JCS ePress online publication date 2 February 2012
Research Article
1
Presenilin-2 regulates the degradation of RBP-Jk
protein through p38 mitogen-activated protein kinase
Su-Man Kim*, Mi-Yeon Kim*, Eun-Jung Ann, Jung-Soon Mo, Ji-Hye Yoon and Hee-Sae Park`
Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, 500-757, Republic of Korea
*These authors contributed equally to this work
`
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 18 October 2011
Journal of Cell Science 125, 1–13
2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.095984
Summary
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 timedependent 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 d (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.
Key words: RBP-Jk, Presenilin 2, p38 MAPK, Proteasome, Lysosome, Protein degradation
Introduction
Notch is a vitally important signaling receptor, which modulates
cell fate determination and pattern formation in a number of ways
during the development of both invertebrate and vertebrate species
(Artavanis-Tsakonas and Simpson, 1991; Duffy and Perrimon,
1996; Weinmaster, 1998; Artavanis-Tsakonas et al., 1999; Robey,
1999; Neumann, 2001; Wang and Sternberg, 2001; Whitford et al.,
2002; Lai, 2004). Mammals express four Notch genes (NOTCH1–
NOTCH4) and five ligands for Notch from two conserved families,
Jagged (JAG1 and JAG2) and Delta (DLL1, DLL3 and DLL4)
in different combinations in most cell types. After ligand
binding, Notch proteins are activated by a series of cleavages
that release its intracellular domain (Notch-IC), followed by its
nuclear translocation (Kopan and Ilagan, 2009). The nuclear
translocation of Notch-IC results in the transcriptional activation of
genes of the HES family [Hes/E(spl) family] and the HEY family
(Hesr/Hey family) through the interaction of Notch-IC with RBPJk [CBF1 or RBP-Jk in vertebrates, Suppressor of Hairless (SuH)
in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans]
and through mastermind-like (MAML) (Weinmaster, 1997; Kopan
and Ilagan, 2009). In the absence of Notch-IC, RBP-Jk functions
as a transcriptional repressor because of its ability to bind
to transcriptional co-repressors and histone deacetylase-1. NotchIC translocates to the nucleus, where it interacts with the RBP-Jk
and displaces co-repressor complexes and recruits co-activators,
thus turning RBP-Jk into a transcriptional activator (Kao et al.,
1998).
Presenilin proteins are integral components of a multiprotein
protease complex, referred to as c-secretase, which is responsible
for the intramembranous cleavage of the amyloid precursor
protein (APP), the Notch receptors, Delta, Jagged, E-cadherin,
p75 neurotrophin receptor, low density lipoprotein-related
protein, nectin-1a, CD44 and Erb-b4, resulting in the release of
intracellular signaling fragments (Koo and Kopan, 2004). These
intracellular fragments of presenilin substrates are translocated
into the nucleus, yet their functional roles in the cellular system
are still not apparent. Presenilins are stably associated with the
c-secretase complex along with three other proteins: nicastrin,
Pen-2 and Aph-1, which collectively constitute the functional
c-secretase (Edbauer et al., 2003). Presenilin-1 (PS1)-knockout
mice have an embryonic lethal phenotype with similarities to that
of the Notch-knockout mouse, as well as dramatically reduced
amyloid-beta (Ab) peptide levels (Shen et al., 1997; Palacino
et al., 2000). By way of contrast, PS2-knockout mice have no
apparent phenotype or change in levels of Ab peptide (Herreman
et al., 1999). These observations indicate that, under normal
circumstances, c-secretase activity resulting in the cleavage of
substrates is associated predominantly with PS1, rather than PS2
complexes.
Recent studies have confirmed the role of ubiquitylation in the
regulation of Notch signaling (Lai, 2002). Several research
groups have demonstrated that the F-box containing protein
Sel10/Fbw7 mediates Notch ubiquitylation in the nucleus, and
targets it for proteasome-dependent degradation (Wu et al., 2001;
Gupta-Rossi et al., 2001; Hubbard et al., 1997; O’Neil et al.,
2007). We demonstrated that ILK, SGK1 and Jagged-1
intracellular domain (JICD) downregulate the protein stability
of Notch1-IC through the ubiquitin-proteasome pathway by
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means of Fbw7 (Mo et al., 2007; Mo et al., 2011; Kim et al.,
2011). Thus far, little has been definitively determined regarding
the regulation of RBP-Jk protein stability. In our recent report,
we proposed that degradation mediated by the amyloid precursor
protein intracellular domain (AICD), might be involved in the
preferential degradation of RBP-Jk by the lysosomal pathway.
Despite this observation, the precise mechanisms underlying the
degradation of the RBP-Jk protein remain to be accurately
delineated.
Therefore, we evaluated the degrdation of RBP-Jk using
proteasome and lysosome inhibitors. In this study, we determined
that RBP-Jk degradation involves both the proteasome and the
lysosome. We also demonstrated that PS2 modulates the protein
stability of RBP-Jk by the p38 mitogen-activated protein kinase d
(MAPK13; referred to here as p38 MAPK) signaling pathway.
Furthermore, we showed that the phosphorylation of RBP-Jk
by p38 MAPK induces RBP-Jk protein degradation and
ubiquitylation. Collectively, our findings indicate that p38
MAPK functions as a regulator of RBP-Jk protein stability by
RBP-Jk phosphorylation.
Journal of Cell Science
Results
Proteasome inhibition increases exogenous and
endogenous RBP-Jk protein levels
Despite the many studies of RBP-Jk thus far conducted, the
mechanism underlying the degradation of the protein remains to
be completely understood. To determine whether the RBP-Jk
protein is degraded by the proteasome, proteasome inhibitors
were used to treat Myc–RBP-Jk-transfected HEK293 cells. These
cells were then treated with ALLN (25 mM), MG132 (5 mM) and
lactacystin (10 mM) for 4 hours (Fig. 1A). Myc–RBP-Jk protein
was detected with the anti-Myc antibody 9E10. As shown in
Fig. 1A, similarly to lactacystin treatment, ALLN and MG132
significantly increased RBP-Jk levels relative to those observed
in control cells treated with vehicle solution only. Protein levels
were normalized to b-actin. Myc–RBP-Jk-transfected HEK293
cells were then treated with different doses of MG132 (0–10 mM)
for 4 hours or at 5 mM for different periods of time (0–6 hours).
MG132 treatment markedly increased RBP-Jk protein levels in a
dose-dependent (Fig. 1B) and time-dependent manner (Fig. 1C).
In order to determine whether treatment with proteasome
inhibitors also induces an increase in endogenous RBP-Jk
proteins, HEK293 and C2C12 cells were treated with the
proteasome inhibitor MG132 at different dosages for 4 hours
(0–10 mM) (Fig. 1D,E). Endogenous RBP-Jk protein was
detected with rabbit anti-RBP-Jk antibody. MG132 markedly
increased endogenous RBP-Jk protein levels in HEK293 and
C2C12 cells.
The half-life of RBP-Jk was determined using cycloheximide,
a protein translation inhibitor. Cycloheximide interacts with the
translocase enzyme and blocks protein synthesis in eukaryotic
cells. After cycloheximide treatment, the level of RBP-Jk protein
dropped gradually, and approximately half of the protein was
degraded after 2 hours compared with levels in control cells
(Fig. 1F,G). Upon treatment with MG132, the level of RBP-Jk
protein dropped slightly and approximately half of the protein
was degraded after 8 hours in the presence of MG132
(Fig. 1F,G). Taken together, these results clearly demonstrate
that treatment with proteasome inhibitor significantly increases
RBP-Jk protein levels, thereby suggesting that the degradation of
RBP-Jk protein is indeed mediated by the proteasome pathway.
Lysosome inhibition increases exogenous and
endogenous RBP-Jk protein levels
In an effort to determine whether RBP-Jk protein is
lysosomally degraded, lysosome inhibitors were administered
to Myc–RBP-Jk-transfected HEK293 cells. The cells were
treated with chloroquine (100 mM) and NH4Cl (50 mM) for
6 hours. Myc–RBP-Jk protein was then detected with the antiMyc antibody 9E10. Chloroquine and NH4Cl significantly
increased RBP-Jk levels in comparison with levels in the
vehicle control cells (Fig. 2A). Protein levels were normalized
to b-actin. The cells were subsequently treated with different
doses of NH4Cl for 10 hours (0–50 mM) or at 50 mM for
different periods of time (0–10 hours). NH4Cl treatment
affected a marked increase in RBP-Jk protein levels in a
dose-dependent (Fig. 2B) and time-dependent manner
(Fig. 2C). To determine whether treatment with lysosome
inhibitors also induces an increase in endogenous RBP-Jk
proteins, HEK293 and C2C12 cells were treated with the
lysosome inhibitor NH4Cl at different doses for 10 hours
(Fig. 2D,E). NH4Cl markedly increased endogenous RBP-Jk
protein levels in HEK293 and C2C12 cells.
To determine the half-life of RBP-Jk in the presence of
lysosome inhibitor, Myc–RBP-Jk-transfected HEK293 cells were
treated with NH4Cl and cycloheximide. After cycloheximide
treatment, the level of RBP-Jk protein declined gradually, with
approximately half of the protein degraded after 2.5 hours
relative to the level in control cells (Fig. 2F,G). Upon NH4Cl
treatment, the level of RBP-Jk protein changed moderately;
approximately half of the protein was degraded after 6 hours in
the presence of NH4Cl (Fig. 2F,G). Taken together, these results
clearly demonstrate that treatment with lysosome inhibitor
moderately increases RBP-Jk protein levels, thereby suggesting
that degradation of RBP-Jk is also mediated by the lysosome
pathway.
PS2 downregulates the level of endogenous RBP-Jk
proteins
To determine the effects of the absence of PS1 and PS2 on RBPJk expression, immunoblot analyses were conducted using wildtype PS1 and PS2 cells and double-knockout cells. The half-life
of endogenous RBP-Jk was determined using cycloheximide, a
protein translation inhibitor. PS1 and PS2 double-knockout and
wild-type control cells were exposed to cycloheximide, and the
amount of RBP-Jk was analyzed using immunoblot. As a result,
the steady-state level and the half-life of endogenous RBP-Jk
were higher in the double-knockout cells than in wild-type cells
(Fig. 3A,B). We subsequently assessed the involvement of PS1
or PS2 in RBP-Jk protein degradation using PS1-knockout and
PS2-knockout cells. The steady-state level and the half-life of
endogenous RBP-Jk were higher in PS2-knockout cells than in
PS1-knockout cells (Fig. 3C,D). We next investigated the highly
specific c-secretase inhibitor DAPT (Dovey et al., 2001) for its
capacity to block RBP-Jk protein degradation and half-life. With
this objective, wild-type PS1 and PS2 cells were treated with or
without DAPT along with cycloheximide (Fig. 3E). As shown in
Fig. 3E, endogenous RBP-Jk levels were increased markedly in
cells treated with DAPT alone and in those with DAPT and
cycloheximide. We also attempted to confirm the effect of PS2
on the protein level of RBP-Jk using PS1 and PS2 doubleknockout cells. We measured protein levels of RBP-Jk in the
presence of wild-type PS2 or an inactive dominant-negative
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Degradation of RBP-Jk by p38 MAPK
Fig. 1. Proteasomal inhibition increases the levels of exogenous and endogenous RBP-Jk proteins. (A) Immunoblots of Myc–RBP-Jk-transfected HEK293
cells treated with vehicle solution control, ALLN (25 mM), MG132 (5 mM), lactacystin (10 mM) for 4 hours. (B) Myc–RBP-Jk transfected HEK293 cells treated
with vehicle solution control or MG132 for 4 hours at 0, 2.5, 5 or 10 mM for a dosage-dependent assay, or (C) with MG132 at 10 mM for 0, 4 or 6 hours for a
time-course assay. (D) HEK293 and (E) C2C12 cells were treated with vehicle solution control or MG132 for 4 hours at 0, 5 or 10 mM for a dosage-dependent
assay. (F,G) Myc–RBP-Jk-transfected HEK293 cells were treated with 200 mM of cycloheximide (CHX) together with vehicle or MG132 for 0, 2, 4, 6 and
8 hours. We quantified the intensity of each band using a densitometer and plotted relative intensities. Data are expressed as means ± s.d. from three independent
experiments. In A–F the cell lysates were also subjected to immunoblotting analysis with the indicated antibodies. Equal amounts of protein from each sample
were immunoblotted with antibody against b-actin. These results were replicated at least three times.
mutant of PS2 [PS2(D366A)] (Fig. 3F). As shown in Fig. 3F,
endogenous levels of RBP-Jk protein were markedly decreased
in cells transfected with wild-type PS2. However, levels were
substantially increased in cells expressing the PS2(D366A)
mutant. Furthermore, we attempted to confirm the effect of
PS1 and PS2 on the half-life of endogenous RBP-Jk using PS1
and PS2 double-knockout cells. We measured levels of RBP-Jk
in the presence of the PS1 or PS2 using cycloheximide, a protein
translation inhibitor. The steady-state level and the half-life of
endogenous RBP-Jk were higher in PS1-transfected cells than in
PS2-transfected cells (Fig. 3G,H). These results indicate that PS2
has a critical role in the regulation of RBP-Jk protein degradation
and half-life and show that the stability of the endogenous RBPJk protein is downregulated by PS2.
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Fig. 2. Lysosomal inhibition increases the levels of exogenous and endogenous RBP-Jk proteins. (A) Immunoblots of Myc–RBP-Jk-transfected HEK293
cells treated with the vehicle solution control, chloroquine (100 mM), NH4Cl (50 mM) for 6 hours. (B) Myc–RBP-Jk-transfected HEK293 cells treated with
vehicle solution control or NH4Cl for 10 hours at 0, 10, 20 or 50 mM for a dosage-dependent assay, or (C) with NH4Cl at 50 mM for 0, 6, 8 or 10 hours for a
time-course assay. (D) HEK293 cells and (E) C2C12 cells treated with vehicle solution control or NH4Cl for 10 hours at 0, 20 or 50 mM for a dosage-dependent
assay. (F,G) Myc–RBP-Jk-transfected HEK293 cells were treated with 200 mM of cycloheximide (CHX) together with vehicle or NH4Cl for 0, 2, 4, 6 and
8 hours. We quantified the intensity of each band using a densitometer and plotted relative intensities. Data are expressed as means ± s.d. from three independent
experiments. The cell lysates in A–F were also subjected to immunoblotting analysis with the indicated antibodies. Equal amounts of protein from each sample
were immunoblotted with antibody against b-actin. These results were replicated at least three times.
PS2 facilitates degradation of RBP-Jk by p38 MAPK
signaling
PS1-knockout cells showed degradation of RBP-Jk, followed by
periods during which we inhibited further protein synthesis by
applying cycloheximide (CHX). In the presence of CHX alone,
RBP-Jk degraded over a 4 hour observation period (Fig. 4A). By
contrast, when CHX and MG132 were applied together, RBP-Jk
levels were unchanged over 6 hours (Fig. 4B,D). Degradation of
RBP-Jk was inhibited significantly by proteasome inhibition.
Collectively, these results clearly show that treatment with
proteasome inhibitor significantly increases RBP-Jk protein
levels, thus suggesting that degradation of RBP-Jk is mediated
by the proteasome pathway. We then attempted to determine
whether lysosomal inhibitors exerted any detectable effects on
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Degradation of RBP-Jk by p38 MAPK
Fig. 3. PS2 downregulates the level of endogenous RBP-Jk. (A) Immunoblots of PS1 and PS2 double-knockout and wild-type control cells treated with
cycloheximide (CHX) at 200 mM for 0, 1, 2, 4 and 6 hours. (B) The intensity of each band was measured using a densitometer and relative intensities are plotted.
(C) PS1-knockout and PS2-knockout cells were treated with cycloheximide (CHX) at 200 mM for 0, 1, 2, 4 and 6 hours. (D) The intensity of each band was
measured using a densitometer and relative intensities are plotted. (E) PS1 and PS2 wild-type cells were treated with 100 mM of DAPT for 4 hours along with
200 mM of cycloheximide for 6 hours. (F) PS1 and PS2 double-knockout cells were transfected with expression vectors encoding FLAG–PS2 and FLAG–
PS2(D366A) (D/A) as indicated. After 48 hours of transfection, the cell lysates were subjected to immunoblotting analysis with the indicated antibodies.
(G,H) PS1 and PS2 double-knockout cells were transfected with expression vectors encoding HA–PS1 and FLAG–PS2 as indicated. Cells were treated with
cycloheximide at 200 mM for 0, 1, 2, 4 and 6 hours. We quantified the intensity of each band using a densitometer and plotted relative intensities. The cell lysates
in A–H were also subjected to immunoblotting analysis with the indicated antibodies. Equal amounts of protein from each sample were immunoblotted with
antibody against b-actin. Data are expressed as means ± s.d. from three independent experiments.
the degradation of RBP-Jk. Our studies showed that lysosomal
inhibitors moderately regulate the steady-state levels of RBP-Jk
proteins in PS1-knockout cells (Fig. 4C,D). These results
demonstrated that the stability of the RBP-Jk protein is
downregulated by PS2 through proteasome- and lysosomedependent pathways.
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Fig. 4. PS2 facilitates the degradation of RBP-Jk by p38 MAPK signaling. (A) Immunoblots of PS1-knockout cells treated with cycloheximide (CHX) at
200 mM for 0, 1, 2, 4 and 6 hours. (B) PS1-knockout cells treated with cycloheximide (CHX) together with vehicle or MG132 for 0, 1, 2, 4 and 6 hours. (C) PS1knockout cells treated with cycloheximide (CHX) together with vehicle or NH4Cl for 0, 1, 2, 4 and 6 hours. (D) The intensity of each band was measured using a
densitometer and relative intensities are plotted. (E) PS1-knockout cells treated with cycloheximide (CHX) along with vehicle or SB203580 for 0, 1, 2, 4
and 6 hours. (F) PS1-knockout cells treated with cycloheximide with vehicle or LiCl for 0, 1, 2, 4 and 6 hours. (G) The intensity of each band was measured using
a densitometer and relative intensities are plotted. The cell lysates were also subjected to immunoblotting analysis with the indicated antibodies. Equal amounts of
protein from each sample were immunoblotted with antibody against b-actin. Data in D and G are expressed as means ± s.d. of three independent experiments.
A previous report has demonstrated that PS2 functions as a
signaling molecule upstream of the p38 MAPK pathway (Sun
et al., 2001). To determine whether treatment with p38 MAPK
inhibitors increases the stability of endogenous RBP-Jk
proteins, PS1-knockout cells were treated with the p38 MAPK
inhibitor SB203580 (Fig. 4E,G). SB203580 markedly increased
endogenous RBP-Jk protein stability in PS1-knockout cells.
Additionally, to determine whether treatment with GSK-3b
inhibitors increases the protein stability of endogenous RBP-Jk
proteins, PS1-knockout cells were treated with the GSK-3b
inhibitor LiCl. LiCl did not increase endogenous RBP-Jk protein
stability in PS1-knockout cells. The results show that the
downregulated level of RBP-Jk protein was not restored to a
sufficient degree by the inhibition of GSK-3b (Fig. 4F,G). These
results demonstrate that the negative regulation of the stability of
RBP-Jk by PS2 occurs in a GSK-3b-independent pathway. Our
results show that the downregulated RBP-Jk protein is restored
by treatment with a p38-MAPK-specific inhibitor in PS1knockout cells, thereby indicating that PS2-mediated degradation
of RBP-Jk occurs in a manner that is dependent on p38 MAPK
signaling.
p38 MAPK downregulates the level of RBP-Jk protein
We investigated whether the presenilins affect p38 MAPK
activity in MEF cells by examining the endogenous activity
of p38 MAPK in wild-type and presenilin-knockout cells.
Endogenous p38 MAPK activity at the basal state was higher
in wild-type PS1 and PS2 cells and PS1-knockout MEF cells than
it was in PS2-knockout MEF cells in normal serum conditions
(Fig. 5A). The basal activity of p38 MAPK activity in wild-type
and PS1-knockout MEF cells was substantially suppressed in the
presence of the p38 MAPK inhibitor SB203580 (Fig. 5A). This
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Degradation of RBP-Jk by p38 MAPK
Fig. 5. p38 MAPK downregulates the level of RBP-Jk protein. (A) Immunoblots of wild-type PS1 and PS2, PS1-knockout and PS2-knockout cells treated with
vehicle or SB203580 at 10 mM for 6 hours. (B) HEK293 cells were transfected for 42 hours with the indicated combinations of expression vectors encoding for
Myc–RBP-Jk and HA-p38 MAPK. The cells were treated with cycloheximide (CHX) at 200 mM for 0, 1, 2, 4 and 6 hours. (C) The intensity of each band was
measured using a densitometer and relative intensities are plotted. Data are expressed as means ± s.d. from three independent experiments. (D) HEK293 cells were
transfected for 42 hours with the indicated combinations of expression vectors encoding for Myc–RBP-Jk and HA-p38 MAPK. The cells were treated with the
indicated quantity of MG132 for 6 hours. (E) HEK293 cells were transfected for 42 hours with the indicated combinations of expression vectors encoding for
Myc–RBP-Jk and HA-p38 MAPK. The cells were treated with the indicated amounts of NH4Cl for 6 hours. The cell lysates were also subjected to
immunoblotting analysis with the indicated antibodies. Equal amounts of protein from each sample were immunoblotted with b-actin antibody for b-actin. These
results were replicated at least three times.
suggests that PS2 might influence the basal activity of
endogenous p38 MAPK. We transfected the HEK293 cells with
Myc–RBP-Jk and HA–p38-MAPK, and the quantity of remaining
RBP-Jk was evaluated after various periods of cycloheximide
treatment. We determined the protein stability of RBP-Jk in
HEK293 cells by cycloheximide treatment with or without p38
MAPK. After cycloheximide treatment, the level of RBP-Jk
protein declined gradually, with approximately half of the protein
being degraded after 4 hours without p38 MAPK (Fig. 5B,C).
Upon cycloheximide treatment, the level of RBP-Jk protein
declined rapidly, with approximately half of the protein being
degraded after 1.5 hours in the presence of p38 MAPK
(Fig. 5B,C). This result demonstrates that RBP-Jk is rapidly
turned over in the presence of p38 MAPK. To determine whether
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Fig. 6. See next page for legend.
Degradation of RBP-Jk by p38 MAPK
Journal of Cell Science
the degradation of RBP-Jk proteins is mediated by the
proteasome pathway, the proteasome inhibitor MG132 was
administered to cells expressing RBP-Jk and p38 MAPK. The
cells were treated with proteasome inhibitor for 6 hours and the
RBP-Jk protein was detected in an immunoblot assay. The results
of our studies showed that the proteasome inhibitor significantly
increased RBP-Jk levels (Fig. 5D). The level of RBP-Jk protein
was reduced in the presence of p38 MAPK, but was significantly
restored by MG132 treatment (Fig. 5D).
We then attempted to determine whether lysosomal inhibitors
exerted any detectable effects on RBP-Jk degradation. We
transfected HEK293 cells with Myc–RBP-Jk and HA–p38MAPK, and the quantity of RBP-Jk remaining was analyzed
after various periods of treatment with NH4Cl. Our findings
revealed that lysosomal inhibitor also increased the steady-state
levels of RBP-Jk proteins (Fig. 5E). These results showed that
Fig. 6. p38 MAPK facilitates the degradation of RBP-Jk by
phosphorylation at Thr339. (A) Recombinant GST or GST–RBP-Jk proteins
were immobilized onto GSH-agarose. HEK293 cells were transfected with an
expression vector encoding for HA-p38 MAPK or an empty vector. After
48 hours of transfection, the cell lysates were subjected to GST pull-down
experiments with immobilized GST or GST–RBP-Jk. Proteins bound to GST or
GST–RBP-Jk were analyzed by immunoblotting with an anti-HA. (B) HEK293
cells transfected with expression vectors encoding for Myc–RBP-Jk and HAp38 MAPK as indicated. The cell lysates were subjected to
immunoprecipitation (IP) with an anti-HA. The immunoprecipitates were then
immunoblotted (IB) with an anti-Myc antibody. Cell lysates were also
immunoblotted with anti-Myc and anti-HA antibodies. (C) HEK293 cells
transfected with expression vectors encoding for HA-p38 MAPK as indicated.
After 48 hours of transfection, the cell lysates were subjected to
immunoprecipitation with an anti-HA antibody, and the resultant precipitates
were evaluated for p38 MAPK activity by an immune complex kinase assay
using GST–RBP-Jk and GST–RBP-Jk(T339A). (D) HEK293 cells transfected
with expression vectors encoding Myc–RBP-Jk(WT), Myc–RBP-Jk(T339A)
and HA–p38-MAPK as indicated. After 48 hours of transfection, the cell lysates
were subjected to immunoblotting (IB) with antibodies against phosphorylated
Ser and Thr or the HA tag. (E) HEK293 cells transfected with expression
vectors encoding Myc–RBP-Jk, Myc–RBP-Jk(T339A) and HA–p38-MAPK as
indicated. After 48 hours of transfection, the cell lysates were subjected to
immunoprecipitation with anti-HA antibody. The immunoprecipitates were
then immunoblotted with an anti-Myc antibody. Cell lysates were also
immunoblotted with an anti-Myc and anti-HA antibody. (F) HEK293 cells were
transfected with expression vectors encoding Myc–RBP-Jk(WT), Myc–RBPJk(T339A) and HA–p38-MAPK as indicated. After 48 hours of transfection, the
cell lysates were subjected to immunoblotting with an anti-Myc or anti-HA
antibody. (G) HEK293 cells transfected with expression vectors for Myc–RBPJk, Myc–RBP-Jk(T339A), FLAG–p38-MAPK and HA–ubiquitin as indicated.
After 42 hours of transfection, the cells were treated with MG132 (10 mM) for
6 hours and the cell lysates immunoprecipitated with anti-Myc antibody.
Immunoprecipitates were immunoblotted with anti-HA antibody. The cell
lysates were subjected to immunoblot analysis with anti-Myc and anti-Flag
antibodies. (H) PS1-knockout cells were transfected with expression vectors
encoding Myc–RBP-Jk and Myc–RBP-Jk(T339A). The cells were treated with
MG132 for 6 hours at 0, 5 or 10 mM for a dosage-dependent assay. The cell
lysates were subjected to immunoprecipitation with an anti-Myc antibody, and
the immunoprecipitates were immunoblotted with an anti-phosphorylated Ser
and Thr antibody. The cell lysates were also subjected to immunoblotting
analysis with anti-Myc antibody. (I) PS1-knockout cells transfected with
expression vectors encoding for Myc–RBP-Jk and Myc–RBP-Jk (T339A). The
cells were treated with NH4Cl for 6 hours at 0, 20 or 50 mM for a dosagedependent assay. The cell lysates were also subjected to immunoblotting
analysis with the indicated antibodies. These results were replicated at least
three times. W, wild type; M, mutant.
9
the stability of the RBP-Jk protein was downregulated by p38
MAPK by a proteasome- and lysosome-dependent pathway.
p38 MAPK facilitates the degradation of RBP-Jk by
phosphorylation at Thr339
Because our results suggested that RBP-Jk is a target of
p38 MAPK, we subsequently attempted to determine whether
these two proteins interact physically in intact cells. In in vitro
binding studies, purified GST and GST–RBP-Jk proteins were
immobilized onto GSH–agarose. HA–p38-MAPK-expressing cell
lysates were incubated either with GST or GST–RBP-Jk
immobilized onto GSH–agarose. The interaction between GST–
RBP-Jk and p38 MAPK was detected on bead complexes
(Fig. 6A). We then evaluated the physical interactions occurring
between RBP-Jk and p38 MAPK. HEK293 cells were cotransfected with vectors encoding Myc–RBP-Jk and HA–p38MAPK, and were then subjected to co-immunoprecipitation
analysis (Fig. 6B). Immunoblot analysis using the anti-Myc
antibody of the anti-HA immunoprecipitates from the transfected
cells revealed that p38 MAPK did indeed physically associate
with RBP-Jk in the cells.
We next conducted an in vitro kinase assay with HA–
p38-MAPK and purified GST–RBP-Jk. HA–p38-MAPK
immunocomplexes prepared from HEK293 cells catalyzed the
phosphorylation of purified recombinant GST–RBP-Jk (Fig. 6C).
p38 MAPK preferentially phosphorylates substrate protein serine
and threonine residues that lie in Pro-x-(Ser/Thr)-Pro or S/T-P
motifs (Alvarez et al., 1991; Court et al., 2004). The results of in
silico studies have shown that RBP-Jk harbors a possible
conserved threonine residue in vertebrates; this threonine
residue is accessible and is located immediately after the DNA
binding domain. Furthermore, using site-directed mutagenesis,
we determined that the replacement of Thr339 of RBP-Jk with
alanine effected a reduction in the in vitro phosphorylation of the
recombinant protein by HA–p38-MAPK immunoprecipitates
(Fig. 6C). p38 MAPK phosphorylated GST–RBP-Jk(WT)
but did not phosphorylate GST-RBP-Jk(T339A). Additionally,
HEK293 cells were transfected with expression vectors
encoding Myc–RBP-Jk(WT), Myc–RBP-Jk(T339A) and HA–
p38-MAPK. After 48 hours of transfection, the cell lysates
were subjected to immunoblotting with an antibodies against
phosphorylated Ser and Thr, or the HA tag (Fig. 6D). p38 MAPK
phosphorylated RBP-Jk(WT) but did not phosphorylate RBPJk(T339A).
We then attempted to characterize the involvement of
phosphorylation in the physical association between p38
MAPK and RBP-Jk by co-immunoprecipitation. HEK293 cells
were cotransfected with vectors coding for HA–p38-MAPK,
Myc–RBP-Jk and Myc–RBP-Jk(T339A) and were then subjected
to co-immunoprecipitation analysis. RBP-Jk and p38 MAPK
were co-immunoprecipitated, but when co-transfected with RBPJk(T339A), the band of RBP-Jk that interacted with p38 MAPK
disappeared (Fig. 6E). These results demonstrate that the
phosphorylation of RBP-Jk by p38 MAPK plays a pivotal
role in its ability to bind with p38 MAPK. Moreover, we
ascertained that RBP-Jk(T339A) is resistant to p38-MAPKinduced degradation, which implies that the p38-MAPKinduced phosphorylation of RBP-Jk is crucially important for
the degradation of the RBP-Jk protein (Fig. 6F). We then
attempted to characterize the involvement of phosphorylation in
the polyubiquitylation of RBP-Jk by p38 MAPK. HEK293 cells
10
Journal of Cell Science 125 (0)
Journal of Cell Science
were co-transfected with vectors coding for wild-type Myc–RBPJk, Myc–RBP-Jk(T339A) mutant and HA–ubiquitin, and were
then subjected to ubiquitylation analysis. The results of
immunoblot analysis of the anti-HA immunoprecipitates from
the transfected cells with an anti-Myc antibody demonstrated
that p38 MAPK facilitated the ubiquitylation of RBP-Jk and that
the ubiquitylation of RBP-Jk(T339A) prevented this (Fig. 6G).
We attempted to characterize the involvement of T339
phosphorylation in the degradation of RBP-Jk. PS1-knockout
cells were transfected with vectors coding for Myc–RBP-Jk
and Myc–RBP-Jk(T339A) and were then subjected to
immunoblotting with antibodies against phosphorylated Ser or
Thr. RBP-Jk protein levels and phosphorylation of RBP-Jk were
increased by the addition of proteasome inhibitor in a dosedependent manner (Fig. 6H). Furthermore, our findings revealed
that lysosomal inhibitor also increased the steady-state levels of
RBP-Jk proteins and phosphorylated RBP-Jk proteins (Fig. 6I).
However, we did not see phosphorylation of the RBP-Jk(T339A)
mutant. These results demonstrate that the phosphorylation of
RBP-Jk by p38 MAPK is crucial to its ability to degrade RBP-Jk.
RBP-Jk is a direct substrate of p38 MAPK, and that the
phosphorylation of RBP-Jk by p38 MAPK is one of the key
factors in the regulation of RBP-Jk protein stability.
Discussion
RBP-Jk functions as a transcription repressor when it is not
associated with Notch1-IC. When associated with the Notch1-IC
protein, RBP-Jk functions as a transcription activator complex
that activates the transcription of Notch1 target genes, including
Enhancer of split [E(spl)] complex genes and the mammalian
homologues of the Hairy and E(spl) genes, HES1 and HES5
(Jennings et al., 1994; Abu-Issa and Cavicchi, 1996; de Celis
et al., 1996; Ligoxygakis et al., 1998; Ohtsuka et al., 1999; Jouve
et al., 2000). The results of one of our recent studies suggested
that the proteasomal and lysosomal degradation of RBP-Jk proteins
could occur in intact cells and plays a crucial role in Notch1
signaling (Kim et al., 2011). However, the precise mechanism by
which RBP-Jk protein degradation occurs remains to be clearly
defined. In this study, we demonstrate that RBP-Jk degradation
involves both the proteasomal and lysosomal pathways. PS2
modulates the protein stability of RBP-Jk through the p38 MAPK
signaling pathway and the phosphorylation of RBP-Jk by p38
MAPK induces RBP-Jk protein degradation and ubiquitylation.
Protein degradation is generally mediated through two major
systems: the proteasome and the lysosome. These different
proteolysis pathways can be differentiated by their sensitivity
to proteasome- or lysosome-specific inhibitors. Proteasomal
proteolysis can be inhibited by ALLN, MG132 and lactacystin.
MG132 can reversibly block all activity of the 26S proteasome
(Rock et al., 1994). ALLN inhibits neutral cysteine proteases and
the proteasome (Sasaki et al., 1990; Debiasi et al., 1999).
Lactacystin is a microbial metabolite isolated from Streptomyces,
which is now used broadly as a selective inhibitor of the 20S
proteasome (Lee and Goldberg, 1998). We determined that
lactacystin, as well as other peptide-based reversible proteasome
inhibitors, including MG132 and ALLN, markedly reduced
exogenous RBP-Jk degradation in RBP-Jk ectopically
expressed cells, and endogenous RBP-Jk protein in HEK293
and C2C12 cells. Our data demonstrate that RBP-Jk is
ubiquitylated, and also clearly show that blockage of the
ubiquitin–proteasome pathway inhibits RBP-Jk degradation.
Degradation of proteins by the lysosome can be inhibited by
NH4Cl and chloroquine. NH4Cl is a very effective inhibitor of
lysosomal function and inhibits the function of lysosomal
proteases by an induced increase in intralysosomal pH
(Ohkuma and Poole, 1978; Dean et al., 1984). Chloroquine is
another lysosomotrophic agent, and is commonly used to inhibit
lysosome function (Welman and Peters, 1977). Chloroquine is a
weak base, which can disrupt lysosomal function by blocking
acidification. Our data also show that RBP-Jk degradation is
mediated by the lysosome. Lysosome inhibition significantly
slows down turnover of RBP-Jk. Our data strongly indicate that
these two proteolytic pathways are involved in RBP-Jk
degradation.
Recent studies have emphasized the role of ubiquitylation in
the regulation of Notch signaling (Lai, 2002). Five known E3
ligases regulate Notch itself, Notch ligands, or known Notch
antagonists. LNX is a positive regulator of Notch signaling that is
responsible for the degradation of Numb, a membrane-associated
protein that inhibits the function of the Notch receptor (Nie et al.,
2002). Neuralized (neur) and Mind bomb (mib) promote the
monoubiquitylation and endocytosis of Delta (Deblandre et al.,
2001; Lai et al., 2001; Pavlopoulos et al., 2001; Itoh et al., 2003).
Itch associates with the intracellular domain of Notch through its
WW domains and promotes the ubiquitylation of Notch1-IC
through its HECT domain (Qiu et al., 2000). Finally, Notch1-IC
is also degraded in the nucleus by the ubiquitin–proteasome
system with Fbw7, an E3 ligase for the ubiquitylation of Notch1IC (Oberg et al., 2001; Wu et al., 2001; Lai, 2002; Minella and
Clurman, 2005; Mo et al., 2007). Ubiquitylation and degradation
might actually act on all Notch1 signaling components, rather
than separately on its individual components.
The transcriptional activation of downstream target genes by
Notch1-IC depends on the association of Notch1-IC with RBP-Jk
within the nucleus. To determine whether Notch1-IC-deficient
conditions also modulate RBP-Jk protein levels through
proteasomal and lysosomal pathways, we constructed PS1 and
PS2 double- and single-knockout cells. Our results show that
disruption of the PS2 gene significantly reduced RBP-Jk protein
turnover. However, knockout of the PS1 gene does not affect
RBP-Jk protein turnover, thereby indicating that RBP-Jk protein
degradation is both PS2 dependent and PS1 independent. The
results of a previous report demonstrated that PS2 functions as a
signaling molecule upstream of the p38 MAPK pathway (Sun
et al., 2001). To determine whether p38 MAPK modulates RBPJk protein levels, we treated PS1-knockout cells with a specific
p38 MAPK inhibitor. Collectively, our findings show that the
stability of the RBP-Jk protein was downregulated by p38 MAPK
by proteasome- and lysosome-dependent pathways. p38 MAPK
preferentially phosphorylates serine and threonine residues
that lie within P-x-S/T-P or S/T-P motifs (Kobayashi et al.,
1999). RBP-Jk harbors one conserved consensus site for
phosphorylation by p38 MAPK and is phosphorylated by p38
MAPK both in vitro and in vivo. The transcriptional activation of
Notch1 signaling plays a crucial role in the regulation of diverse
cell fate determinations. RBP-Jk protein performs a central role
in Notch1 signaling for the induction of Notch1 target genes.
Therefore, the protein turnover of RBP-Jk is critically relevant
to the regulation of Notch1 signaling. Taking this into
consideration, the mechanism underlying the downregulation of
RBP-Jk proteins by p38 MAPK holds promise in controlling
developmental programs or reducing the functions of Notch1
Journal of Cell Science
Degradation of RBP-Jk by p38 MAPK
11
Fig. 7. Schematic diagram of phosphorylation-dependent degradation of RBP-Jk by p38 MAPK. The cellular expression of PS2 shows increased activation
of p38 MAPK by an as-yet-unknown mechanism. p38 MAPK facilitates RBP-Jk phosphorylation and results in a reduction in the RBP-Jk protein level. RBP-Jk is
capable of forming a complex with p38 MAPK; p38 MAPK enhances the protein degradation of RBP-Jk by proteasomal and lysosomal pathways. p38 MAPK
downregulates the protein levels of phosphorylated RBP-Jk.
proteins (Fig. 7). To achieve this, the precise mechanisms
underlying the functions of p38 MAPK and other kinase(s) in
the regulation of RBP-Jk protein turnover should be investigated
intensively in the future.
In summary, our results show that RBP-Jk degradation
involves both the proteasome and the lysosome pathways. We
also demonstrate that PS2 modulates the protein stability of RBPJk through the p38 MAPK signaling pathway. Furthermore, we
show that phosphorylation of RBP-Jk by p38 MAPK induces
RBP-Jk protein degradation and ubiquitylation. Henceforth, the
findings of this study might begin to shed some light onto what
might be a signal crosstalk mechanism of RBP-Jk and p38
MAPK, or might point to the existence of phosphorylationdependent regulation of RBP-Jk.
Antibodies
For immunoprecipitation and immunoblotting, antibodies against HA (12CA5
1:1000), Myc (9E10, 1:1000) and FLAG (Sigma, 1:1000) were used. RBP-Jk was
detected with the RBP-Jk (H-50) rabbit polyclonal antibody (Santa Cruz, 1:1000),
phosphorylated RBP-Jk with antibodies against phosphorylated Ser and Thr
(Upstate, 1:1000). For detection of b-actin (C-4), the b-actin mouse monoclonal
antibody was used (Santa Cruz, 1:5000). Phosphorylated p38 was detected with the
antibody against phosphorylated Ser and Thr (Upstate, 1:1000).
Pharmacological treatment
Cells were treated with the proteasomal inhibitors ALLN (25 mM), MG132 (5 mM)
or Lactacystin (10 mM) or the lysosomal inhibitors Chloroquine (100 mM) or
NH4Cl (50 mM). MG132 was used at 0, 2.5, 5 and 10 mM for 4 hours for the
dosage assay, and at 10 mM for 0, 4 and 6 hours for the time-course assay of the
proteasomal inhibitor. NH4Cl was used at 0, 10, 20 and 50 mM for 10 hours for
the dosage, and at 50 mM for the 0, 6, 8 and 10 hours for the time-course assay of
the lysosomal inhibitor. Cycloheximide (CHX) was used at 200 mM at 0, 2, 4, 6
and 8 hours for the time-course assay of the translational inhibitor (Mo et al.,
2011).
Materials and Methods
Cell culture and transfection
HEK293 (Human embryonic kidney 293) cells, C2C12 (Mouse myoblast), PS1/2+/+,
PS1/2–/–, PS1–/– and PS2–/– MEF 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. For transient transfection, cells were grown to ,80%
confluence. The cultured cells were transiently transfected by the calcium
phosphate method or with Lipofectamine (Invitrogen) (Kim et al., 2007).
GST pull-down assay
The recombinant GST–RBP-Jk protein was expressed in E. coli BL21 strain using
the pGEX system. The GST fusion protein was then purified with GSH–agarose
beads (Sigma) in accordance with the manufacturer’s instructions. An equal
amount of GST or GST–RBP-Jk fusion protein was incubated with the lysates of
the HEK293 cells, which had been transfected for 4 hours with combinations of
expression vectors at 4 ˚C, with rotation. After incubation, the beads were washed
three times in ice-cold phosphate-buffered saline (PBS, pH 7.4) and boiled with
12
Journal of Cell Science 125 (0)
20 ml of Laemmli sample buffer. The precipitates were then separated by SDSPAGE, and the pull-down proteins were detected by immunoblotting with specific
antibodies (Mo et al., 2007).
Coimmunoprecipitation and western blotting
The cells were transfected with pCS4 Myc–RBP-Jk or the ubiquitin plasmid. Cells
were lysed 48 hours post transfection in 1 ml of RIPA buffer for 30 minutes
at 4 ˚C. After 20 minutes of centrifugation at 12,000 g, the supernatants
were subjected to immunoprecipitation with appropriate antibodies coupled to
Protein-A–agarose beads. The resultant immunoprecipitates were washed three
times in phosphate-buffered saline (PBS, pH 7.4). The samples were then treated
with 56 protein sample buffer and boiled before immunoblotting. Western
blotting was then conducted with the indicated antibodies (Mo et al., 2008).
Site-directed mutagenesis
The site-directed mutagenesis of cDNA encoding RBP-Jk was conducted using
a QuikChange kit (Stratagene), and the mutagenic primer was T339A (59CCTTGCCCCAGTCgCTCCTGTGCCTGT-39) (mismatch with the RBP-Jk cDNA
template is indicated by lowercase letter). The mutations were confirmed by
automatic DNA sequencing (Kim et al., 2008).
Journal of Cell Science
Immunocomplex kinase assay
The cultured cells were harvested and lysed in buffer A containing 50 mM TrisHCl (pH 7.5), 150 mM NaCl, 1 mM PMSF, 2 mg/ml of leupeptin, 2 mg/ml of
aprotinin, 25 mM glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM
sodium fluoride, 1% NP-40, 0.5% deoxycholate and 0.1% SDS for 30 minutes
at 4 ˚C. The cell lysates were then subjected to 20 minutes of centrifugation at
12,000 g at 4 ˚C. The soluble fraction was incubated for 3 hours with appropriate
antibodies against the indicated protein kinase at 4 ˚C. The immunocomplexes were
then coupled to Protein-A–agarose for 1 hour at 4 ˚C, after which they were
pelleted by centrifugation. The immunopellets were rinsed three times in lysis
buffer and then twice with 20 mM HEPES (pH 7.4). The immunocomplex kinase
assays were conducted by incubation of the immunopellets for 30 minutes at 30 ˚C
with 2 mg of substrate protein in 20 ml of reaction buffer containing 0.2 mM
sodium orthovanadate, 10 mM MgCl2, 2 mCi [32P]ATP and 20 mM HEPES
(pH 7.4). The phosphorylated substrates were separated by SDS-PAGE and
quantified with a Fuji BAS 2500 PhosphoImager (Mo et al., 2008). The GST
fusion proteins to be used as substrates were expressed in E. coli using pGEX-4T
(Pharmacia) and purified with GSH–Sepharose, as previously described. The
protein concentrations were determined using the Bradford method (Shimadzu).
Acknowledgements
We would like to thank T. Honjo (Kyoto University, Japan) for the
RBP-Jk-related constructs, Jiahuai Han (The Scripps Research
Institute, La Jolla, CA) for providing the p38 MAPK constructs
and Jin Tae Hong (Chungbuk National University, South Korea) for
providing PS1 and PS2 constructs.
Funding
This work was supported by a Mid-career Researcher Program
through NRF funded by the MEST [grant number 2010-0014122].
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