anti-Myc and anti-Flag
Flag-RBP-Jkor Myc-SNCA were analyzed via immunoblotting using anti-
Myc and anti-Flag antibody, respectively. Data are shown as mean ± antibody, respectively. (D) HEK293 cells lysates were subjected to
SD(n = 3), *p < 0.05. immunoprecipitationusing IgG or anti-Fbw7 antibodies, as indicated. The
immunoprecipitates were immunoblotted with anti-SNCA antibody. The
expressions of SNCA, Notch1 or Fbw7 wereanalyzed via immunoblotting
using anti-SNCA, anti-Notch1 and anti-Fbw7 antibody, respectively. (A–
D) The data presented here are representative from three
independentexperiments.
Fig. 2. SNCA down-regulates Notch1-IC protein
binding protein stability through proteasomal pathway.
Fig. 4. SNCA regulates ubiquitination of
Notch1-IC by Fbw7.
(A) HEK293 cells were transiently transfected with expression vectors
encoding for Myc- Notch1-IC and 200 ng (+), 400 ng (+ +), 600 ng (+ +
+) of Myc-SNCA, as indicated.48 h after transfection, the cells were
lysed and lysates were immunoblotted with anti-Myc antibody. The
expressions of Myc-SNCA were analyzed via immunoblottingusing anti-
transgenic mice, and Myc antibody. (B) HEK293 cells were transfected for 48 h with
expression vectors encoding for control shRNA and SNCA shRNA, and
treated with 100 M DMSOor 100 M cycloheximide for the indicated
periods of time. The lysates were immunoblotted with anti-Notch1 (A) HEK293 cells were transiently transfected with shCon or shSNCA as
antibody. (C) HEK293 cells were transfected for 48 h withexpression indicated. 48 h after transfection, the cells were lysed, and lysates
vectors encoding for Flag-Notch1-IC and Myc-SNCA. HEK293 cells were
treated with 25 M of ALLN, 5 M of MG132 and 100 nM of Epoxomicin were subjected to immunopre-cipitation using IgG or anti-Fbw7
antibodies, as indicated. The immunoprecipitates were immunoblotted
for 6 h. The lysateswere immunoblotted with anti-Flag antibody. The with anti-Notch1 antibody. The expressions of Notch1-IC, Fbw7or
expressions of Myc-SNCA were analyzed via immunoblotting using anti-
Myc antibody. (D) HEK293 cells were transfectedfor 48 h with SNCA were analyzed via immunoblotting using anti-Notch1, anti-Fbw7
and anti-SNCA antibody, respectively. (B) HEK293 cells were
expression vectors encoding for Myc-Notch1-IC and Myc-SNCA. transfected with expression vectorsencoding for Myc-tagged Notch1-IC,
HEK293 cells were treated with 100 M of Chloroquine and 50 mM of shFbw7, shSNCA and HA-tagged ubiquitin, as indicated. 48 h after
NH4Cl for 6 h. The lysateswere immunoblotted with anti-Myc antibody.
The expressions of Myc-SNCA were analyzed via immunoblotting using transfection, the cells lysates were subjected to
immunoprecipitationanalysis with anti-Myc. The immunoprecipitates were
anti-Myc antibody. (A–D) The data presented here arerepresentative then immunoblotted with anti-HA. (C) HEK293 cells were transfected
from three independent experiments.
with expression vectors encoding for 4xCSL-Lucand -galatosidase, along
with Notch1-IC, SNCA and Fbw7 F, as indicated. Data are shown as
mean ± SD(n = 3), *p ≤ 0.005; NS: non-significant.
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