Presenilin-2 regulates the degradation of RBP-Jkprotein 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 (proteome@jnu.ac.kr)

Accepted 18 October 2011Journal of Cell Science 125, 1296–1308� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.095984

SummaryTranscriptional 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-Jkremains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cellstreated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout

cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found thatPS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase d (MAPK) had significantly increased levelsof 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 throughphosphorylation by p38 MAPK.

Key words: RBP-Jk, Presenilin 2, p38 MAPK, Proteasome, Lysosome, Protein degradation

IntroductionNotch 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 RBP-

Jk [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. Notch-

IC 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

1296 Research Article

Journ

alof

Cell

Scie

nce

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 precursorprotein intracellular domain (AICD), might be involved in the

preferential degradation of RBP-Jk by the lysosomal pathway.Despite this observation, the precise mechanisms underlying thedegradation of the RBP-Jk protein remain to be accuratelydelineated.

Therefore, we evaluated the degrdation of RBP-Jk usingproteasome and lysosome inhibitors. In this study, we determined

that RBP-Jk degradation involves both the proteasome and thelysosome. We also demonstrated that PS2 modulates the proteinstability 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-Jkby p38 MAPK induces RBP-Jk protein degradation andubiquitylation. Collectively, our findings indicate that p38

MAPK functions as a regulator of RBP-Jk protein stability byRBP-Jk phosphorylation.

ResultsProteasome inhibition increases exogenous andendogenous RBP-Jk protein levels

Despite the many studies of RBP-Jk thus far conducted, themechanism underlying the degradation of the protein remains to

be completely understood. To determine whether the RBP-Jkprotein is degraded by the proteasome, proteasome inhibitorswere used to treat Myc–RBP-Jk-transfected HEK293 cells. These

cells were then treated with ALLN (25 mM), MG132 (5 mM) andlactacystin (10 mM) for 4 hours (Fig. 1A). Myc–RBP-Jk proteinwas detected with the anti-Myc antibody 9E10. As shown inFig. 1A, similarly to lactacystin treatment, ALLN and MG132

significantly increased RBP-Jk levels relative to those observedin control cells treated with vehicle solution only. Protein levelswere 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 proteasomeinhibitors also induces an increase in endogenous RBP-Jkproteins, 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 wasdetected with rabbit anti-RBP-Jk antibody. MG132 markedly

increased endogenous RBP-Jk protein levels in HEK293 andC2C12 cells.

The half-life of RBP-Jk was determined using cycloheximide,

a protein translation inhibitor. Cycloheximide interacts with thetranslocase enzyme and blocks protein synthesis in eukaryoticcells. After cycloheximide treatment, the level of RBP-Jk protein

dropped gradually, and approximately half of the protein wasdegraded 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 proteinwas degraded after 8 hours in the presence of MG132(Fig. 1F,G). Taken together, these results clearly demonstrate

that treatment with proteasome inhibitor significantly increasesRBP-Jk protein levels, thereby suggesting that the degradation ofRBP-Jk protein is indeed mediated by the proteasome pathway.

Lysosome inhibition increases exogenous andendogenous RBP-Jk protein levels

In an effort to determine whether RBP-Jk protein islysosomally degraded, lysosome inhibitors were administeredto Myc–RBP-Jk-transfected HEK293 cells. The cells were

treated with chloroquine (100 mM) and NH4Cl (50 mM) for6 hours. Myc–RBP-Jk protein was then detected with the anti-Myc antibody 9E10. Chloroquine and NH4Cl significantly

increased RBP-Jk levels in comparison with levels in thevehicle control cells (Fig. 2A). Protein levels were normalizedto b-actin. The cells were subsequently treated with different

doses of NH4Cl for 10 hours (0–50 mM) or at 50 mM fordifferent periods of time (0–10 hours). NH4Cl treatmentaffected a marked increase in RBP-Jk protein levels in adose-dependent (Fig. 2B) and time-dependent manner

(Fig. 2C). To determine whether treatment with lysosomeinhibitors also induces an increase in endogenous RBP-Jkproteins, 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-Jkprotein 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 weretreated with NH4Cl and cycloheximide. After cycloheximidetreatment, the level of RBP-Jk protein declined gradually, with

approximately half of the protein degraded after 2.5 hoursrelative to the level in control cells (Fig. 2F,G). Upon NH4Cltreatment, the level of RBP-Jk protein changed moderately;

approximately half of the protein was degraded after 6 hours inthe presence of NH4Cl (Fig. 2F,G). Taken together, these resultsclearly demonstrate that treatment with lysosome inhibitor

moderately increases RBP-Jk protein levels, thereby suggestingthat degradation of RBP-Jk is also mediated by the lysosomepathway.

PS2 downregulates the level of endogenous RBP-Jkproteins

To determine the effects of the absence of PS1 and PS2 on RBP-Jk expression, immunoblot analyses were conducted using wild-

type PS1 and PS2 cells and double-knockout cells. The half-lifeof endogenous RBP-Jk was determined using cycloheximide, aprotein translation inhibitor. PS1 and PS2 double-knockout and

wild-type control cells were exposed to cycloheximide, and theamount 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 PS1or PS2 in RBP-Jk protein degradation using PS1-knockout andPS2-knockout cells. The steady-state level and the half-life of

endogenous RBP-Jk were higher in PS2-knockout cells than inPS1-knockout cells (Fig. 3C,D). We next investigated the highlyspecific c-secretase inhibitor DAPT (Dovey et al., 2001) for its

capacity to block RBP-Jk protein degradation and half-life. Withthis objective, wild-type PS1 and PS2 cells were treated with orwithout DAPT along with cycloheximide (Fig. 3E). As shown in

Fig. 3E, endogenous RBP-Jk levels were increased markedly incells treated with DAPT alone and in those with DAPT andcycloheximide. We also attempted to confirm the effect of PS2

on the protein level of RBP-Jk using PS1 and PS2 double-knockout cells. We measured protein levels of RBP-Jk in thepresence of wild-type PS2 or an inactive dominant-negative

Degradation of RBP-Jk by p38 MAPK 1297

Journ

alof

Cell

Scie

nce

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 RBP-

Jk protein is downregulated by PS2.

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 6 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.

Journal of Cell Science 125 (5)1298

Journ

alof

Cell

Scie

nce

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

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 6 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.

Degradation of RBP-Jk by p38 MAPK 1299

Journ

alof

Cell

Scie

nce

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 lysosome-

dependent pathways.

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 6 s.d. from three independent experiments.

Journal of Cell Science 125 (5)1300

Journ

alof

Cell

Scie

nce

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-3binhibitors increases the protein stability of endogenous RBP-Jk

proteins, PS1-knockout cells were treated with the GSK-3binhibitor 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 PS1-

knockout 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

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) PS1-

knockout 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 6 s.d. of three independent experiments.

Degradation of RBP-Jk by p38 MAPK 1301

Journ

alof

Cell

Scie

nce

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

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 6 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.

Journal of Cell Science 125 (5)1302

Journ

alof

Cell

Scie

nce

Fig. 6. See next page for legend.

Degradation of RBP-Jk by p38 MAPK 1303

Journ

alof

Cell

Scie

nce

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–p38-

MAPK, 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

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 byphosphorylation at Thr339

Because our results suggested that RBP-Jk is a target ofp38 MAPK, we subsequently attempted to determine whetherthese two proteins interact physically in intact cells. In in vitro

binding studies, purified GST and GST–RBP-Jk proteins wereimmobilized onto GSH–agarose. HA–p38-MAPK-expressing celllysates were incubated either with GST or GST–RBP-Jkimmobilized 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 occurringbetween RBP-Jk and p38 MAPK. HEK293 cells were co-

transfected with vectors encoding Myc–RBP-Jk and HA–p38-MAPK, and were then subjected to co-immunoprecipitationanalysis (Fig. 6B). Immunoblot analysis using the anti-Myc

antibody of the anti-HA immunoprecipitates from the transfectedcells revealed that p38 MAPK did indeed physically associatewith RBP-Jk in the cells.

We next conducted an in vitro kinase assay with HA–

p38-MAPK and purified GST–RBP-Jk. HA–p38-MAPKimmunocomplexes prepared from HEK293 cells catalyzed thephosphorylation of purified recombinant GST–RBP-Jk (Fig. 6C).

p38 MAPK preferentially phosphorylates substrate protein serineand threonine residues that lie in Pro-x-(Ser/Thr)-Pro or S/T-Pmotifs (Alvarez et al., 1991; Court et al., 2004). The results of in

silico studies have shown that RBP-Jk harbors a possibleconserved threonine residue in vertebrates; this threonineresidue is accessible and is located immediately after the DNAbinding domain. Furthermore, using site-directed mutagenesis,

we determined that the replacement of Thr339 of RBP-Jk withalanine effected a reduction in the in vitro phosphorylation of therecombinant 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 lysateswere subjected to immunoblotting with an antibodies againstphosphorylated Ser and Thr, or the HA tag (Fig. 6D). p38 MAPK

phosphorylated RBP-Jk(WT) but did not phosphorylate RBP-Jk(T339A).

We then attempted to characterize the involvement of

phosphorylation in the physical association between p38MAPK and RBP-Jk by co-immunoprecipitation. HEK293 cellswere 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 MAPKwere co-immunoprecipitated, but when co-transfected with RBP-Jk(T339A), the band of RBP-Jk that interacted with p38 MAPK

disappeared (Fig. 6E). These results demonstrate that thephosphorylation of RBP-Jk by p38 MAPK plays a pivotalrole in its ability to bind with p38 MAPK. Moreover, we

ascertained that RBP-Jk(T339A) is resistant to p38-MAPK-induced degradation, which implies that the p38-MAPK-induced phosphorylation of RBP-Jk is crucially important for

the degradation of the RBP-Jk protein (Fig. 6F). We thenattempted to characterize the involvement of phosphorylation inthe polyubiquitylation of RBP-Jk by p38 MAPK. HEK293 cells

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 HA-

p38 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–RBP-

Jk(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–RBP-

Jk, 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 dosage-

dependent 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.

Journal of Cell Science 125 (5)1304

Journ

alof

Cell

Scie

nce

were co-transfected with vectors coding for wild-type Myc–RBP-Jk, Myc–RBP-Jk(T339A) mutant and HA–ubiquitin, and were

then subjected to ubiquitylation analysis. The results ofimmunoblot analysis of the anti-HA immunoprecipitates fromthe transfected cells with an anti-Myc antibody demonstratedthat 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 T339phosphorylation in the degradation of RBP-Jk. PS1-knockout

cells were transfected with vectors coding for Myc–RBP-Jkand Myc–RBP-Jk(T339A) and were then subjected toimmunoblotting with antibodies against phosphorylated Ser or

Thr. RBP-Jk protein levels and phosphorylation of RBP-Jk wereincreased by the addition of proteasome inhibitor in a dose-dependent manner (Fig. 6H). Furthermore, our findings revealedthat 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 thephosphorylation of RBP-Jk by p38 MAPK is one of the key

factors in the regulation of RBP-Jk protein stability.

DiscussionRBP-Jk functions as a transcription repressor when it is not

associated with Notch1-IC. When associated with the Notch1-ICprotein, RBP-Jk functions as a transcription activator complexthat activates the transcription of Notch1 target genes, including

Enhancer of split [E(spl)] complex genes and the mammalianhomologues 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; Jouveet al., 2000). The results of one of our recent studies suggestedthat the proteasomal and lysosomal degradation of RBP-Jk proteins

could occur in intact cells and plays a crucial role in Notch1signaling (Kim et al., 2011). However, the precise mechanism bywhich RBP-Jk protein degradation occurs remains to be clearlydefined. In this study, we demonstrate that RBP-Jk degradation

involves both the proteasomal and lysosomal pathways. PS2modulates the protein stability of RBP-Jk through the p38 MAPKsignaling pathway and the phosphorylation of RBP-Jk by p38

MAPK induces RBP-Jk protein degradation and ubiquitylation.

Protein degradation is generally mediated through two majorsystems: the proteasome and the lysosome. These different

proteolysis pathways can be differentiated by their sensitivityto proteasome- or lysosome-specific inhibitors. Proteasomalproteolysis 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 andthe 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 20Sproteasome (Lee and Goldberg, 1998). We determined thatlactacystin, as well as other peptide-based reversible proteasome

inhibitors, including MG132 and ALLN, markedly reducedexogenous RBP-Jk degradation in RBP-Jk ectopicallyexpressed cells, and endogenous RBP-Jk protein in HEK293

and C2C12 cells. Our data demonstrate that RBP-Jk isubiquitylated, and also clearly show that blockage of theubiquitin–proteasome pathway inhibits RBP-Jk degradation.

Degradation of proteins by the lysosome can be inhibited byNH4Cl and chloroquine. NH4Cl is a very effective inhibitor of

lysosomal function and inhibits the function of lysosomalproteases by an induced increase in intralysosomal pH(Ohkuma and Poole, 1978; Dean et al., 1984). Chloroquine isanother lysosomotrophic agent, and is commonly used to inhibit

lysosome function (Welman and Peters, 1977). Chloroquine is aweak base, which can disrupt lysosomal function by blockingacidification. Our data also show that RBP-Jk degradation is

mediated by the lysosome. Lysosome inhibition significantlyslows down turnover of RBP-Jk. Our data strongly indicate thatthese two proteolytic pathways are involved in RBP-Jk

degradation.

Recent studies have emphasized the role of ubiquitylation inthe regulation of Notch signaling (Lai, 2002). Five known E3ligases regulate Notch itself, Notch ligands, or known Notch

antagonists. LNX is a positive regulator of Notch signaling that isresponsible for the degradation of Numb, a membrane-associatedprotein that inhibits the function of the Notch receptor (Nie et al.,

2002). Neuralized (neur) and Mind bomb (mib) promote themonoubiquitylation 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 itsWW domains and promotes the ubiquitylation of Notch1-ICthrough its HECT domain (Qiu et al., 2000). Finally, Notch1-ICis also degraded in the nucleus by the ubiquitin–proteasome

system with Fbw7, an E3 ligase for the ubiquitylation of Notch1-IC (Oberg et al., 2001; Wu et al., 2001; Lai, 2002; Minella andClurman, 2005; Mo et al., 2007). Ubiquitylation and degradation

might actually act on all Notch1 signaling components, ratherthan separately on its individual components.

The transcriptional activation of downstream target genes by

Notch1-IC depends on the association of Notch1-IC with RBP-Jkwithin the nucleus. To determine whether Notch1-IC-deficientconditions also modulate RBP-Jk protein levels through

proteasomal and lysosomal pathways, we constructed PS1 andPS2 double- and single-knockout cells. Our results show thatdisruption of the PS2 gene significantly reduced RBP-Jk proteinturnover. However, knockout of the PS1 gene does not affect

RBP-Jk protein turnover, thereby indicating that RBP-Jk proteindegradation is both PS2 dependent and PS1 independent. Theresults of a previous report demonstrated that PS2 functions as a

signaling molecule upstream of the p38 MAPK pathway (Sunet al., 2001). To determine whether p38 MAPK modulates RBP-Jk protein levels, we treated PS1-knockout cells with a specific

p38 MAPK inhibitor. Collectively, our findings show that thestability of the RBP-Jk protein was downregulated by p38 MAPKby proteasome- and lysosome-dependent pathways. p38 MAPKpreferentially 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 forphosphorylation by p38 MAPK and is phosphorylated by p38

MAPK both in vitro and in vivo. The transcriptional activation ofNotch1 signaling plays a crucial role in the regulation of diversecell 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 relevantto the regulation of Notch1 signaling. Taking this into

consideration, the mechanism underlying the downregulation ofRBP-Jk proteins by p38 MAPK holds promise in controllingdevelopmental programs or reducing the functions of Notch1

Degradation of RBP-Jk by p38 MAPK 1305

Journ

alof

Cell

Scie

nce

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 RBP-

Jk 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 phosphorylation-

dependent regulation of RBP-Jk.

Materials and MethodsCell 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 C̊ in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and

1% penicillin-streptomycin, in a humidified incubator with an atmospherecontaining 5% CO2. For transient transfection, cells were grown to ,80%

confluence. The cultured cells were transiently transfected by the calciumphosphate method or with Lipofectamine (Invitrogen) (Kim et al., 2007).

Antibodies

For immunoprecipitation and immunoblotting, antibodies against HA (12CA51:1000), Myc (9E10, 1:1000) and FLAG (Sigma, 1:1000) were used. RBP-Jk wasdetected 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 monoclonalantibody was used (Santa Cruz, 1:5000). Phosphorylated p38 was detected with theantibody 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) orNH4Cl (50 mM). MG132 was used at 0, 2.5, 5 and 10 mM for 4 hours for thedosage assay, and at 10 mM for 0, 4 and 6 hours for the time-course assay of theproteasomal inhibitor. NH4Cl was used at 0, 10, 20 and 50 mM for 10 hours forthe dosage, and at 50 mM for the 0, 6, 8 and 10 hours for the time-course assay ofthe lysosomal inhibitor. Cycloheximide (CHX) was used at 200 mM at 0, 2, 4, 6and 8 hours for the time-course assay of the translational inhibitor (Mo et al.,2011).

GST pull-down assay

The recombinant GST–RBP-Jk protein was expressed in E. coli BL21 strain usingthe pGEX system. The GST fusion protein was then purified with GSH–agarosebeads (Sigma) in accordance with the manufacturer’s instructions. An equalamount of GST or GST–RBP-Jk fusion protein was incubated with the lysates ofthe HEK293 cells, which had been transfected for 4 hours with combinations ofexpression vectors at 4 C̊, with rotation. After incubation, the beads were washedthree times in ice-cold phosphate-buffered saline (PBS, pH 7.4) and boiled with

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.

Journal of Cell Science 125 (5)1306

Journ

alof

Cell

Scie

nce

20 ml of Laemmli sample buffer. The precipitates were then separated by SDS-PAGE, and the pull-down proteins were detected by immunoblotting with specificantibodies (Mo et al., 2007).

Coimmunoprecipitation and western blotting

The cells were transfected with pCS4 Myc–RBP-Jk or the ubiquitin plasmid. Cellswere lysed 48 hours post transfection in 1 ml of RIPA buffer for 30 minutesat 4 C̊. After 20 minutes of centrifugation at 12,000 g, the supernatantswere subjected to immunoprecipitation with appropriate antibodies coupled toProtein-A–agarose beads. The resultant immunoprecipitates were washed threetimes in phosphate-buffered saline (PBS, pH 7.4). The samples were then treatedwith 56 protein sample buffer and boiled before immunoblotting. Westernblotting 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 usinga QuikChange kit (Stratagene), and the mutagenic primer was T339A (59-CCTTGCCCCAGTCgCTCCTGTGCCTGT-39) (mismatch with the RBP-Jk cDNAtemplate is indicated by lowercase letter). The mutations were confirmed byautomatic DNA sequencing (Kim et al., 2008).

Immunocomplex kinase assay

The cultured cells were harvested and lysed in buffer A containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM PMSF, 2 mg/ml of leupeptin, 2 mg/ml ofaprotinin, 25 mM glycerophosphate, 0.1 mM sodium orthovanadate, 1 mMsodium fluoride, 1% NP-40, 0.5% deoxycholate and 0.1% SDS for 30 minutesat 4 C̊. The cell lysates were then subjected to 20 minutes of centrifugation at12,000 g at 4 C̊. The soluble fraction was incubated for 3 hours with appropriateantibodies against the indicated protein kinase at 4 C̊. The immunocomplexes werethen coupled to Protein-A–agarose for 1 hour at 4 C̊, after which they werepelleted by centrifugation. The immunopellets were rinsed three times in lysisbuffer and then twice with 20 mM HEPES (pH 7.4). The immunocomplex kinaseassays were conducted by incubation of the immunopellets for 30 minutes at 30 C̊with 2 mg of substrate protein in 20 ml of reaction buffer containing 0.2 mMsodium orthovanadate, 10 mM MgCl2, 2 mCi [32P]ATP and 20 mM HEPES(pH 7.4). The phosphorylated substrates were separated by SDS-PAGE andquantified with a Fuji BAS 2500 PhosphoImager (Mo et al., 2008). The GSTfusion proteins to be used as substrates were expressed in E. coli using pGEX-4T(Pharmacia) and purified with GSH–Sepharose, as previously described. Theprotein concentrations were determined using the Bradford method (Shimadzu).

AcknowledgementsWe would like to thank T. Honjo (Kyoto University, Japan) for theRBP-Jk-related constructs, Jiahuai Han (The Scripps ResearchInstitute, La Jolla, CA) for providing the p38 MAPK constructsand Jin Tae Hong (Chungbuk National University, South Korea) forproviding PS1 and PS2 constructs.

FundingThis work was supported by a Mid-career Researcher Programthrough NRF funded by the MEST [grant number 2010-0014122].

ReferencesAbu-Issa, R. and Cavicchi, S. (1996). Genetic interactions among vestigial, hairy, and

Notch suggest a role of vestigial in the differentiation of epidermal and neural cells of

the wing and halter of Drosophila melanogaster. J. Neurogenet. 10, 239-246.

Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C.,

Curran, T. and Davis, R. J. (1991). Pro-Leu-Ser/Thr-Pro is a consensus primary

sequence for substrate protein phosphorylation. Characterization of the phosphorylation

of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669

protein kinase. J. Biol. Chem. 266, 15277-15285.

Artavanis-Tsakonas, S. and Simpson, P. (1991). Choosing a cell fate: a view from the

Notch locus. Trends Genet. 7, 403-408.

Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell

fate control and signal integration in development. Science 284, 770-776.

Court, N. W., Kuo, I., Quigley, O. and Bogoyevitch, M. A. (2004). Phosphorylation of

the mitochondrial protein Sab by stress-activated protein kinase 3. Biochem. Biophys.

Res. Commun. 319, 130-137.

de Celis, J. F., de Celis, J., Ligoxygakis, P., Preiss, A., Delidakis, C. and Bray, S.

(1996). Functional relationships between Notch, Su(H) and the bHLH genes of the

E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during

imaginal development. Development 122, 2719-2728.

Dean, R. T., Jessup, W. and Roberts, C. R. (1984). Effects of exogenous amines on

mammalian cells, with particular reference to membrane flow. Biochem. J. 217, 27-

40.

Debiasi, R. L., Squier, M. K., Pike, B., Wynes, M., Dermody, T. S., Cohen, J. J. and

Tyler, K. L. (1999). Reovirus-induced apoptosis is preceded by increased cellular

calpain activity and is blocked by calpain inhibitors. J. Virol. 73, 695-701.

Deblandre, G. A., Lai, E. C. and Kintner, C. (2001). Xenopus neuralized is a ubiquitinligase that interacts with XDelta1 and regulates Notch signaling. Dev. Cell 1, 795-

806.

Dovey, H. F., John, V., Anderson, J. P., Chen, L. Z., de Saint Andrieu, P., Fang, L. Y.,

Freedman, S. B., Folmer, B., Goldbach, E., Holsztynska, E. J. et al. (2001).

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J.

Neurochem. 76, 173-181.

Duffy, J. B. and Perrimon, N. (1996). Recent advances in understanding signaltransduction pathways in worms and flies. Curr. Opin. Cell Biol. 8, 231-238.

Edbauer, D., Winkler, E., Regula, J. T., Pesold, B., Steiner, H. and Haass, C. (2003).

Reconstitution of gamma-secretase activity. Nat. Cell Biol. 5, 486-488.

Gupta-Rossi, N., Le Bail, O., Gonen, H., Brou, C., Logeat, F., Six, E., Ciechanover,

A. and Israel, A. (2001). Functional interaction between SEL-10, an F-box protein,

and the nuclear form of activated Notch1 receptor. J. Biol. Chem. 276, 34371-34378.

Herreman, A., Hartmann, D., Annaert, W., Saftig, P., Craessaerts, K., Serneels, L.,

Umans, L., Schrijvers, V., Checler, F., Vanderstichele, H. et al. (1999). Presenilin

2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursorprotein processing but enhances the embryonic lethal phenotype of presenilin 1deficiency. Proc. Natl. Acad. Sci. USA 96, 11872-11877.

Hubbard, E. J., Wu, G., Kitajewski, J. and Greenwald, I. (1997). sel-10, a negativeregulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4family of proteins. Genes Dev. 11, 3182-3193.

Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust, D., Yeo, S. Y.,

Lorick, K., Wright, G. J., Ariza-McNaughton, L. et al. (2003). Mind bomb is aubiquitin ligase that is essential for efficient activation of Notch signaling by Delta.Dev. Cell 4, 67-82.

Jennings, B., Preiss, A., Delidakis, C. and Bray, S. (1994). The Notch signallingpathway is required for Enhancer of split bHLH protein expression duringneurogenesis in the Drosophila embryo. Development 120, 3537-3548.

Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler, A., Ish-Horowicz, D.

and Pourquie, O. (2000). Notch signalling is required for cyclic expression of thehairy-like gene HES1 in the presomitic mesoderm. Development 127, 1421-1429.

Kao, H. Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner,

C. R., Evans, R. M. and Kadesch, T. (1998). A histone deacetylase corepressorcomplex regulates the Notch signal transduction pathway. Genes Dev. 12, 2269-2277.

Kim, M. Y., Ann, E. J., Kim, J. Y., Mo, J. S., Park, J. H., Kim, S. Y., Seo, M. S. and

Park, H. S. (2007). Tip60 histone acetyltransferase acts as a negative regulator ofNotch1 signaling by means of acetylation. Mol. Cell. Biol. 27, 6506-6519.

Kim, M. Y., Park, J. H., Mo, J. S., Ann, E. J., Han, S. O., Baek, S. H., Kim, K. J., Im,

S. Y., Park, J. W., Choi, E. J. et al. (2008). Downregulation by lipopolysaccharide

of Notch signaling, via nitric oxide. J. Cell Sci. 121, 1466-1476.

Kim, M. Y., Mo, J. S., Ann, E. J., Yoon, J. H., Jung, J., Choi, Y. H., Kim, S. M.,

Kim, H. Y., Ahn, J. S., Kim, H. et al. (2011). Regulation of Notch1 signaling by the

APP intracellular domain facilitates degradation of the Notch1 intracellular domainand RBP-Jk. J. Cell Sci. 124, 1831-1843.

Kobayashi, T., Deak, M., Morrice, N. and Cohen, P. (1999). Characterization of the

structure and regulation of two novel isoforms of serum- and glucocorticoid-inducedprotein kinase. Biochem. J. 344, 189-197.

Koo, E. H. and Kopan, R. (2004). Potential role of presenilin-regulated signaling

pathways in sporadic neurodegeneration. Nat. Med. 10 Suppl. S26-S33.

Kopan, R. and Ilagan, M. X. (2009). The canonical Notch signaling pathway:unfolding the activation mechanism. Cell 137, 216-233.

Lai, E. C. (2002). Protein degradation: four E3s for the notch pathway. Curr. Biol. 12,R74-R78.

Lai, E. C. (2004). Notch signaling: control of cell communication and cell fate.Development 131, 965-973.

Lai, E. C., Deblandre, G. A., Kintner, C. and Rubin, G. M. (2001). Drosophilaneuralized is a ubiquitin ligase that promotes the internalization and degradation ofdelta. Dev. Cell 1, 783-794.

Lee, D. H. and Goldberg, A. L. (1998). Proteasome inhibitors: valuable new tools forcell biologists. Trends Cell Biol. 8, 397-403.

Ligoxygakis, P., Yu, S. Y., Delidakis, C. and Baker, N. E. (1998). A subset of notchfunctions during Drosophila eye development require Su(H) and the E(spl) genecomplex. Development 125, 2893-2900.

Minella, A. C. and Clurman, B. E. (2005). Mechanisms of tumor suppression by theSCF(Fbw7). Cell Cycle 4, 1356-1359.

Mo, J. S., Kim, M. Y., Han, S. O., Kim, I. S., Ann, E. J., Lee, K. S., Seo, M. S., Kim,

J. Y., Lee, S. C., Park, J. W. et al. (2007). Integrin-linked kinase controls Notch1signaling by down-regulation of protein stability through Fbw7 ubiquitin ligase. Mol.

Cell. Biol. 27, 5565-5574.

Mo, J. S., Kim, M. Y., Ann, E. J., Hong, J. A. and Park, H. S. (2008). DJ-1 modulatesUV-induced oxidative stress signaling through the suppression of MEKK1 and celldeath. Cell Death Differ. 15, 1030-1041.

Mo, J. S., Ann, E. J., Yoon, J. H., Jung, J., Choi, Y. H., Kim, H. Y., Ahn, J. S., Kim,

S. M., Kim, M. Y., Hong, J. A. et al. (2011). Serum- and glucocorticoid-induciblekinase 1 (SGK1) controls Notch1 signaling by downregulation of protein stabilitythrough Fbw7 ubiquitin ligase. J. Cell Sci. 124, 100-112.

Neumann, C. J. (2001). Pattern formation in the zebrafish retina. Semin. Cell. Dev. Biol.

12, 485-490.

Degradation of RBP-Jk by p38 MAPK 1307

Journ

alof

Cell

Scie

nce

Nie, J., McGill, M. A., Dermer, M., Dho, S. E., Wolting, C. D. and McGlade, C. J.(2002). LNX functions as a RING type E3 ubiquitin ligase that targets the cell fatedeterminant Numb for ubiquitin-dependent degradation. EMBO J. 21, 93-102.

O9Neil, J., Grim, J., Strack, P., Rao, S., Tibbitts, D., Winter, C., Hardwick, J.,

Welcker, M., Meijerink, J. P., Pieters, R. et al. (2007). FBW7 mutations inleukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors. J. Exp. Med. 204, 1813-1824.

Oberg, C., Li, J., Pauley, A., Wolf, E., Gurney, M. and Lendahl, U. (2001). TheNotch intracellular domain is ubiquitinated and negatively regulated by themammalian Sel-10 homolog. J. Biol. Chem. 276, 35847-35853.

Ohkuma, S. and Poole, B. (1978). Fluorescence probe measurement of theintralysosomal pH in living cells and the perturbation of pH by various agents.Proc. Natl. Acad. Sci. USA 75, 3327-3331.

Ohtsuka, T., Ishibashi, M., Gradwohl, G., Nakanishi, S., Guillemot, F. and

Kageyama, R. (1999). Hes1 and Hes5 as notch effectors in mammalian neuronaldifferentiation. EMBO J. 18, 2196-2207.

Palacino, J. J., Berechid, B. E., Alexander, P., Eckman, C., Younkin, S., Nye, J. S.

and Wolozin, B. (2000). Regulation of amyloid precursor protein processing bypresenilin 1 (PS1) and PS2 in PS1 knockout cells. J. Biol. Chem. 275, 215-222.

Pavlopoulos, E., Pitsouli, C., Klueg, K. M., Muskavitch, M. A., Moschonas, N. K.and Delidakis, C. (2001). neuralized Encodes a peripheral membrane proteininvolved in delta signaling and endocytosis. Dev. Cell 1, 807-816.

Qiu, L., Joazeiro, C., Fang, N., Wang, H. Y., Elly, C., Altman, Y., Fang, D., Hunter,T. and Liu, Y. C. (2000). Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase. J. Biol. Chem. 275, 35734-35737.

Robey, E. (1999). Regulation of T cell fate by Notch. Annu. Rev. Immunol. 17, 283-295.

Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and

Goldberg, A. L. (1994). Inhibitors of the proteasome block the degradation of most

cell proteins and the generation of peptides presented on MHC class I molecules. Cell

78, 761-771.

Sasaki, T., Kishi, M., Saito, M., Tanaka, T., Higuchi, N., Kominami, E., Katunuma,

N. and Murachi, T. (1990). Inhibitory effect of di- and tripeptidyl aldehydes on

calpains and cathepsins. J. Enzyme Inhib. 3, 195-201.

Shen, J., Bronson, R. T., Chen, D. F., Xia, W., Selkoe, D. J. and Tonegawa, S. (1997).

Skeletal and CNS defects in Presenilin-1-deficient mice. Cell 89, 629-639.

Sun, J., Li, M., Han, J. and Gu, J. (2001). Sensitization of differentiated PC12 cells to

apoptosis by presenilin-2 is mediated by p38. Biochem. Biophys. Res. Commun. 287,

536-541.

Wang, M. and Sternberg, P. W. (2001). Pattern formation during C. elegans vulval

induction. Curr. Top. Dev. Biol. 51, 189-220.

Weinmaster, G. (1997). The ins and outs of notch signaling. Mol. Cell. Neurosci. 9, 91-

102.

Weinmaster, G. (1998). Notch signaling: direct or what? Curr. Opin. Genet. Dev. 8,

436-442.

Welman, E. and Peters, T. J. (1977). Prevention of lysosome disruption in anoxic

myocardium by chloroquine and methyl prednisolone. Pharmacol. Res. Commun. 9, 29-38.

Whitford, K. L., Dijkhuizen, P., Polleux, F. and Ghosh, A. (2002). Molecular control

of cortical dendrite development. Annu. Rev. Neurosci. 25, 127-149.

Wu, G., Lyapina, S., Das, I., Li, J., Gurney, M., Pauley, A., Chui, I., Deshaies, R. J.

and Kitajewski, J. (2001). SEL-10 is an inhibitor of notch signaling that targets notch

for ubiquitin-mediated protein degradation. Mol. Cell. Biol. 21, 7403-7415.

Journal of Cell Science 125 (5)1308

Journ

alof

Cell

Scie

nce