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NFB Activation Is Associated with Its O-GlcNAcylation State under Hyperglycemic Conditions Author(s): Won Ho Yang, Sang Yoon

Park, Hyung Wook Nam, Do Hyun Kim, Jeong Gu Kang, Eun Seok Kang, Yu Sam Kim, Hyun Chul Lee, Kwan Soo Kim and Jin Won Cho Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 45 (Nov. 11, 2008), pp. 17345-17350 Published by: National Academy of Sciences Stable URL: http://www.jstor.org/stable/25465280 . Accessed: 12/11/2013 21:26
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NFkB activation

O-GlcNAcylation hyperglycemic conditions

is associated state under

with

its

Won Ho Yangab, Sang Yoon Parkab, Hyung Wook Nambc, Do Hyun Kimab, Jeong Gu Kangab, Eun Seok Kangd, Won Choabe'1 Yu Sam Kimbce, Hyun Chul Leed, Kwan Soo Kimf, and Jin
Departments of aBiology, biochemistry, and fChemistry and Center for Bioactive Molecular Hybrids, and bProtein Network Research Center, Yonsei of Internal Medicine, Yonsei University College of Medicine, Seoul 120-749, Korea; department University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea; and eProteomeTech, Yonsei Dairy Building, Seodaemun-gu, Edited byWilliam The J. Lennarz, Stony Brook University, Stony Brook, NY, and approved September 25, 2008 (received for review June 27, 2008)

acetylation of

and factor NFkB is activated by phosphorylation transcription and plays important roles in inflammatory and immune in the cell. Additionally, modification responses posttranslational the NFkB p65 has been subunit reported, its exact function that (O by O-linked A/-acetylglucosamine but the modification site of O-GlcNAc have not been elucidated. In this NFkB p65 decreases activity under hyper and

mutation

(21) increase NFkB transcriptional activity.Additionally, we Thr-322 and Thr-352 as O-GlcNAcylation sites in a identify
study and with mass spectrometry analysis. Our data

GlcNAc) on NFkB p65 and show work, we binding glycemic cation

to IkBc* and conditions. on

of O-GlcNAcylation increases transcriptional demonstrate

Thr-352of NFkB p65 can be modified with O-GlcNAc, but modifi


activation. may not Thr-322, is important for transcriptional Thr-352, Our findings suggest that site-specific O-GlcNAcylation be a reason why NFkB activity increases under continuously conditions.

Also, we

that both Thr-322

show that an increased amount of O-GlcNAcylation on NFkB p65 increases the transcriptionalactivityofNFkB. Furthermore, O-GlcNAcylation on Thr-352 inhibits the interaction between NFkB and IkB. This O-GlcNAcylated NFkB is translocated to the nucleus and has a longer half-life in the nucleus than unmodified protein. This finding may partly answer why NFkB is continuously activated in diabetic conditions. Results
OGA Overexpression-Mediated Inhibits Hyperglycemia-lnduced of O-GlcNAcylation Down-Regulation NFkB Activation. We first confirmed

hyperglycemic diabetes

|O-GlcNAc transferase

|O-GlcNAcase

that hyperglycemic conditions induced NFkB activation in rat VSMCs (22). To examine NFKB-mediated gene regulation,we structunder normal glucose (5mM) and high-glucose (25 mM) conditions. When cells were exposed to high glucose for 24 h,
an analyzed the expression of a KB-luciferase reporter gene con

matory, Transcription mammals,

combinations of Rel proteins such as p65 (RelA), RelB, c-Rel, NFkB is composed p50/pl05, and p52/pl00. Inmost cell types, of p65 and p50 and is localized in the cytosol where it binds inhibitor(IkB). Treatment with NFKB-activating agents such as tumor necrosis factor a (TNFa) activates IkB kinase (IKK)
phosphorylation event induces in the N terminus of IkB. a Free uitin-dependent proteolysis. and activates nucleus the expression and acetylation (10, 11) regulate NFkB IkB degradation via translocates of target genes ubiq to the

and immune, is present NFkB

factorNFkB plays important roles in inflam


antiapoptotic as a dimer responses composed (1-3). of various In

was observed

increased

amount

of KB-luciferase

(Fig. L4). At this time point, exposure to levels increased levels of O-GlcNAcylation on high-glucose total protein and NFkB p65 (Fig. IB, 1st and 3rd panels from top); however, O-GlcNAcylation of NFkB p50 was not de
tected

reporter

gene

expression

> cc ? E UJ X u o m

complexes, inducing The phosphorylation

Posttranslational modifications such as phosphorylation (4-9)


which

(1, 2).

cell adhesion molecule 1 (VCAM-1), which is known to be controlled by NFkB, increased under high-glucose conditions (Fig. IB, 5th panel). To determine the role of O-GlcNAcylation inhyperglycemia
induced NFkB

(data

not

shown).

Furthermore,

expression

of vascular

NFkB. The activity of NFkB


osamine biosynthetic pathway,

is influenced also by the hex


produces a substrate

the transcriptional

activity

of of

FLAG-tagged human OGA


overexpression as measured pression reduced by luciferase decreased the

activation,

we

intoVSMCs

transfected

a vector

and found thatOGA


NFkB activation overex in

expressing

hyperglycemia-induced

(9-GlcNAcylation,UDP-GlcNAc (12). Many nucleocytoplasmic proteins are known to be dynamicallymodified with O-GlcNAc. This modification is modulated byO-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (13-18). O-GlcNAcylation levelsplay
an important protein role port,

can be increased under hyperglycemic conditions caused by diabetes (16-18). Although it is known thatO-GlcN Acylation of NFkB is involved in hyperglycemia-induced NFkB activa tion (12) and is required for lymphocyte activation (19), the specific sites and the function of O-GlcN Acylation on NFkB
are not well understood. we In this work, show that OGA overexpression down

stability,

in transcription, nuclear translation, and protein-protein interactions

trans

crease in VCAM-1 expression (Fig. ID, 5th panel). As expected, OGA overexpression decreased levels of O-GlcNAcylation on total proteins and NFkB p65 (Fig. ID, 1stand 3rd panels). Taken
Author contributions: W.H.Y., S.Y.P., and J.W.C. W.H.Y., S.Y.P.,H.W.N., designed research; and E.S.K. performed research; D.H.K., J.G.K., W.H.Y., S.Y.P., H.W.N., Y.S.K., and J.W.C. analyzed data; Y.S.K., H.C.L.,and K.S.K. contributednew reagents/analytic tools; and J.W.C. wrote the paper. The authors declare no conflict of interest. This article isa PNASDirect Submission. Data deposition: The sequences reported in thispaper have been deposited in theGenBank database [accession nos. M62399 (NFkBp65), BC014434 (OGT), and NP036347 (OGA)]. 1To whom correspondence should be addressed at: Department of Biology,Yonsei Uni versity,134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea. E-mail: chojw311@ yonsei.ac.kr. This article contains supporting informationonline at www.pnas.org/cgi/content/full/ 0806198105/DCSupplemental. ? 2008 by The National Academy of Sciences of the USA

OGA assays (Fig. 1C). Also, observed hyperglycemia-induced

and

regulatesO-GlcN Acylation and inhibitshyperglycemia-induced NFkB activation in rat vascular smoothmuscle cells (VSMCs).
In contrast, up-regulation and treatment expression itors streptozotocin (STZ) of O-GlcN after OGT Acylation of cells with the O-GlcNAcase (20) and over inhib

(9-(2-acetamido-2-deoxy-D

glucopyranosylidene)amino-7V-phenylcarbamate
www.pnas.org/cgi/doi/10.1073/pnas.0806198105

(PUGNAc)
PNAS |

November

11, 2008

| vol.105

| no. 45

| 17345-17350

This content downloaded from 181.15.183.213 on Tue, 12 Nov 2013 21:26:27 PM All use subject to JSTOR Terms and Conditions

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inhibits the reduction in NFkB p65-l/<Ba inter Fig. 2. OGA overexpression actions and nuclear translocation of NFkB p65 under high-glucose conditions. VSMCs were transfected with either vector or a plasmid expressing FLAG tagged OGA and exposed to high glucose for 24 h. (A) Immunoblotting (IB) for NFkB p65, IkB?, and FLAG-tagged OGA was performed with an anti-NFKB p65 antibody, an anti-kBa antibody, and an anti-FLAG antibody (1st, 5th, and 6th (IP) were analyzed for panels, respectively). NFkB p65 immunoprecipitates IkBcx and NFkB p65 expression by using immunoblotting with the correspond ing antibodies (2nd and 4th panels, respectively). Actin was used as a loading control (7th panel). (B) (Upper) VSMCs were immunostained by using anti bodies against NFkB p65 followed by anti-rabbit Ig covalently conjugated to rhodamine (R) to determine the subcellular localization. (Lower) OGA over

IP : Actin HHHHHH ^^^^^^^^ 25 5 25

were

cotransfected with the KB-luciferase reporter gene plasmid and the plasmid expressing FLAG-tagged human OGA, and incubated for 24 h under normal- or high-glucose conditions. The luciferase activitywas measured and normalized to )3-galactosidase activity, and the data shown represent the mean ? SD (n = 3); *, P < 0.01 by Student's ttest. (D) VSMCs transfected with empty vector (lanes 1 and 3) or that encoding FLAG-tagged OGA (lanes 2 and (lanes 3 (lanes 1 and 2) or high-glucose 4) were exposed to normal-glucose and 4) OGA, and

(lanes 1 and 2) or high-glucose (lanes 3 and 4) levels for 24 h. The levels of O-GlcNAc, NFkB p65, VCAM-1, OGT, and OGA were determined in total cell lysates by immunoblotting (IB; 1st, 2nd, and 5th-7th panels, respectively). NFkB p65 immunoprecipitates (IP) obtained from cellular extracts were ana lyzed by immunoblotting for O-GlcNAc and NFkB p65 (3rd and 4th panels, respectively). Actin was included as a loading control (8th panel). (Q VSMCs

in O-GlcNAcylation decrease OGA overexpression-mediated sup Fig. 1. NFkB activation. (4) Rat VSMCs were trans presses hyperglycemia-induced fected with the KB-luciferase reporter gene plasmid and then incubated for 24 h under normal (5 mM) or high-glucose (25 mM) conditions. The luciferase and normalized to /3-galactosidase activity from a activity was measured cotransfected control plasmid. The data shown represent the mean ? SD (n = 3); *, P < 0.01 by Student's ttest. (B) VSMCs were exposed to normal-glucose

expression was detected with an anti-FLAG antibody conjugated to FITC. The percentages of cells showing NFkB p65 localization are derived from at least 150 transfected cells with FLAG-OGA in microscopic fields. (Q Immunoblotting of nuclear or cytosolic extracts of VSMCs by using antibodies against NFkB p65 was performed to determine NFkB p65 nuclear translocation (1st row). The purity of the nuclear and cytosolic extracts was determined by immunoblot ting of histone H2A fractions, 3rd row). (nuclear fractions, 2nd row) and a-tubulin (cytosolic

conditions (Fig. 24, 1st panel). To investigatehow high-glucose conditions influence the interaction between IkBc* and NFkB,
we

levels for 24 h. The levels of O-GlcNAc, NFkB p65, VCAM-1, OGT, in total cell lysates by FLAG-tagged OGA were determined (1st, 2nd, and 5th-8th panels, respectively). NFkB p65 immunoblotting immunoprecipitates obtained from the cellular extracts were analyzed by immunoblotting for O-GlcNAc and NFkB p65 (3rd and 4th panels, respec tively). Actin was included as a loading control (9th panel).

with NFkB p65 and found that the interactionbetween NFkB p65 and IkBcxwas decreased dramatically under high-glucose conditions (Fig. 2A, 2nd and 3rd panels). We then investigated how OGA overexpression affects the OGA interaction between NFkB p65 and IkB?. Interestingly, overexpression inhibited the hyperglycemia-induced decrease in NFkB p65-lKBa interactionsbut did not significantly affect total IkBc* levels (Fig. 2A, 1st 3 panels). Next, we investigated the nuclear translocation of NFkB p65 in cells under high-glucose conditions by using immunostaining and immunoblotting.NFkB p65 was present primarily in the
of cytosol translocated the cells under to the nucleus

analyzed

the

amount

of

IkBc*

that

coimmunoprecipitated

together, induced

these NFkB

results activation.

indicate

that

an

increased

amount

of

O-GlcNAcylation

has an

important role in hyperglycemia

OGA

NFkB

uitination, and proteolytic degradation of IkB releases NFkB, which can then enter thenucleus, bind toDNA, and activate the the expression level of IkB, the interactionbetween NFkB and
the nuclear translocation of NFkB under high-glucose transcription of various genes (1-3). Therefore, we investigated

in Reduction Blocks Hyperglycemia-induced Nuclear Interactions and Hyperglycemia-induced p65-IkB? the phosphorylation, Translocation of NFkB p65. Generally, ubiq Overexpression

NFkB

2B). Interestingly,the translocated NFkB p65 was found in the cytosolwhen FLAG-tagged OGA was transfected into the cells under high-glucose conditions (Fig. 2B). This observation was confirmed by a Western blotting experiment using nuclear extracts (Fig. 2C). Therefore, O-GlcNAcylation is involved in hyperglycemia-inducedNFkB activation by inducinga change in
nuclear interactions, p65-IkBc* translocation of NFkB resulting p65. of O-GlcNAcylation Up-Regulation of NFkB. To investigate expressing FLAG-tagged In the in an increase in the

conditions but was normal-glucose conditions under high-glucose (Fig.

OGT Overexpression-Mediated creases the Transcriptional activity, we transfected

IkB, and conditions.

influence of increased intracellularO-GlcNAcylation on NFkB


a vector human

Activity

When VSMCs were exposed to high glucose for24 h, the IkBq


expression 17346 level was similar to that found under normal-glucose Yang etal.

OGT into VSMCs. As expected, OGT overexpression increased levels of total protein and NFkB p65 O-GlcNAcylation levels OGT overexpression (Fig. 3B, 1st and 3rd panels). Interestingly,

| www.pnas.org/cgi/doi/10.1073/pnas.0806198105

This content downloaded from 181.15.183.213 on Tue, 12 Nov 2013 21:26:27 PM All use subject to JSTOR Terms and Conditions

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^^^

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NFkB p65 mutant proteinswere immunoprecipitated from cells overexpressing OGT with an anti-NFKB p65 antibody (Fig. 4, with an 2nd row), and O-GlcNAcylation levelswere identified anti-O-GlcNAc antibody (Fig. 4, 3rd row). The specificityof O-GlcNAcylation on NFkB p65 mutant proteins was confirmed by adding 10 mM GlcNAc during immunoblotting (Fig. 4, 4th His-NFkB p65 (1?551) was mod row).As expected, full-length ifiedwith O-GlcNAc, but the modification of truncated His NFkB p65 (1-304) was greatly reduced (Fig. 4, 3rd row).
Therefore, and 551. constructs occurs between amino O-GlcNAcylation To map the site further, we tested other and found that O-GlcNAcylation occurred acids 305 truncated on His

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NFkB p65 (1-330), but the amount of modification was greatly decreased on PHs-NFkB p65 (1-320) (Fig. 4, 3rd row). These results show thatO-GlcNAcylation occurs on Thr-322 of NFkB p65 because this is the only possible modification site inNFkB p65 between amino acids 321 and 330. we replaced Thr-322 with Ala and found To test this further, that the amount of O-GlcNAcylation of HIs-NFkB p65 (1-330) was greatly decreased (Fig. 4, 3rd row). However, full-length NFkB p65 T322A was modified still with O-GlcNAc (Fig. 4, 3rd the full-lengthHIs-NFkB p65 T322A to find additional O GlcNAcylation sites. From this investigation,we found that His-NFKB p65 (1-360) T322A was largelymodified by O GlcNAc compared with HIs-NFkB p65 (1-330) T322A and His-NFKB p65 (1-350) T322A (Fig. 4,3rd row).This result shows 360 ofNFkB p65. There are 5 Ser or Thr residues in this region:
Thr-352, that O-GlcNAcylation Ser-353, also occurs between amino acids 351 and and Thr-357. To define row). Therefore, more deletion constructs were made by using

+~~

increase inO-GlcNAcylation induces Fig. 3. OGT overexpression-mediated the up-regulation of NFkB activity. (>A)VSMCs were transfected with the KB-luciferase reporter gene plasmid and the plasmid encoding FLAG-tagged OGT and incubated for 24 h under normal conditions. The luciferase activity was measured and normalized to /3-galactosidase activity, and the data shown = represent the mean ? SD (n 3); *, P< 0.01 by Student's ttest. (B) VSMCs were cotransfected with the plasmid encoding FLAG-tagged OGT (+) or vector control (-) and incubated for 24 h under normal glucose conditions. Immu noblotting (IB) forO-GlcNAc, NFkB p65, IkB?, VCAM-1, OGT, FLAG-OGT, and OGA was performed with the corresponding antibodies (1st, 2nd, 5th, and 8th-11th panels, respectively). NFkB p65 immunoprecipitates (IP) were ana

furtherthe modification site in351-360 ofNFkB p65, additional mutations T352A, S353A, S354A, Ser or Thr toAla substitution S356A, and T357A were made in the HIs-NFkB p65 (1-360) T322A protein. O-GlcNAcylation of His-NFKB p65 (1-360) T322A/T352A was greatly decreased compared with the other
substitution mutants (Fig. 4, 3rd row). From these results, we

Ser-354,

Ser-356,

> k ? i/>
IUJ x u o D3

lyzed forO-GlcNAc, NFkB p65, and IkB? (3rd, 4th, and 6th panels, respectively). Actin was used as a loading control (12th panel). (Q (Upper) VSMCs were immunostained by using antibodies against NFkB p65 to determine the sub cellular localization. (Lower) OGT overexpression was detected with an anti FLAG antibody conjugated to FITC The percentages of cells showing NFkB p65 localization are derived from at least 150 transfected cells with FLAG-OGT in microscopic fields. (D) Immunoblotting of nuclear or cytosolic extracts of VSMCs by using antibodies against NFkB p65 was performed to determine NFkB p65 nuclear translocation (1 st and 4th panels) or total levels of NFkB p65 (7th panel). The purity of the nuclear and cytosolic extracts was determined by immunoblotting of histone H2A (nuclear fractions, 2nd and 5th panels) a-tubulin (cytosolic fractions, 3rd and 6th panels). and

conclude that Thr-352 ofNFkB p65 is modified with O-GlcNAc. Additional O-GlcNAcylation sites other thanThr-322 and Thr 352 may exist because O-GlcNAcylation of the full-lengthand HIs-NFkB p65 (1-520) proteins occurs despite mutations in the identified sites (T322A/T352A) (Fig. 4, 3rd row).Also, by using mass spectrometry (25, 26), we identified thatO-GlcNAcylation occurs in peptide 337-360 of NFkB p65 (Fig. S2).
of NFkB p65 O-GlcNAcylation Important for O-GlcNAc-lnduced on Is Thr-352, but Not on Thr-322, NFkB Transcriptional Activation. We

also

increased

a KB-luciferase

and the expression of VCAM-1 (Fig. 3B, 8th panel). Further more, OGT overexpression inhibitedNFkB p65-IkB? interac tions (Fig. 3B, 6th and 7th panels) and increased the nuclear translocationofNFkB p65 (Fig. 3 C and D) in a manner similar to thatobserved under hyperglycemia-inducedNFkB activation. Interestingly, O-GlcNAcylation of NFkB p65 and its transcrip tional activity are increased in STZ-induced diabetes inmice [supporting information (SI) Fig. SI] (23). Therefore, these
that an increased suggest NFkB transcriptional up-regulates results amount

reporter

gene

expression

(Fig.

3A)

then investigatedthe functional importance ofO-GlcNAcylation on both Thr-322 and Thr-352. We used Thr toAla substitution mutants ofNFkB p65 expressed inNFkB p65 KO MEFs to rule out endogenous NFkB p65 activity (24). Also, we used OGT
overexpression and treatment of cells with O-GlcNAcase inhib

itors (STZ and PUGNAc) to increase NFkB p65 O-GlcNAcy NFkB p65 lation.First,we investigated whether the mutations in the NFkB of by using the changed transcriptional activity
luciferase assay. Plasmids encoding the His-tagged wild-type

NFkB

tion-inducedNFkB activation is closely involved in diabetes.

activity,

of O-GlcNAcylation and 0-GlcNAcyla

WT

p65 (HIs-NFkB p65 WT) or NFkB p65 containing site-specificThr toAla substitutionmutations (HIs-NFkB p65 T322A or T352A) were cotransfectedwith FLAG-tagged IkBc* intoNFkB p65 KO MEFs. In the case of eitherHIs-NFkB p65 porter gene expression, but in the case ofT352A, itdid not (Fig. SA). Additionally, in an electrophoretic mobility-shift assay (EMSA) experiment using nuclear extracts,O-GlcNAcylation increased theDNA-binding affinity of both His-NFKB p65WT and T322A, but O-GlcNAc did not increase theDNA-binding affinityof T352A (Fig. SB). p65 mutants and FLAG-tagged
Yang etal. PNAS Next, we examined the interactions between the HIs-NFkB or T322A, O-GlcNAcylation increased KB-luciferase re

of NFkB p65 Occurs at Multiple Sites. To identify O-GlcNAcylation O-GlcNAcylated sites on NFkB p65, we studied His-tagged
NFkB

mutants ofNFkB p65, in which Ser or Thr was changed stitution intoAla, expressed in NFkB p65 knockout murine embryo fibroblasts (NFkB p65 KO MEFs) (24) (Fig. A, 1st row). His

p65

C-terminal

deletion

mutants

and

site-directed

sub

IkBc*.When NFkB p65 KO


| vol.105 | no. 45 | 17347

| November 11,2008

This content downloaded from 181.15.183.213 on Tue, 12 Nov 2013 21:26:27 PM All use subject to JSTOR Terms and Conditions

kDa

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89101112

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23242526 2728293031 16171819 202122

3233343536

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O-GlcNAcylation of NFkB p65 occurs at Thr-322 and Thr-352. O-GlcNAcylation of NFkB p65 C-terminal deletion mutant proteins and Ser or Thr to Ala Fig. 4. substitution mutant proteins. NFkB p65 KO MEFs were cotransfected with the vector alone or plasmids encoding the Hjs-NFkB p65 deletion (lanes 1-12) or substitution mutants (lanes 13-36), along with the plasmid encoding FLAG-tagged OGT for 12 h. Hjs-NFkB p65 immunoprecipitates (2nd row)were obtained from cellular extracts by using the anti-NFKB p65 polyclonal antibody and were analyzed by immunoblotting for O-GlcNAc (3rd row). Total levels of Hjs-NFkB p65 mutants inthese extracts were examined by using the anti-NFKB p65 monoclonal antibody (1 st row), and the specificity of O-GlcNAcylation on NFkB p65 mutants was confirmed by decreasing the modification via the addition of 10 mM GlcNAc (4th row).

MEFs were cultured in thepresence of STZ or PUGNAc orwere transfectedwith a plasmid expressing OGT, interactions be tween either NFkB p65 WT or T322A and FLAG-IkBc* were reduced dramatically, but those between HIs-NFkB p65 T352A and FLAG-IkBq: did not change to the same degree (Fig. 5C). Under these conditions, the total levels of HIs-NFkB p65 and FLAG-IkBq: did not change, but (9-GlcNAcylation on HIs-NFkB p65 was increased dramatically afterSTZ or PUGNAc treatment or upon OGT transfection (Fig. 5C). We then studied the nuclear translocation of FHs-NFkB p65
and its mutated forms

modulating hyperglycemia-induced NFkB activation. Further more, we also found that O-GlcNAcylation on Thr-352 ofNFkB p65 interrupts the interactionbetween NFkB and IkB?, which
increases the nuclear These portant observations role NFkB. of O-GlcNAcylated an im that O-GlcNAcylation plays we showed interactions. in protein-protein Finally, translocation suggest

betic mice. were

that an increased amount of O-GlcNAcylation on NFkB p65 under hyperglycemic conditions might explain, in part, the transcriptional activation of NFkB in STZ-induced type 1 dia In our studies, increased levels ofO-GlcNAcylated-NFKB
observed in cells under high-glucose conditions and in cells

eitherHIs-NFkB p65 WT
cated into the nucleus

by using

in the case of FHs-NFkB p65 T352A, most of theproteinwas not translocated (Fig. 5D). These resultswere confirmed with im munoblotting experiments by using an anti-NFKB p65 antibody
on

under

or T322A, the proteins were translo


elevated O-GlcNAc conditions, but

immunostaining.

In the case

of

p65

Thr-352, but not on Thr-322, can inhibitthe interactionbetween NFkB p65 and IkBc*.Taken together, these results show that inhibitionof the interactionbetween NFkB p65 and IkBc*caused by O-GlcNAcylation on Thr-352 of NFkB p65 induces the nuclear translocation of freeNFkB p65, resulting in increased
transcriptional conditions activity. Also, these results show

nuclear

extracts

(Fig.

5E).

Therefore,

O-GlcNAcylation

on

GlcNAcylation on NFkB p50 and IkBcx,but we could not find any O-GlcNAcylation on these 2 proteins (data not shown). Thr toAla substitution mutants ofNFkB p65, Also, by studying we found that O-GlcNAcylation ofThr-352 played a crucial role in the interactionbetween NFkB and IkBc*.
As

under

treatedwith STZ, PUGNAc, or overexpressing OGT, and the interactionbetween NFkB p65 and IkBcxdramatically decreased
these conditions. These results could be caused by O

O-GlcNAcylation
caused

can regulate NFkB


by diabetes.

activity in hyperglycemic

that site-specific

Discussion Since the discovery of O-GlcNAcylation (27), it has been re ported thatO-GlcNAcylation on Ser or Thr residues plays an important role in the function of nucleocytoplasmic proteins as Sp-1 and FoxOl can be modulated by O-GlcNAcylation (28-31), and the stabilityand activityof p53 are also controlled by O-GlcNAcylation (26). Although it is known thatNFkB is modification is importantfor O-GlcNAcylated (12) and that this T and B lymphocyteactivation (19), the specific sites of modi we confirmed that the p65 subunit ofNFkB isO-GlcNAcylated, and we found that this modification plays an important role in
17348 | www.pnas.org/cgi/doi/10.1073/pnas.0806198105 Yang etal. fication and the functions are not well understood. In this work, (16-18). For example, the activity of transcription factors such

receptor /3are reciprocally modified with O-GlcNAc and O phosphate (26, 32). Because NFkB p65 is also modified with O-phosphate and thismodification plays an important role in modulating its activity (2), it is possible that the changes in O-GlcNAcylation level affect not only its activity but also its even though its level on NFkB p65 did not change significantly with O-GlcNAcylation levelwas largely increased by treatment STZ, PUGNAc, or OGT (Fig. S3). Also, there are no existing reports of modification of theO-GlcNAcylation sites,Thr-322 and Thr-352, by (9-phosphate, and we could not identifyany phosphorylation on these sites with MS analysis. Therefore, these results indicate thatO-GlcNAcylation on NFkB p65 is a GlcNAcylated states such as hyperglycemic conditions. By using C-terminal deletion and substitutionmutants of NFkB p65, we identified O-GlcNAcylation of amino acids Thr-322 and Thr-352 of NFkB p65, and the modification of
key regulator of NFkB activation, at least in hyper-O phosphorylation states. In our experiments, the phosphorylation

reported,

some

proteins

such

as

p53

and murine

estrogen

This content downloaded from 181.15.183.213 on Tue, 12 Nov 2013 21:26:27 PM All use subject to JSTOR Terms and Conditions

12 3 4 5 6 7 8 910111213

WT

T322A

T352A

A .-._?_,
f

2 5-

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"STZ

iB:iKBaHHHHHHHHH
IP:p65H^BHHHHHHHHB|

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|fe|8lfei?l?^8l 5 ??o ?Og

WT |T322A|T352A Control Nucieus 32% 54% 50% 96.8% 94.6% 95.0% Cytosol Nudeus58.3% 57.4% 27.4% <5T7 41.7% 42.6% 72.6% Cytosoi o?o fr^^4*% "56^%181%
PUGNAc c^toJ5T"45l%l3T%7:r4%

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saM^^^^M^? IB: Histone-H2A||^^^^^^^^^H

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WT T322AT352A -a-Tut^M^^

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WT T322A T352A EV WT T322A

T352A EV

by using immunoblotting (3rd, 4th, and 6th panels, respectively). Actin was used as a loading control (10th panel). (D) Plasmids encoding His-NFKB p65 (WT or into NFkB p65 KO MEFs and incubated for 12 h in the absence (control) or presence of STZ or PUGNAc. mutants) and FLAG-kBa were cotransfected Immunostaining for NFkB p65 was performed to determine the subcellular localization of Hjs-NFkB p65 WT and mutants. The table indicates percentage localization in each compartment, and the percentages are derived from at least 200 transfected cells inmicroscopic fields. (E) NFkB p65 KO MEFs were cotransfected with empty vector (EV) or plasmids containing Hjs-NFkB p65 (WT or mutants) and FLAG-tagged kBa, and incubated for 12 h in the presence or absence of STZ, PUGNAc, or transfected with a plasmid expressing FLAG-tagged OGT. Immunoblotting of nuclear or cytosolic extracts by using antibodies against NFkB p65 was performed to determine NFkB p65 nuclear translocation (1st row). The purity of the nuclear and cytosolic extracts was determined by immunoblotting of histone H2A (nuclear fractions, 2nd row) and a-tubulin (cytosolic fractions, 3rd row).

and plasmids encoding theWT or mutant His-tagged NFkB p65 proteins were incubated for 12 h inthe absence (control) plasmids encoding FLAG-tagged IkBck, or presence of STZ or PUGNAc. Nuclear NFkB DNA-binding affinitywas analyzed by EMSA using 32P-radiolabeled KB-enhancer probes. The supershift assay was were cotransfected intoNFkB examined with antibodies against NFkB p65 (rabbit polyclonal). (Q Plasmids encoding Hjs-NFkB p65 (WT or mutants) and FLAG-IkBcx p65 KO MEFs. After a 12-h incubation inthe absence (control) or presence of STZ, PUGNAc, or after transfection with the plasmid expressing FLAG-tagged OGT, immunoblotting for NFkB p65, IkB?,VCAM-1, OGA, and OGA was performed by using the corresponding antibodies (1st, 2nd, and 7th-9th panels, respectively). NFkB p65 WT and mutant proteins were also immunoprecipitated from cell lysates, and the immunoprecipitates were analyzed forkBa, NFkB p65, and O-GlcNAc

Fig. 5. Mutation of the NFkB p65 O-GlcNAcylation site at Thr-352 abrogates O-GlcNAc-induced NFkB transcriptional activation. (>A)NFkB p65 KO MEFs were transfected with empty vector, the KB-luciferase reporter gene plasmid, the plasmid encoding wild-type FLAG-tagged kBa, and theWT or mutant NFkB p65 (T322A and T352A) His-tagged expression vectors. The transfected cells were then incubated for 12 h inthe absence (control) or presence of STZ (2mM), PUGNAc (100 pM), or transfected with the plasmid expressing FLAG-tagged OGT as indicated, and the luciferase activitywas measured and normalized to j3-galactosidase = 3); *, P < 0.01 by Student's ttest. (B) NFkB p65 KO MEFs were cotransfected with empty vector (EV) or activity. The data shown represent the mean ? SD (n

t/i 5 UJ X u o m

> oe

Thr-352 was confirmed with MS analysis. By studying the site-directed mutants, we found thatO-GlcNAcylation ofNFkB p65 on Thr-352, but not on Thr-322, was important for the transcriptionalactivation of NFkB through the inhibitionof the interactionbetween NFkB p65 and IkB?. However, ithas been reported that the binding domain, or Rel homology domain (RHD), to IkBcxexists in the 300 aa residues at theN terminus of NFkB p65 (33, 34). Interestingly,although Thr-322 is closer to the RHD than Thr-352, O-GlcNAcylation on Thr-352 of with IkBcx,butmodification NFkB p65 decreases the interaction on Thr-322 does not likelyinfluence the interaction.It ispossible that the O-GlcNAcylation on Thr-352 is closer to the IkBq: studies of the 3-dimensional structure of NFkB p65 and the modification-induced changes of the interaction with other proteinswould be beneficial. duce NFkB activation via the degradation of IkBc* inVSMCs, and thedegradation occurs in relativelyquickly (^180 min) (22). was similar to that timepoint (^24 h), although the levelof IkBcx
According to previous reports, hyperglycemic conditions in binding site than Thr-322 in a solution state. Therefore, future

observed Moreover,

under

normal

glucose

conditions NFkB

in our activation

ited by OGA

hyperglycemia-induced Therefore,

experiments. was inhib the possi

overexpression-induced down-regulation of O
this observation supports

GlcNAcylation.

mice.

bility that0-GlcNAcylation-induced inhibitionof NFkB-IkBck interactions is involved in the sustained activation ofNFkB that is associated with diabetes (35). Additionally, in the STZ induced diabetic mouse model, we found thatNFkB p65 O GlcNAcylation largely increased, and less IkB? was bound to NFkB p65 in each organ indiabetic mice compared with normal activation is involved in complications developed by type 1 or 2 NFkB was activated dramatically diabetic patients (36). In fact, in peripheral blood cells isolated from diabetic nephropathy patients (35). Therefore, O-GlcNAcylation on p65 of NFkB appears to be related to its sustained activation in diabetic conditions and the development of complications in diabetic patients. It is likely that other factors also contribute to the
diabetes-associated subunit, NFkB activation. Furthermore, according to a previous report, NFkB

in our experiments, the nuclear localization of NFkB However, was at a relatively under high-glucose conditions later increased

In our proposed model, O-GlcNAcylation


particularly at Thr-352, induces

NFkB

of theNFkB p65
nuclear translo

cation through the inhibitionof the interaction between NFkB


Yang etal. PNAS | November 11,2008 | vol.105 | no. 45 | 17349

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and IkBcx,resulting in NFkB transcriptionalactivation (Fig. S4). We observed thatO-GlcNAcylation stilloccurred on NFkB p65 when Thr to Ala substitutionswere present at Thr-322 and Thr-352. Justas each phosphorylation of p65 ofNFkB modulates its activity (2, 37), it is possible thatO-GlcNAcylation on other
sites also can modulate NFkB

says; (v) EMSA; (vi) mapping diabetic mouse models.

O-GlcNAc

sites using

ESI-MS/MS;

and

(vii)

O-GlcNAcylation
investigated

at other sites on NFkB

activity.

Therefore,

the effects

of

in the future.

activity should be

Methods
Please see SI Methods DNA transfection, noblotting, for the following detailed methods: (/) cell culture, and plasmids; (ii) reagents and antibodies; (Hi) immu and immunostaining; (iv) luciferase as immunoprecipitation,

We thank Dr. Alexander Hoffmann (University of ACKNOWLEDGMENTS. California at San Diego, La Jolla, CA) for the NFkB p65 knockout mouse embryonic fibroblasts cell lines, and Dr. Tae Ho Lee (Yonsei University, Seoul, Korea) for various vectors. This work was supported by grants from the Korea Science and Engineering Foundation (KOSEF) funded by the Ministry of SRC pro Education, Science and Technology Grant ROA-2007-000-20011-0, Korea Re and R11200301901001, grams of MEST/KOSEF R112000078020010 search Foundation Grant KRF-2004-005-C00112, and, in part, by Grant A030003 from the Health 21 research and development project, via the Ministry of Health and Welfare, Republic of Korea (to J.W.C.). Additionally, W.H.Y., S.Y.P., and D.H.K. are fellowship awardees of the Brain Korea 21 program. This work was made possible through the use of research facilities in the Yonsei Center for Biotechnology. 21. Haltiwanger RS, Grove K, Philipsberg GA (1998) Modulation of O-linked N on nuclear and cytoplasmic vivo using the peptide acetylglucosamine levels proteins in inhibitor 0-GlcNAc-j3-/V-acetylglucosaminidase 0-(2-acetamido-2-deoxy-D-glucopyr JBiol Chem 273:3611-3617. anosylidene)amino-/V-phenylcarbamate. 22. Ramana KV, Friedrich B, SrivastavaS, BhatnagarA, Srivastava SK (2004) Activation of nuclear factor-KB by hyperglycemia invascular smoothmuscle cells is regulated by aldose reductase.Diabetes 53:2910-2920. 23. Parker and inhibition of glycogen G, TaylorR, Jones D, McClain D (2004) Hyperglycemia mice: Role of O-linked A/-acetylglucosamine. JBiol synthase instreptozotocin-treated Chem 279:20636-20642. 24. Gapuzan ME, Schmah O, PollockAD, HoffmannA, Gilmore TD (2005) Immortalized fibroblasts from NF-kB RelA knockout mice show phenotypic heterogeneity and to tumornecrosis factora aftertransformation maintain increasedsensitivity by v-Ras. Oncogene 24:6574-6583. of novel sites of 0-A/-acety(glu 25. Chalkley RJ,Burlingame AL (2003) Identification mass cosaminemodification of serum response factorusingquadrupole time-of-flight Mol Cell Proteomics 2:182-190. spectrometry. 26. YangWH, etal. (2006)Modification of p53 with O-linked /V-acetylglucosamine regu Nat Cell Biol 8:1074-1083. latesp53 activityand stability. 27. Torres CR, Hart GW (1984) Topography and polypeptide distribution of terminal Evidence for residues on the surfaces of intact lymphocytes: A/-acetylglucosamine O-linked GlcNAc. JBiol Chem 259:3308-3317. to Sp1 activation domain 28. Yang X, et al. (2001) O-linkage of A/-acetylglucosamine inhibitsitstranscriptional capability.ProcNatl Acad Sci USA 98:6611-6616. 29. Kang HT, JuJW, Cho JW, Hwang ES (2003) Down-regulation of Sp1 activitythrough with a low-glucose modulation of O-glycosylationby treatment mimetic, 2-deoxyglu cose. JBiol Chem 278:51223-51231. 30. Majumdar G, et al. (2004) Insulin stimulates and diabetes inhibitsO-linked N acetylglucosamine transferaseand O-glycosylationof Sp1. Diabetes 53:3184-3192. 31. HousleyMP, etal. (2008) O-GlcNAc regulates FoxO activation in response to glucose. JBiol Chem 283:16283-16292. of serine-16 32. Cheng X, HartGW (2001) AlternativeO-glycosylation/O-phosphorylation in murine estrogen receptor ]3: Posttranslational regulationof turnoverand transac JBiol Chem 276:10570-10575. tivationactivity. of an IkBo/NF-kB 33. Jacobs MD, Harrison SC (1998) Structure complex. Cell 95:749-758. structure of the IkBo/NF-kB 34. HuxfordT, Huang DB,Malek S,Ghosh G (1998) The crystal Cell 95:759-770. complex revealsmechanisms of NF-kB inactivation. 35. BierhausA, etal. (2001) Diabetes-associated sustained activationof the transcription Diabetes 50:2792-2808. factornuclear factor-KB. to type 1 diabetes. 36. Hegazy DM, et al. (2001) NFkB polymorphismsand susceptibility Genes Immun2:304-308. 37. Viatour P,Merville MP, BoursV, ChariotA (2005) Phosphorylationof NF-kB and IkB TrendsBiochem Sci 30:43-52. proteins: Implicationsincancer and inflammation.

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