This article was downloaded by: [Northwestern University] On: 20 December 2014, At: 03:58 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
The E3 ligase APC/C-Cdh1 regulates MEF2A-dependent transcription by targeting SUMO-specific protease 2 for ubiquitination and degradation a
b
a
c
a
a
a
Han Lu , Bin Liu , Fu-Jun Zhang , Jin Zhang , Rong Dong , Lei Chen , Dong-Mei Qu , Yan Lu
d
a
& Bu-Wei Yu a
Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai, People's Republic of China b
Key Laboratory of Kidney Disease Pathogenesis and Intervention of Hubei Province, College of Medicine, Hubei Polytechnic University, Huangshi, Hubei, PR China c
Click for updates
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People's Republic of China d
Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Accepted author version posted online: 31 Oct 2014.Published online: 17 Oct 2014.
To cite this article: Han Lu, Bin Liu, Fu-Jun Zhang, Jin Zhang, Rong Dong, Lei Chen, Dong-Mei Qu, Yan Lu & Bu-Wei Yu (2014): The E3 ligase APC/C-Cdh1 regulates MEF2A-dependent transcription by targeting SUMO-specific protease 2 for ubiquitination and degradation, Cell Cycle, DOI: 10.4161/15384101.2014.973302 To link to this article: http://dx.doi.org/10.4161/15384101.2014.973302
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
Downloaded by [Northwestern University] at 03:58 20 December 2014
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
The E3 ligase APC/C-Cdh1 regulates MEF2A-dependent transcription by targeting SUMO-specific protease 2 for ubiquitination and degradation Han Lu1*, Bin Liu2*, Fu-Jun Zhang1*, Jin Zhang3, Rong Dong1, Lei Chen1, Dong-Mei Qu1, Yan
ip t
Lu4, Bu-Wei Yu1# 1. Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong University School of
cr
us
Disease Pathogenesis and Intervention of Hubei Province, College of Medicine, Hubei
an
Polytechnic University, Huangshi, Hubei, PR China. 3. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China. 4. Department of
M
Endocrine and Metabolic Diseases, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao
pt
ed
Tong University School of Medicine, Shanghai, China.
ce
*These people contributed equally.
#Corresponding author
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Medicine (SJTU-SM), Shanghai, People’s Republic of China. 2. Key Laboratory of Kidney
Bu-Wei Yu, E-mail: [email protected]
Keywords: Activity-dependent stimuli; MEF2A; APC-Cdh1; SENP2
1
Abstract Activity-dependent stimuli induced a
calcineurin-mediated dephosphorylation of the
transcriptional factor MEF2A at serine408 and promoted a switch from SUMOylation to
ip t
acetylation at lysine403 which led to MEF2A transcriptional activation. We previously identified SENP2 is the de-SUMOylation enzyme for MEF2A and promotes MEF2A-dependent
cr
us
MEF2A transcriptional activation. APCCdh1 interacts with and targets SENP2 for ubiquitination
an
and destruction in the cytoplasm by recognizing a conserved canonical D-box motif in SENP2. Moreover, Cdh1 regulates the transcriptional activity of MEF2A in a SENP2 dependent manner.
M
Activity-dependent stimuli prevented APCCdh1-induced SENP2 ubiquitination, promoted SENP2 nuclear accumulations, and caused MEF2A de-SUMOylation and MEF2A acetylation, leading to
ed
MEF2A transcriptional activation. Thus, our findings defined a post-transcriptional mechanism
ce
pt
underlying activity-dependent stimuli-induced MEF2A transcriptional activation.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
transcription. We report here a requirement for APCCdh1-SENP2-MEF2A axis in the regulation of
2
Introduction Activity-dependent stimuli can modulate neuronal plasticity, which is a result of the brain’s ability to convert transient stimuli into long-lasting alterations in neuronal structure and function.
ip t
This results in an increase (potentiation) or a decrease (depression) in synaptic strength1. This process involves changes in receptor trafficking, protein–protein interactions, local mRNA
cr
us
phosphorylation, acetylation, ubiquitination and SUMOylation2-5.
an
SUMOylation is the covalent linkage of a small ubiquitin-related modifier (SUMO) to the -amine of lysine residues in target proteins. It is an important regulatory mechanism for
M
modifying protein activity, stability, and cellular localization6. Mammalian cells express four SUMO paralogs: SUMO1–SUMO4. Human SUMO2 and SUMO3 are about 95% identical to
ed
each other, whereas they share only 45% identity with SUMO1. SUMO4 is the least well
pt
characterized isoform and is expressed mainly in the kidney, lymph node and spleen, whereas
ce
SUMO1, SUMO2 and SUMO3 are ubiquitously expressed7. SUMOylation is a high dynamic process that is catalyzed by the activating (E1), conjugating (E2) and ligating (E3) enzymes and reversed by a family of Sentrin/SUMO-specific proteases (SENPs)8. In mammalian cells, six
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
translation, new gene synthesis and protein post-translational modifications including
SENPs have been identified, which have different substrate specificity and subcellular localization. SENPs are divided into three subfamilies on the basis of their sequence homology, cellular location and substrate specificity9. The first subfamily includes SENP1 and SENP2, which have broad substrate specificity
10, 11
. SENP2 is a nucleocytoplasmic shuttling protease
that appears to have a activity similar to that of SENP1 when overexpressed12. Loss of mouse 3
SENP2 caused embryonic lethality
13
. SENP2 is essential for embryonic cardiac development
through regulation of the SUMOylation status of Pc2/CBX4 and has a specific role in the G-S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast
ip t
proliferation and differentiation 13, 14. The second subfamily includes SENP3 and SENP5, both of which are nucleolar proteins with preference for SUMO-2/315. The third subfamily includes
cr
us
The MEF2A transcriptional factor is highly expressed in the brain and plays a key role in
for
postsynaptic
differentiation
an
synaptic dendritic development in the cerebellar cortex17. The modifications of MEF2A required occur
within
a
phosphorylation-regulated
M
SUMOylation-acetylation switch (SAS) peptide motif18. Activity-dependent stimuli increased MEF2A transcriptional activity by activating a highly programmed post-transcriptional event
ed
taken place on MEF2A, including MEF2A de-phosphorylation, MEF2A de-SUMOylation and
pt
MEF2A acetylation19. Calcineurin dephosphorylates MEF2A Ser408 which resulted in Lys403 de-SUMOylation,
sequential
Lys403
acetylation
and
de-represses
MEF2A-induced
ce
transcription18. Further study discovered that PIASx promoted postsynaptic dendritic morphogenesis by enhancing the SUMOylation of MEF2A20. We previously identified SENP2
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP6 and SENP7, which have an extra loop in their catalytic domains16.
was the predominant de-SUMOylation enzyme for MEF2A by using unbiased functional loss of function screen21. We found that SENP2 regulates the transcriptional function of MEF2A via direct de-SUMOylation. In conformity with these clues, MEF2A was heavily SUMOylated in SENP2-/- embryo21. However, how endogenous SENP2 is regulated during this process is still undefined. 4
Results Activity-dependent stimuli prevent SENP2 ubiquitination and degradation We first investigated whether endogenous SENP2 was regulated under KCl-induced
ip t
depolarization manipulation by using primary rat cortex neuron cells. KCl-induced depolarization promote the transcriptional activity of MEF2A has been well documented18, 19.
cr
us
the accumulation of SynGAP, one of the downstream target genes of MEF2A 18(Fig.1A). Under
an
the same circumstances, endogenous SENP2 protein was also accumulated in a time-dependent manner (Fig.1A). However, the mRNA level of SENP2 was almost unchanged, suggesting a
M
post-transcriptional regulation was taking place (Fig.S1). SENP2 has been reported to be a short-lived protein and destructed via ubiquitin-proteasome system12. We proposed that
ed
KCl-induced endogenous SENP2 accumulation could be due to impaired SENP2 destruction.
pt
Indeed, the half-life of endogenous SENP2 was significantly increased in KCl-treated SHSY5Y
ce
cells (Fig.1B). Moreover, KCl stimuli dramatically decreased the ubiquitination form of SENP2 (Fig.1C). Consistent with these data, KCl stimuli further enhanced the de-SUMOylation activity of SENP2 towards MEF2A and caused MEF2A SAS transition (Fig.1D) and SENP2-mediated
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Indeed, KCl treatment significantly enhanced MEF2A transcriptional activation as evidenced by
MEF2A transcriptional activation (Fig.S2). Furthermore, silencing the expression of SENP2 by siRNA largely abolished KCl-induced MEF2A activation (Fig.1E, 1F). Taken together, these data indicated that activity-dependent stimuli prevents SENP2 ubiquitination and degradation accompanied with MEF2A activation. Activity-dependent stimuli promote SENP2 nuclear translocation 5
SENP2 is reported to be a nucleocytoplasmic shuttling protein and is ubiquitinated in the cytoplasm12. We then asked that whether activity-dependent stimuli promoted SENP2 nuclear translocation. To test this possibility, primary rat cortex neuron cells were treated with 50mM
ip t
KCl to induce SENP2 accumulation. The nuclear-cytoplasmic fraction of primary rat cortex neuron cells was conducted. As depicted in Fig.2A, KCl stimuli significantly promoted SENP2
cr
12
(Fig.2B). A cytoplasm-localized
us
CRM1-dependent nuclear export signal (NES) in SENP2
an
SENP2 made by introducing mutations in the NLS (SENP2 mNLS) was unable to accumulate in response to KCL stimuli in SHSY5Y cells (Fig.2C, Fig.S3). KCl stimuli could also not decrease
M
the ubiquitination form of SENP2 mNLS (Fig.S4). Moreover, overexpression of SENP2 mNLS could not increase the transcriptional activity of MEF2A when compared with SENP2 WT
ed
(Fig.2D). leptomycin B, an inhibitor of CRM1, could specifically inhibit the CRM1-dependent
pt
nuclear export activity of the NES22. LMB treatment did not affect the mRNA level of SENP2
ce
(Fig.S5). However, LMB treatment inhibited the nuclear export of SENP2 (Fig.2E), prevented SENP2 ubiquitination (Fig.2F), promoted MEF2A de-SUMOylation (Fig.2G), transcriptional activation (Fig.2H) and downstream target gene SynGAP expression (Fig.2I). Conclusively,
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
nuclear translocation. There is a bipartite nuclear localization signal (NLS) and a
these data suggested that activity-dependent stimuli prevent SENP2 ubiquitination and degradation by promoting its nuclear translocation. SENP2 contains a canonical D-box motif. To identify which E3 ligase is required for the degradation of SENP2, we first checked the protein sequence of SENP2 for potential degrons. We noted that SENP2 contains a canonical 6
‘destruction box’ (D-box) motif (R-X-X-L-X-X-X-X-N/D/E) (Fig.3A). D-box and KEN are two degradation motifs have been identified in the substrates of Anaphase-Promoting Complex (APC), which is a multi-subunit E3 protein23. APC is responsible for ubiquitination of cell cycle
ip t
regulators in non-neuronal cells but also highly expressed and has additional function in the post-mitotic neurons24. Most APC substrates have D box and some have either a KEN box or
cr
us
mention that, this D-box motif of SENP2 is evolutionarily conserved among different species
an
including Mus musculus, Rattus norvegicus, Pongo abelii and Homo sapiens (Fig.3B). Tosyl-L-arginine methyl ester (TAME), which is a potent inhibitor of APC, could directly bind to
M
APC and disrupts the interactions between APC and its activator proteins Cdc20 or Cdh125, 26. Administration of proTAME resulted in the accumulation of Cyclin B1, a well-known substrate
ed
of APC (Fig.3C). Administration of TAME also caused SENP2 accumulation and slightly
pt
decrease global SUMOylation (Fig.3C, 3D). However, the mRNA level of SENP2 was almost unchanged (Fig.3E). Collectively, these data suggested that SENP2 might be a substrate of APC.
ce
SENP2 is a substrate of APCCdh1
To investigate whether SENP2 is a substrate of APC, we first tested the interaction between
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
both D and KEN boxes. In some cases two D boxes appear in the same protein. It is worth to
Cdh1 and SENP2 by coimmunoprecipitation of tagged Cdh1 and SENP2overexpressed in 293T cells. Cdh1 was able to bind to SENP2, whereas Cdc20 another coactivator of APC complex did not show any detectable association with SENP2 (Fig.4A, Fig.S6). Endogenous Cdh1 and SENP2 could also be efficiently co-immunoprecipitated with each other in SHSY5Y cells (Fig.4B). We further confirmed the interaction between Cdh1 and SENP2 by in vitro 7
precipitation assays in which substantial HA-tagged Cdh1 was precipitated specifically by glutathione S-transferase (GST)-SENP2 but not by GST alone (Fig.4C). Expression of Cdh1 but not that of Cdc20 dramatically reduced SENP2 protein level which was prevented by MG132
ip t
administration (Fig.4D, Fig.S7). Moreover, SENP2 was degraded at a faster rate in the presence of Cdh1 (Fig. 4E). Consistent with these observations, silencing the expression of Cdh1 by
cr
us
accumulation (Fig.4F, Fig.4G). Furthermore, the half-life of SENP2 was significantly increased
an
when Cdh1 was silenced (data not shown). Lastly, Cdh1 significantly promoted the ubiquitination of SENP2 in vivo (Fig.4H). Taken together, our data indicated that SENP2 is a
M
bona fide substrate of APCCdh1.
D-box-dependent degradation of SENP2 by APCCdh1
ed
To investigate whether the degradation of SENP2 was dependent on its D-box motif, we
pt
generated mutant SENP2 protein (SENP2 Mut) in which the key arginine and leucine residues
ce
were changed into alanine and valine, respectively (Fig.5A). SENP2 Mut was expressed at level higher than SENP2 WT and was resistant to Cdh1-mediated destabilization (Fig.5B). Furthermore, GST–SENP2 WT but not GST–SENP2 Mut captured HA-tagged Cdh1 from
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
siRNA or overexpression of a domination negative form of Cdh1 (DN-Cdh1) resulted in SENP2
SHSY5y cell extract, suggesting the D box is essential for the recognition of SENP2 by APCCdh1(Fig.5C). To determine the importance of the D-box-mediated degradation of SENP2, we monitored the degradation rate of SENP2 WT and SENP2 Mut. In transiently transfected SHSY5y cells, the half-life of SENP2 Mut was significantly extended when compared with SENP2 WT (Fig.5D). In agreement with these data, TAME has little effect on SENP2 Mut 8
protein when compared with SENP2 WT protein (Fig.5E). Cdh1 also significantly promoted the ubiquitination of SENP2WT but not SENP2 Mut in vivo (Fig.5F). Together, these data suggested that APCCdh1 can polyubiquitinate SENP2 in a D-box-dependent manner.
ip t
Cdh1 regulates MEF2A transcriptional activity via SENP2 destruction To investigate the functional significance of APCCdh1-mediated SENP2 destruction, we test
cr
us
dramatically repressed MEF2A transcriptional activity (Fig.6A). Indeed, Cdh1 repressed MEF2A
an
transcriptional activity in a dose dependent manner (Fig.6B). Furthermore, we found that overexpression of Cdh1 also decreased KCl-induced MEF2A de-SUMOylation, decreased
M
KCL-induced MEF2A acetylation, and KCl-induced MEF2A activation (Fig.6C, 6D). Indeed, using SynGAP and Nur77 neuronal specific promoters, we found that overexpressing Cdh1
ed
significantly decreased SynGAP- and Nur77-luciferase reporter genes activity in SHSY5Y cells
pt
treated with KCl (Fig.6E, Fig.S8). Furthermore, silencing the expression of Cdh1 by siRNA or
ce
overexpression of DN-Cdh1 promotes the transcriptional activity of MEF2A and decreases MEF2A SUMOylation (Fig.6F, Fig.S9). Co-expression of SENP2 Mut but not SENP2 WT significantly restored the transcriptional activity of MEF2A repressed by Cdh1 (Fig.6G).
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
whether Cdh1 regulates MEF2A transcriptional activity. Overexpression of Cdh1 but not Cdc20
Previous studies have showed that membrane depolarization by KCl promotes the survival of neurons, and this process is dependent on MEF2 transactivation activity27, 28. Thus, we proposed that APC/Cdh1–Senp2 axis may play a role in activity-dependent neuronal survival. To this end, we found that overexpression of Cdh1 or silencing the expression of SENP2 could prevent activity dependent neuronal survival, whereas overexpression of SENP2 or DN-Cdh1 could 9
promote
activity
dependent
neuronal
survival
(Fig.6H,
Fig.6I),
suggesting
that
APC/Cdh1–Senp2-MEF2 axis at least plays a role in activity dependent neuronal survival. Discussion
ip t
By checking the protein level of SENP2 in response to activity-dependent stimuli, we have found that exogenous SENP2 was rapidly stabilized to facilitate MEF2A de-SUMOylation and
cr
us
stimuli. SENP2 was reported to be a nucleocytoplasmic shuttling protein and ubiquitinated in the
an
cytoplasm. We found that activity-dependent stimuli could increase the half-life of SENP2 by preventing SENP2 ubiquitination and degradation. Our earlier work revealed that
M
activity-dependent stimuli could significantly increase global SUMOylation and alter the subcellular distribution of SUMO conjugated proteins4. Ce´ line et al. also demonstrated that in hippocampal neurons, the SUMOylation enzyme UBC9 and
ed
primary cultured rat
pt
deSUMOylation enzymes SENP1 and SENP6 are differentially redistributed in and out of
ce
synapses upon activity-dependent stimulation29. We then asked whether neuronal depolarization could also alter the distribution of SENP2. Indeed, SENP2 was translocated from cytosol to nucleus in response to activity-dependent stimuli, suggesting that activity-dependent stimuli
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
activation. In this study, we mainly focused on the regulation of SENP2 by activity-dependent
prevent SENP2 ubiquitination and degradation by promoting its nuclear translocation. We next asked which E3 ligase is required for the degradation of SENP2. Bioinformational
analysis found that SENP2 contains a canonical D-box degron which is also presented in the well-known substrates of APCCdh1. This clue led us to investigate the relationship between APCCdh1 and SENP2. By using various biochemistry studies, we found that Cdh1 but not Cdc20, 10
another APC/C activator, physically interacted with SENP2 and targeted SENP2 for ubiquitin-dependent degradation. We also test the functional significance of APCCdh1-mediated SENP2 destruction. We found that Cdh1 could enhance MEF2A SUMOylation and decrease the
ip t
MEF2A transcriptional activity. Overexpression of stable SENP2 form (SENP2 Mut) could significantly restore Cdh1-repressed MEF2A activation. We also found that both Cdh1 and
cr
us
previous unknown Cdh1-SENP2-MEF2A axis that mediated activity-dependent stimuli-induced
an
MEF2A transcriptional activation and activity-dependent neuronal survival.
Cdh1 is required to initiate APC activity during late mitosis and G1 and is the master of 30, 31
. Recently, SENP2 was reported to play a role in the
M
G0/G1 phase in non-neuronal cells
regulation of cell cycle progress 32. However, whether SENP2 itself is regulated during cell cycle
ed
progress is largely unknown. Further studies are needed to investigate the functional relevance
pt
between Cdh1 with SENP2 in the cell cycle regulation in the future.
ce
MEF2A has been reported to be able to regulate postsynaptic dendritic morphogenesis 18. It will be interesting to test whether SENP2 or Cdh1 could play a role in postsynaptic dendritic morphogenesis. APCCdh1 complex is highly expressed in the post-mitotic neurons, which
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP2 were participated in activity-dependent neuronal survival. Thus, our data clarified a
restrains the intrinsic axon growth potential33. APCCdh1 complex is required for associative fear memory and long-term potentiation in the amygdala of adult mice34, 35. APCCdh1 complex targets both SnoN and Id2 for destruction to elevate the expression of growth inhibitory molecules results in neurite outgrowth36, 37. It will be reasonable to test whether Cdh1 plays a role in postsynaptic dendritic morphogenesis in the future. SENP2 was also reported to be highly 11
expressed in central neuron system13. However, the biology function of SENP2 is completely unknown in central neuron system due to the embryonic lethal of SENP2-/- mice. Indeed, several neuronal SUMOylation substrates have been identified while the de-SUMOylases specific for
ip t
these substrates were remain to be identified38. Systematic identification of the neuron specific SUMOylation substrates of SENP2 might also be required to clarify the neuronal function of
cr
us
the neuronal function of SENP2 in the future.
an
Material and methods Isolating Rat Neurons
M
All the reagents used for neurons were purchased form Invitrogen. The cortex was dissected from ten E-18 rat embryo brains and put in a conical tube containing Hibernate®-E
ed
supplemented with 2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I at 4°C.
pt
Enzymatically digest the tissue in 4 mL of Hibernate®-E medium without Ca2+ containing 2
ce
mg/mL papain for 30 minutes at 30°C. 6 mL complete Hibernate®-E medium was added to the tube and centrifuge for 5 minutes at 150 × g. The supernatant was removed and the cells were transferred to a new tube in Neurobasal® medium with 2% B-27® Serum-Free Supplement and
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP2. Neuron- or glial-specific knockout of SENP2 mice will be a powerful tool to investigate
0.5 mM GlutaMAX™-I for culturing. Cell culture and treatments Primary rat cortex neuron cells were cultured in poly-D-lysine coated 6-well plate and cultured with in Neurobasal® medium. SHSY5Y neuroblastoma cell in high passages was purchased from ATCC, and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) 12
containing 10 % fetal bovine serum (FBS), 2 mM glutamine and 1 % antibiotics (penicillin–streptomycin). Cells were maintained at 37°C in a humidified 5 % CO2 atmosphere. The cells were depolarized by KCl in the medium with addition of 31 % depolarization buffer
ip t
(170 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES) at a final concentration of 50 mM KCl for the indicated lengths of time. MG132 was purchased from Calbiochem, TAME
cr us
Plasmids and Transfection
an
The 3 X MRE-luc and Gal4-MEF2A expression plasmids were gifts from Dr. Michael Greenberg. The pCDNA3-MEF2A-Flag expression plasmid was a gift from Dr. Rhonda Bassel-Duby.
M
Flag-SENP2, HA-SUMO1, His-Ubiquitin, Myc-SUMO1 and luciferase renilla reporter constructs, have been described previously. Cdh1 and Cdc20 were amplified from 293T cells by
ed
PCR and cloned into the pcDNA 3.1 vector. SENP2 MUT, SENP2 mNLS, DN-Cdh1 and
pt
DN-Cdc20 were generated using QuickChange Site-Directed Mutagenesis Kit (Stratagene). All cDNAs were completely sequenced. Cells were transiently transfected using Lipofectamine 2000
ce
(Invitrogen), according to the manufacturer’s instructions. RNAi
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
hydrochloride was purchased from sigma.
siRNA against SENP2 (J-006033-05) and nonspecific siRNA (D-001810-01) were purchased from Dharmacon RNA Technologies as previously described. The siRNA oligonucleotides targeting
human
Cdh1
(GAAGGGUCUGUUCACGUAUTT)
and
(CGGCAGGACUCCGGGCCGATT) was chemically synthesized by Dharmacon.
13
Cdc20
Nuclear-cytoplasmic fraction The nuclear-cytoplasmic fraction of rat primary cortex neurons and SHSY5Y cells were conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher
ip t
Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. SUMOylation assays
cr
us
cotransfected with expression plasmids for Flag-MEF2A and HA-SUMO1 were lysed in RIPA
an
buffer (150mM NaCl, 10mM Na2HPO4, pH 7.2, 2mM EDTA, 50mM NaF, 1mM NaVO4, 1%NP-40, 0.1% SDS, 0.75% sodium deoxycholate, 1mMPMSF, 10mM N-ethylmaleimide). Five
M
percent of this starting material was retained for detection of input proteins, and the remainder was subjected to immunoprecipitation overnight at 4°C. Flag M2 antibody (sigma) was used
ed
together with protein A-Sepharose beads. Immune complexes were washed eight times with
pt
RIPA buffer at 4°C and resuspended in 2XSDS loading buffer. Immune complexes and input
ce
samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with HA or Flag antibodies.
Immunoprecipitation (IP) assay
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
In vivo SUMOylation assays were performed as described previously21. Briefly, cells
Immunoprecipitation experiments were performed as described previously with minor modifications21. Briefly, cells cotransfected with tagged plasmids were lysed in IP buffer (150 mM NaCl, 50 mM TrisHCl, pH 7.5, 1 mM EDTA, 50 mMNaF, 1mMNaVO4, 1%NP-40, 1mMPMSF, 10mM N-ethylmaleimide). Five percent of this starting material was retained for detection of input proteins, and the remainder was subjected to immunoprecipitation with 14
anti-FLAG antibodies overnight at 4°C. Immune complexes were bound to protein A-beads for 1 h at 4°C, washed twice with IP buffer, once with PBS (pH 7.4), and resuspended in 2XSDS loading buffer. Immune complexes and input samples were subjected to SDS-PAGE, transferred
ip t
to nitrocellulose membranes, and probed with appropriate antibodies GST pull-down assay
cr
us
GST-SENP2 WT or GST-SENP2 MUT were expressed in BL21 cells and purified using
an
glutathione-Sepharose beads (Amersham Pharmacia) in binding buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 100 μg/ml
M
phenylmethylsulfonyl fluoride). After 1 h at 4 °C, equal amounts of GST or GST fusion proteins were resuspended in reaction buffer (200 mM NaCl, 50 mM HEPES pH 7.5, 1 mM MgCl2, and
ed
0.2% Triton X-100) containing 0.2 mg/ml bovine serum albumin (BSA) (Fraction V,
pt
Sigma-Aldrich) and incubated for 2 h at 4 °C . Then, 1 mg HA-Cdh1-transfected cell lysate were
ce
added to each mixture followed by rotation at room temperature for 1 h, centrifugation, and three washes. The beads were boiled in sodium dodecyl sulfate (SDS) sample buffer to elute the bound proteins, which were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Interaction between SENP2 and Cdh1 were assessed by GST-pull down assay. Briefly, GST or
by Western blot analysis. Western blotting and antibodies Protein extracts were equally loaded on 12 % SDS–polyacrylamide gels and subsequently transferred to nitrocellulose membrane by electrophoresis. The membrane was blocked in Tris–buffered saline with 0.1 % Tween-20 (TBST) containing 5 % nonfat dried milk for 1 h at 15
room temperature. Primary antibodies were incubated overnight at 4°C and washed three times each in TBST. Horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG as secondary antibody was added to TBST and membrane was incubated for 1 h followed by three washes in
ip t
TBST (5 min each). The images were visualized by FUJIFILM LAS-4000 Luminescent image analyzer. Western blot was performed using antibodies as indicated: SENP2 (H-300, Santa Cruz),
cr
us
Abcam), Lamin B (C-20, Santa Cruz), Cyclin B1 Antibody (GNS1, Santa Cruz), Cdh1 (ab89535,
an
Abcam), Cdc20 (ab26483, Abcam), Flag (M2, Sigma), Myc (ab9106, Abcam), HA (Y-11, Santa Cruz) and β-Actin (4967L, Cell Signaling Technology).
M
Luciferase assays
Luciferase experiments were performed as described previously with minor modifications21.
ed
Cells were transfected with firefly luciferase reporter plasmids 3XMRE-luc, MEF2A and other
pt
plasmids as indicated. For all reporter gene experiments, firefly luciferase plasmids were
ce
cotransfected with the TK-pRL vector, which expresses renilla luciferase and allows for normalization. Fresh growth media were added within 24 h of transfection. Cells were lysed 48 h after transfection. Luciferase activity was determined using the dual luciferase assay system from
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP2 (ab3660, Abcam), SynGAP (3200s, Cell Signaling Technology), beta Tubulin (ab6046,
Promega.
Quantitative Real-Time PCR Assays Total RNA was isolated with use of TRIzol reagent (Invitrogen). Quantitative PCR was performed with Light Cycler 480 (Roche, Switzerland) and SYBR Green (Molecular Probes, Eugene, OR) used as a fluorescent probe. Target gene mRNA levels were normalized to that of 16
β-actin in the same sample. The following sets of oligonucleotides were used as primers: SENP2 5’-primer
(5’-
GATTCCCATTCCAGCTGACCAC-3’)
(5’-CACTCTGATCTTTGGATAGTCA-3’)
β-Actin: and
3’-primer
5’-primer
3’-primer
(5
(5’’
-
ip t
TCATGAAGTGTGACGTTGACATCC-3’)
and
CCTAGAAGCATTTGCGGTGCACGA-3’). Thermal cycling parameters were 2 min at 50℃,
cr
us
denaturation, 30 sec at 60℃ for anneal. For each sample, average threshold (Ct) value assays
an
were repeated in triplicate, and the ΔCt value was determined by subtracting the average β-actin Ct value from the average SENP2 Ct value.
M
Immunofluorescence analysis.
Cells transfected with indicated plasmids were grown on cover slips. After NaCl or KCl
ed
treatment, cells were washed with PBS, fixed with 4% formaldehyde in PBS for 10 min and
pt
permeabilized with 0.5% Triton X-100 in PBS for 10 min. Then cells were incubated with
ce
anti-Flag or HA antibodies overnight. After washing with PBS three times, cells were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:1000; Invitrogen, Carlsbad, CA) for 1 h. DNA was stained with DAPI. Images were captured on Nikon DS-Ri1-U2 Digital Camera.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
and 10 min at 95℃, for cDNA denaturation followed by 40 cycles of 15 sec at 95℃ for
Apoptosis assay
Apoptosis was measured by the Annexin V Fluos Apoptosis detection kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer’s instructions. Fluorescent intensities of probes were determined by flow cytometry.
17
Statistical analysis The results are presented as mean ± SEM. The Student’s t test was used to compare the difference between two different groups. A value of p < 0.05 was considered to be statistically
ip t
significant. Funding
cr
and Specialized Research Fund for Outstanding Young Teachers of Higher
Education, Shanghai (to Dr. Han Lu).
M
Conflict of interest statement
an
Dr. Buwei Yu)
us
Republic of China, grant No. 81102513 (to Dr. Han Lu), grant No. 81072703 ＆ 81373492 (to
ce
pt
ed
None declared.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
This work was supported by the National Natural Science Foundation, Beijing, People’s
18
References 1. Bertrand SS, Cazalets JR. Activity-dependent Synaptic Plasticity and Metaplasticity in Spinal
ip t
Motor Networks. Curr Pharm Des 2013; 19:4498-508. 2. Cerda O, Trimmer JS. Activity-dependent phosphorylation of neuronal Kv2.1 potassium
cr
us
3. Renthal W, Maze I, Krishnan V, Covington HE, 3rd, Xiao G, Kumar A, Russo SJ, Graham A,
an
Tsankova N, Kippin TE, et al. Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli. Neuron 2007; 56:517-29.
M
4. Lu H, Liu B, You S, Xue Q, Zhang F, Cheng J, Yu B. The activity-dependent stimuli increase SUMO modification in SHSY5Y cells. Biochemical and biophysical research communications 2009;
ed
390:872-6.
pt
5. Carulli D, Foscarin S, Rossi F. Activity-dependent plasticity and gene expression modifications in the adult CNS. Front Mol Neurosci 2011; 4:50.
ce
6. Muller S, Hoege C, Pyrowolakis G, Jentsch S. SUMO, ubiquitin's mysterious cousin. Nature reviews Molecular cell biology 2001; 2:202-10.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
channels by CDK5. J Biol Chem 2011; 286:28738-48.
7. Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nature reviews Molecular cell biology 2007; 8:947-56. 8. Shin EJ, Shin HM, Nam E, Kim WS, Kim JH, Oh BH, Yun Y. DeSUMOylating isopeptidase: a second class of SUMO protease. EMBO reports 2012; 13:339-46. 9. Wang Y, Dasso M. SUMOylation and deSUMOylation at a glance. Journal of cell science 19
2009; 122:4249-52. 10. Cheng J, Wang D, Wang Z, Yeh ET. SENP1 enhances androgen receptor-dependent transcription through desumoylation of histone deacetylase 1. Molecular and cellular biology
ip t
2004; 24:6021-8. 11. Cheng J, Perkins ND, Yeh ET. Differential regulation of c-Jun-dependent transcription by
cr
us
12. Itahana Y, Yeh ET, Zhang Y. Nucleocytoplasmic shuttling modulates activity and
an
ubiquitination-dependent turnover of SUMO-specific protease 2. Mol Cell Biol 2006; 26:4675-89.
M
13. Kang X, Qi Y, Zuo Y, Wang Q, Zou Y, Schwartz RJ, Cheng J, Yeh ET. SUMO-specific protease 2 is essential for suppression of polycomb group protein-mediated gene silencing during embryonic
ed
development. Molecular cell 2010; 38:191-201.
pt
14. Chiu SY, Asai N, Costantini F, Hsu W. SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages. PLoS biology 2008;
ce
6:e310.
15. Gong L, Yeh ET. Characterization of a family of nucleolar SUMO-specific proteases with
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SUMO-specific proteases. The Journal of biological chemistry 2005; 280:14492-8.
preference for SUMO-2 or SUMO-3. The Journal of biological chemistry 2006; 281:15869-77. 16. Bawa-Khalfe T, Yeh ET. SUMO Losing Balance: SUMO Proteases Disrupt SUMO Homeostasis to Facilitate Cancer Development and Progression. Genes & cancer 2010; 1:748-52. 17. Rashid AJ, Cole CJ, Josselyn SA. Emerging roles for MEF2 transcription factors in memory. Genes Brain Behav 2013. 20
18. Shalizi A, Gaudilliere B, Yuan Z, Stegmuller J, Shirogane T, Ge Q, Tan Y, Schulman B, Harper JW, Bonni A. A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation. Science 2006; 311:1012-7.
ip t
19. Flavell SW, Cowan CW, Kim TK, Greer PL, Lin Y, Paradis S, Griffith EC, Hu LS, Chen C,
us
20. Shalizi A, Bilimoria PM, Stegmuller J, Gaudilliere B, Yang Y, Shuai K, Bonni A. PIASx is a MEF2
an
SUMO E3 ligase that promotes postsynaptic dendritic morphogenesis. The Journal of neuroscience : the official journal of the Society for Neuroscience 2007; 27:10037-46.
M
21. Lu H, Liu B, You S, Chen L, Dongmei Q, Gu M, Lu Y, Chen Y, Zhang F, Yu B. SENP2 regulates MEF2A de-SUMOylation in an activity dependent manner. Molecular biology reports 2013;
ed
40:2485-90.
pt
22. Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drug resistance in cancer. Biochem Pharmacol 2012; 83:1021-32.
ce
23. Barford D. Structure, function and mechanism of the anaphase promoting complex (APC/C). Q Rev Biophys 2011; 44:153-90.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
excitatory synapse number. Science 2006; 311:1008-12.
cr
Greenberg ME. Activity-dependent regulation of MEF2 transcription factors suppresses
24. Almeida A. Regulation of APC/C-Cdh1 and its function in neuronal survival. Mol Neurobiol 2012; 46:547-54.
25. Zeng X, Sigoillot F, Gaur S, Choi S, Pfaff KL, Oh DC, Hathaway N, Dimova N, Cuny GD, King RW. Pharmacologic
inhibition
of
the
anaphase-promoting
complex
induces
a
spindle
checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell 2010; 21
18:382-95. 26. Zeng X, King RW. An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination. Nat Chem Biol 2012; 8:383-92.
ip t
27. Wang X, She H, Mao Z. Phosphorylation of neuronal survival factor MEF2D by glycogen synthase kinase 3beta in neuronal apoptosis. J Biol Chem 2009; 284:32619-26.
cr
us
survival mediated by transcription factor MEF2. Science 1999; 286:785-90.
an
29. Loriol C, Khayachi A, Poupon G, Gwizdek C, Martin S. Activity-dependent regulation of the sumoylation machinery in rat hippocampal neurons. Biology of the cell / under the auspices of
M
the European Cell Biology Organization 2013; 105:30-45.
30. Hu D, Qiao X, Wu G, Wan Y. The emerging role of APC/CCdh1 in development. Semin Cell
ed
Dev Biol 2011; 22:579-85.
pt
31. Manchado E, Eguren M, Malumbres M. The anaphase-promoting complex/cyclosome (APC/C): cell-cycle-dependent and -independent functions. Biochem Soc Trans 2010; 38:65-71.
ce
32. Zhang XD, Goeres J, Zhang H, Yen TJ, Porter AC, Matunis MJ. SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
28. Mao Z, Bonni A, Xia F, Nadal-Vicens M, Greenberg ME. Neuronal activity-dependent cell
Mol Cell 2008; 29:729-41. 33. Yang Y, Kim AH, Bonni A. The dynamic ubiquitin ligase duo: Cdh1-APC and Cdc20-APC regulate neuronal morphogenesis and connectivity. Curr Opin Neurobiol 2010; 20:92-9. 34. Pick JE, Malumbres M, Klann E. The E3 ligase APC/C-Cdh1 is required for associative fear memory and long-term potentiation in the amygdala of adult mice. Learn Mem 2013; 20:11-20. 22
35. Pick JE, Wang L, Mayfield JE, Klann E. Neuronal expression of the ubiquitin E3 ligase APC/C-Cdh1 during development is required for long-term potentiation, behavioral flexibility, and extinction. Neurobiol Learn Mem 2013; 100:25-31.
ip t
36. Wan Y, Liu X, Kirschner MW. The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction. Mol Cell 2001; 8:1027-39.
cr
us
L, Pagano M, Bonni A, Iavarone A. Degradation of Id2 by the anaphase-promoting complex
an
couples cell cycle exit and axonal growth. Nature 2006; 442:471-4.
38. Krumova P, Weishaupt JH. Sumoylation in neurodegenerative diseases. Cell Mol Life Sci
ce
pt
ed
M
2013; 70:2123-38.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
37. Lasorella A, Stegmuller J, Guardavaccaro D, Liu G, Carro MS, Rothschild G, de la Torre-Ubieta
23
ip t cr us an M
ed
ubiquitination and degradation
pt
A. Lysates of primary rat cortex neuron cells depolarized with 50 mM KCl for the indicated
ce
time were immunoblotted for SynGAP and SENP2. β-actin was used as loading control. The number on the bottom of Western blot lanes indicated signal intensity of SENP2 or SynGAP protein against β-actin. The signal intensity of SENP2 or SynGAP protein against β-actin at 0
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.1. Activity-dependent stimuli activate MEF2A activation by preventing SENP2
min was set as 1.
B. SHSY5Y cells pretreated with 50 mM KCl or 150 mM NaCl (corresponds to physiological conditions: 0.9% NaCl) for 1h and 20mM CHX was added for the indicated time. Lysates from these cells were immunoblotted for SENP2. Histograms represent the ratio of the signal intensity of SENP2 protein quantified against β-actin. 24
C. SHSY5Y cells were transfected with Flag-SENP2 and HA-ubiquitin plasmids for 36h. Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h. Cells were lysed and subjected to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted
ip t
with anti-HA antibody. D. SHSY5Y cells were transfected with indicated plasmids for 36h. Cells were then treated
cr
us
immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted
an
with anti-HA antibody.
E. SHSY5Y cells were transfected with non-specific siRNA or siRNA against SENP2 for 48h.
immunoblotted for SENP2.
SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with
ed
F.
M
Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h. The whole cell lysates were
pt
non-specific siRNA or siRNA against SENP2 for 36h. Cells were then treated with 50 mM KCl
ce
or 150 mM NaCl for 2h, the luciferase activity was measured. Transfection efficiency was normalized by Renilla luciferase expression, and the results are presented as activation over that for non-specific siRNA transfected group. The y axis represents normalized luciferase activity ±
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
with 50 mM KCl or 150 mM NaCl for 2h. Cells were lysed and subjected to
SEM (n = 3). **represents p < 0.01, *** represents p < 0.001.
25
ip t cr us an M ed
pt
A. SHSY5Y cells were treated with 50 mM KCl or 150 mM NaCl for 2h and the
ce
nuclear-cytoplasmic fraction of these cells was conducted. Lysates from each fraction were immunoblotted for SENP2. Tubulin and Lamin B were used to indicate cytoplasmic or nuclear fraction, respectively. The number on the bottom of Western blot lanes indicated signal intensity
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.2. Activity-dependent stimuli promote SENP2 nuclear translocation
of SENP2 protein against β-Tubulin or Lamin B. The signal intensity of SENP2 protein against β-Tubulin or Lamin B without KCl treatment was set as 1. B. Schematic diagrams of the NLS, NES and Protease domain of SENP2. C. SHSY5Y cells were transfected with Flag-SENP2 WT or Flag-SENP2 mNLS for 36h. Cells were treated with 50 mM KCl or 150 mM NaCl for 2h. The whole cell lysates were 26
immunoblotted with Flag antibody. D. SHSY5Y cells were transfected with indicated plasmids for 36h. Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h, the luciferase activity was measured. The y axis
ip t
represents normalized luciferase activity ± SEM (n = 3). **represents p < 0.01. E. SHSY5Y cells were transfected with Flag-SENP2 for 32h and treated with 10nM LMB or
cr
us
each fraction were immunoblotted for Flag-SENP2. Tubulin and Lamin B were used to indicate
F.
an
cytoplasmic or nuclear fraction, respectively.
SHSY5Y cells were transfected with Flag-SENP2 and His-ubiquitin plasmids for 36h. Cells
M
were then treated with 10nM LMB. Cells were lysed and subjected to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted with anti-His antibody.
ed
G. SHSY5Y cells were transfected with Flag-MEF2A and HA-SUMO1 for 36h. Cells were
pt
then treated with 10nM LMB for 2h. Cells were lysed and subjected to immunoprecipitation with
ce
Flag M2 beads. Immunoprecipitated proteins were immunoblotted with anti-HA and Flag antibodies. H.
SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids for 36h.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
DMSO for 2h and the nuclear-cytoplasmic fraction of these cells was conducted. Lysates from
Cells were then treated with 10nM LMB for 2h, the luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). **represents p < 0.01 I.
Lysates of primary rat cortex neuron cells treated with 10nM LMB or DMSO were
immunoblotted for SynGAP and SENP2.
27
ip t cr us an
M
A. Alignment of D boxes in Skp2, Cyclin B1, Securin and SENP2.
pt
Homo sapiens
ed
B. Alignment of D boxes in SENP2 from Mus musculus, Rattus norvegicus, Pongo abelii and
ce
C. SHSY5Y cells were treated with 10μM proTAME for 12h. The whole cell lysates were immunoblotted with SENP2 and Cyclin B1 antibodies. D. SHSY5Y cells were treated with 10μM proTAME for 12h. The whole cell lysates were
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.3. SENP2 contains a canonical D-box motif.
immunoblotted with SUMO1 antibody. E. SENP2 mRNA level from SHSY5Y cells treated with 10μM proTAME for 12h was detected by Realtime-PCR. The y axis represents mean ± SEM (n = 3).
28
ip t cr us an M
ed
A. 293T cells were transfected with indicated plasmids for 36h. Cells were lysed and subjected
pt
to immunoprecipitation with Flag M2 or HA beads. Immunoprecipitated proteins were
ce
immunoblotted with indicated antibodies. B. Lysates of SHSY5Y cells depolarized with 50 mM KCl for 2h subjected to immunoprecipitation with SENP2, Cdh1 or IgG antibodies.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.4. SENP2 is a substrate of APCCdh1
Immunoprecipitated proteins were
immunoblotted with indicated antibodies. C. Beads coated with bacterially expressed GST or GST-SENP2 were incubated with purified HA-Cdh1 protein. Beads were washed, and the bound proteins were analyzed by Western blotting with indicated antibodies. The number on the bottom of Western blot lanes indicated 29
signal intensity of Cdh1. The signal intensity of input Cdh1 protein was set as 1. D. SHSY5Y cells were transfected with indicated increased HA-Cdh1 plasmid for 36h. In MG132 group, 10μM was added 6h before cell harvested. The whole cell lysates were
ip t
immunoblotted with indicated antibodies. E. SHSY5Y cells transfected with empty vector or HA-Cdh1 plasmids were treated with 20 μM
cr
us
The number on the bottom of Western blot lanes indicated signal intensity of SENP2 protein
F.
an
against β-actin. The signal intensity of SENP2 protein against β-actin at 0 min was set as 1. SHSY5Y cells were transfected with non-specific siRNA or siRNAs targeting Cdh1 for 48h.
M
The whole cell lysates were immunoblotted with SENP2 and Cdh1 antibodies. G. SHSY5Y cells were transfected with empty vector or HA-DN-Cdh1 for 48h. The whole cell
ed
lysates were immunoblotted with SENP2 and HA antibodies.
pt
H. 293T cells were transfected with indicated plasmids for 36h. Cells were lysed and subjected
ce
to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted with indicated antibodies.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
CHX for the indicated time. The whole cell lysates were immunoblotted with SENP2 antibody.
30
us
cr
ip t B.
an
A. Schematic diagrams of SENP2 WT and Mut
SHSY5Y cells were transfected with indicated plasmids for 48h. The whole cell lysates
were immunoblotted with Flag and HA antibodies.
M
C. Beads coated with bacterially expressed GST-SENPWT or GST-SENP2Mut were incubated with purified HA-Cdh1 protein. Beads were washed, and the bound proteins were analyzed by
D
ed
western blotting with indicated antibodies.
SHSY5Y cells transfected with Flag-SENP2 WT or Flag-SENP2 Mut for 36h were treated
pt
with 20 μM CHX for the indicated time. In MG132 group, 10μM was added 6h before cell
ce
harvested. The whole cell lysates were immunoblotted with indicated antibodies. E. SHSY5Y cells transfected with Flag-SENP2 WT or Flag-SENP2 Mut for 36h were treated
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.5. D-box-dependent degradation of SENP2 by APCCdh1
with 10μM TAME for 12h. The whole cell lysates were immunoblotted with indicated antibodies. F.
293T cells were transfected with indicated plasmids for 36h. Cells were lysed and subjected
to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted with indicated antibodies. 31
ip t cr us an M ed pt
Downloaded by [Northwestern University] at 03:58 20 December 2014
ce
Ac
Figure.6. Cdh1 regulates MEF2A transcriptional activity via SENP2 destruction A. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with Cdh1 or Cdc20 plasmids for 36h. The luciferase activity was measured. Transfection efficiency was normalized by Renilla luciferase expression, and the results are presented as activation over that for empty vector transfected group. The y axis represents normalized luciferase activity ± 32
SEM (n = 3). **represents p < 0.01 compared with Con group. B. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with indicated increased Cdh1 for 36h. The luciferase activity was measured. The y axis represents
ip t
normalized luciferase activity ± SEM (n = 3). C. SHSY5Y cells were transfected with indicated plasmids for 36h. Cells were then treated
cr
us
immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted
an
with indicated antibodies.
D. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with
M
indicated plasmids for 36h. Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h. The luciferase activity was measured. The y axis represents normalized luciferase activity ±
ed
SEM (n = 3). *represents p < 0.05, **represents p < 0.01.
pt
E. SHSY5Y cells were transfected with MEF2A and SynGap reporter plasmids with indicated
ce
plasmids for 36h. Cells were then treated with 50 mM KCl for 2h. The luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). *represents p < 0.05, **represents p < 0.01.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
with 50 mM KCl or 150 mM NaCl for 2h. Cells were lysed and subjected to
F. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with siRNAs specific targeting Cdh1 for 36h. The luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). **represents p < 0.01 compared with Con-siRNA group. G. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with 33
indicated plasmids for 36h. The luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). *** represents p < 0.001 CDH1+ SENP2 Mut compared with CDH1 group.
ip t
H. Rat Primary Cerebellar Granule Neurons (CGNs) cultured in medium containing serum and depolarizing concentrations of KCl (29mM) were transfected with indicated plasmids for 30h.
cr
us
triggered CGN apoptosis model. The percentage of apoptotic cells were determined by
an
Annexin-V/PI staining assay. The y axis represents normalized luciferase activity ± SEM (n = 3). * represents p < 0.05 compared with Con group.
M
I. Rat Primary Cerebellar Granule Neurons (CGNs) cultured in medium containing serum and depolarizing concentrations of KCl (29mM) were transfected with indicated siRNAs for 30h.
ed
CGNs were switched to medium containing 5 mM KCl for 8h to generate KCl withdrawal
pt
triggered CGN apoptosis model. The percentage of apoptotic cells were determined by
ce
Annexin-V/PI staining assay. The y axis represents normalized luciferase activity ± SEM (n = 3). *represents p < 0.05 compared with Con-siRNA group.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
CGNs were switched to medium containing 5 mM KCl for 8h to generate KCl withdrawal
34
Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
The E3 ligase APC/C-Cdh1 regulates MEF2A-dependent transcription by targeting SUMO-specific protease 2 for ubiquitination and degradation a
b
a
c
a
a
a
Han Lu , Bin Liu , Fu-Jun Zhang , Jin Zhang , Rong Dong , Lei Chen , Dong-Mei Qu , Yan Lu
d
a
& Bu-Wei Yu a
Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai, People's Republic of China b
Key Laboratory of Kidney Disease Pathogenesis and Intervention of Hubei Province, College of Medicine, Hubei Polytechnic University, Huangshi, Hubei, PR China c
Click for updates
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People's Republic of China d
Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Accepted author version posted online: 31 Oct 2014.Published online: 17 Oct 2014.
To cite this article: Han Lu, Bin Liu, Fu-Jun Zhang, Jin Zhang, Rong Dong, Lei Chen, Dong-Mei Qu, Yan Lu & Bu-Wei Yu (2014): The E3 ligase APC/C-Cdh1 regulates MEF2A-dependent transcription by targeting SUMO-specific protease 2 for ubiquitination and degradation, Cell Cycle, DOI: 10.4161/15384101.2014.973302 To link to this article: http://dx.doi.org/10.4161/15384101.2014.973302
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
Downloaded by [Northwestern University] at 03:58 20 December 2014
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
The E3 ligase APC/C-Cdh1 regulates MEF2A-dependent transcription by targeting SUMO-specific protease 2 for ubiquitination and degradation Han Lu1*, Bin Liu2*, Fu-Jun Zhang1*, Jin Zhang3, Rong Dong1, Lei Chen1, Dong-Mei Qu1, Yan
ip t
Lu4, Bu-Wei Yu1# 1. Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong University School of
cr
us
Disease Pathogenesis and Intervention of Hubei Province, College of Medicine, Hubei
an
Polytechnic University, Huangshi, Hubei, PR China. 3. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China. 4. Department of
M
Endocrine and Metabolic Diseases, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao
pt
ed
Tong University School of Medicine, Shanghai, China.
ce
*These people contributed equally.
#Corresponding author
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Medicine (SJTU-SM), Shanghai, People’s Republic of China. 2. Key Laboratory of Kidney
Bu-Wei Yu, E-mail: [email protected]
Keywords: Activity-dependent stimuli; MEF2A; APC-Cdh1; SENP2
1
Abstract Activity-dependent stimuli induced a
calcineurin-mediated dephosphorylation of the
transcriptional factor MEF2A at serine408 and promoted a switch from SUMOylation to
ip t
acetylation at lysine403 which led to MEF2A transcriptional activation. We previously identified SENP2 is the de-SUMOylation enzyme for MEF2A and promotes MEF2A-dependent
cr
us
MEF2A transcriptional activation. APCCdh1 interacts with and targets SENP2 for ubiquitination
an
and destruction in the cytoplasm by recognizing a conserved canonical D-box motif in SENP2. Moreover, Cdh1 regulates the transcriptional activity of MEF2A in a SENP2 dependent manner.
M
Activity-dependent stimuli prevented APCCdh1-induced SENP2 ubiquitination, promoted SENP2 nuclear accumulations, and caused MEF2A de-SUMOylation and MEF2A acetylation, leading to
ed
MEF2A transcriptional activation. Thus, our findings defined a post-transcriptional mechanism
ce
pt
underlying activity-dependent stimuli-induced MEF2A transcriptional activation.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
transcription. We report here a requirement for APCCdh1-SENP2-MEF2A axis in the regulation of
2
Introduction Activity-dependent stimuli can modulate neuronal plasticity, which is a result of the brain’s ability to convert transient stimuli into long-lasting alterations in neuronal structure and function.
ip t
This results in an increase (potentiation) or a decrease (depression) in synaptic strength1. This process involves changes in receptor trafficking, protein–protein interactions, local mRNA
cr
us
phosphorylation, acetylation, ubiquitination and SUMOylation2-5.
an
SUMOylation is the covalent linkage of a small ubiquitin-related modifier (SUMO) to the -amine of lysine residues in target proteins. It is an important regulatory mechanism for
M
modifying protein activity, stability, and cellular localization6. Mammalian cells express four SUMO paralogs: SUMO1–SUMO4. Human SUMO2 and SUMO3 are about 95% identical to
ed
each other, whereas they share only 45% identity with SUMO1. SUMO4 is the least well
pt
characterized isoform and is expressed mainly in the kidney, lymph node and spleen, whereas
ce
SUMO1, SUMO2 and SUMO3 are ubiquitously expressed7. SUMOylation is a high dynamic process that is catalyzed by the activating (E1), conjugating (E2) and ligating (E3) enzymes and reversed by a family of Sentrin/SUMO-specific proteases (SENPs)8. In mammalian cells, six
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
translation, new gene synthesis and protein post-translational modifications including
SENPs have been identified, which have different substrate specificity and subcellular localization. SENPs are divided into three subfamilies on the basis of their sequence homology, cellular location and substrate specificity9. The first subfamily includes SENP1 and SENP2, which have broad substrate specificity
10, 11
. SENP2 is a nucleocytoplasmic shuttling protease
that appears to have a activity similar to that of SENP1 when overexpressed12. Loss of mouse 3
SENP2 caused embryonic lethality
13
. SENP2 is essential for embryonic cardiac development
through regulation of the SUMOylation status of Pc2/CBX4 and has a specific role in the G-S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast
ip t
proliferation and differentiation 13, 14. The second subfamily includes SENP3 and SENP5, both of which are nucleolar proteins with preference for SUMO-2/315. The third subfamily includes
cr
us
The MEF2A transcriptional factor is highly expressed in the brain and plays a key role in
for
postsynaptic
differentiation
an
synaptic dendritic development in the cerebellar cortex17. The modifications of MEF2A required occur
within
a
phosphorylation-regulated
M
SUMOylation-acetylation switch (SAS) peptide motif18. Activity-dependent stimuli increased MEF2A transcriptional activity by activating a highly programmed post-transcriptional event
ed
taken place on MEF2A, including MEF2A de-phosphorylation, MEF2A de-SUMOylation and
pt
MEF2A acetylation19. Calcineurin dephosphorylates MEF2A Ser408 which resulted in Lys403 de-SUMOylation,
sequential
Lys403
acetylation
and
de-represses
MEF2A-induced
ce
transcription18. Further study discovered that PIASx promoted postsynaptic dendritic morphogenesis by enhancing the SUMOylation of MEF2A20. We previously identified SENP2
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP6 and SENP7, which have an extra loop in their catalytic domains16.
was the predominant de-SUMOylation enzyme for MEF2A by using unbiased functional loss of function screen21. We found that SENP2 regulates the transcriptional function of MEF2A via direct de-SUMOylation. In conformity with these clues, MEF2A was heavily SUMOylated in SENP2-/- embryo21. However, how endogenous SENP2 is regulated during this process is still undefined. 4
Results Activity-dependent stimuli prevent SENP2 ubiquitination and degradation We first investigated whether endogenous SENP2 was regulated under KCl-induced
ip t
depolarization manipulation by using primary rat cortex neuron cells. KCl-induced depolarization promote the transcriptional activity of MEF2A has been well documented18, 19.
cr
us
the accumulation of SynGAP, one of the downstream target genes of MEF2A 18(Fig.1A). Under
an
the same circumstances, endogenous SENP2 protein was also accumulated in a time-dependent manner (Fig.1A). However, the mRNA level of SENP2 was almost unchanged, suggesting a
M
post-transcriptional regulation was taking place (Fig.S1). SENP2 has been reported to be a short-lived protein and destructed via ubiquitin-proteasome system12. We proposed that
ed
KCl-induced endogenous SENP2 accumulation could be due to impaired SENP2 destruction.
pt
Indeed, the half-life of endogenous SENP2 was significantly increased in KCl-treated SHSY5Y
ce
cells (Fig.1B). Moreover, KCl stimuli dramatically decreased the ubiquitination form of SENP2 (Fig.1C). Consistent with these data, KCl stimuli further enhanced the de-SUMOylation activity of SENP2 towards MEF2A and caused MEF2A SAS transition (Fig.1D) and SENP2-mediated
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Indeed, KCl treatment significantly enhanced MEF2A transcriptional activation as evidenced by
MEF2A transcriptional activation (Fig.S2). Furthermore, silencing the expression of SENP2 by siRNA largely abolished KCl-induced MEF2A activation (Fig.1E, 1F). Taken together, these data indicated that activity-dependent stimuli prevents SENP2 ubiquitination and degradation accompanied with MEF2A activation. Activity-dependent stimuli promote SENP2 nuclear translocation 5
SENP2 is reported to be a nucleocytoplasmic shuttling protein and is ubiquitinated in the cytoplasm12. We then asked that whether activity-dependent stimuli promoted SENP2 nuclear translocation. To test this possibility, primary rat cortex neuron cells were treated with 50mM
ip t
KCl to induce SENP2 accumulation. The nuclear-cytoplasmic fraction of primary rat cortex neuron cells was conducted. As depicted in Fig.2A, KCl stimuli significantly promoted SENP2
cr
12
(Fig.2B). A cytoplasm-localized
us
CRM1-dependent nuclear export signal (NES) in SENP2
an
SENP2 made by introducing mutations in the NLS (SENP2 mNLS) was unable to accumulate in response to KCL stimuli in SHSY5Y cells (Fig.2C, Fig.S3). KCl stimuli could also not decrease
M
the ubiquitination form of SENP2 mNLS (Fig.S4). Moreover, overexpression of SENP2 mNLS could not increase the transcriptional activity of MEF2A when compared with SENP2 WT
ed
(Fig.2D). leptomycin B, an inhibitor of CRM1, could specifically inhibit the CRM1-dependent
pt
nuclear export activity of the NES22. LMB treatment did not affect the mRNA level of SENP2
ce
(Fig.S5). However, LMB treatment inhibited the nuclear export of SENP2 (Fig.2E), prevented SENP2 ubiquitination (Fig.2F), promoted MEF2A de-SUMOylation (Fig.2G), transcriptional activation (Fig.2H) and downstream target gene SynGAP expression (Fig.2I). Conclusively,
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
nuclear translocation. There is a bipartite nuclear localization signal (NLS) and a
these data suggested that activity-dependent stimuli prevent SENP2 ubiquitination and degradation by promoting its nuclear translocation. SENP2 contains a canonical D-box motif. To identify which E3 ligase is required for the degradation of SENP2, we first checked the protein sequence of SENP2 for potential degrons. We noted that SENP2 contains a canonical 6
‘destruction box’ (D-box) motif (R-X-X-L-X-X-X-X-N/D/E) (Fig.3A). D-box and KEN are two degradation motifs have been identified in the substrates of Anaphase-Promoting Complex (APC), which is a multi-subunit E3 protein23. APC is responsible for ubiquitination of cell cycle
ip t
regulators in non-neuronal cells but also highly expressed and has additional function in the post-mitotic neurons24. Most APC substrates have D box and some have either a KEN box or
cr
us
mention that, this D-box motif of SENP2 is evolutionarily conserved among different species
an
including Mus musculus, Rattus norvegicus, Pongo abelii and Homo sapiens (Fig.3B). Tosyl-L-arginine methyl ester (TAME), which is a potent inhibitor of APC, could directly bind to
M
APC and disrupts the interactions between APC and its activator proteins Cdc20 or Cdh125, 26. Administration of proTAME resulted in the accumulation of Cyclin B1, a well-known substrate
ed
of APC (Fig.3C). Administration of TAME also caused SENP2 accumulation and slightly
pt
decrease global SUMOylation (Fig.3C, 3D). However, the mRNA level of SENP2 was almost unchanged (Fig.3E). Collectively, these data suggested that SENP2 might be a substrate of APC.
ce
SENP2 is a substrate of APCCdh1
To investigate whether SENP2 is a substrate of APC, we first tested the interaction between
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
both D and KEN boxes. In some cases two D boxes appear in the same protein. It is worth to
Cdh1 and SENP2 by coimmunoprecipitation of tagged Cdh1 and SENP2overexpressed in 293T cells. Cdh1 was able to bind to SENP2, whereas Cdc20 another coactivator of APC complex did not show any detectable association with SENP2 (Fig.4A, Fig.S6). Endogenous Cdh1 and SENP2 could also be efficiently co-immunoprecipitated with each other in SHSY5Y cells (Fig.4B). We further confirmed the interaction between Cdh1 and SENP2 by in vitro 7
precipitation assays in which substantial HA-tagged Cdh1 was precipitated specifically by glutathione S-transferase (GST)-SENP2 but not by GST alone (Fig.4C). Expression of Cdh1 but not that of Cdc20 dramatically reduced SENP2 protein level which was prevented by MG132
ip t
administration (Fig.4D, Fig.S7). Moreover, SENP2 was degraded at a faster rate in the presence of Cdh1 (Fig. 4E). Consistent with these observations, silencing the expression of Cdh1 by
cr
us
accumulation (Fig.4F, Fig.4G). Furthermore, the half-life of SENP2 was significantly increased
an
when Cdh1 was silenced (data not shown). Lastly, Cdh1 significantly promoted the ubiquitination of SENP2 in vivo (Fig.4H). Taken together, our data indicated that SENP2 is a
M
bona fide substrate of APCCdh1.
D-box-dependent degradation of SENP2 by APCCdh1
ed
To investigate whether the degradation of SENP2 was dependent on its D-box motif, we
pt
generated mutant SENP2 protein (SENP2 Mut) in which the key arginine and leucine residues
ce
were changed into alanine and valine, respectively (Fig.5A). SENP2 Mut was expressed at level higher than SENP2 WT and was resistant to Cdh1-mediated destabilization (Fig.5B). Furthermore, GST–SENP2 WT but not GST–SENP2 Mut captured HA-tagged Cdh1 from
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
siRNA or overexpression of a domination negative form of Cdh1 (DN-Cdh1) resulted in SENP2
SHSY5y cell extract, suggesting the D box is essential for the recognition of SENP2 by APCCdh1(Fig.5C). To determine the importance of the D-box-mediated degradation of SENP2, we monitored the degradation rate of SENP2 WT and SENP2 Mut. In transiently transfected SHSY5y cells, the half-life of SENP2 Mut was significantly extended when compared with SENP2 WT (Fig.5D). In agreement with these data, TAME has little effect on SENP2 Mut 8
protein when compared with SENP2 WT protein (Fig.5E). Cdh1 also significantly promoted the ubiquitination of SENP2WT but not SENP2 Mut in vivo (Fig.5F). Together, these data suggested that APCCdh1 can polyubiquitinate SENP2 in a D-box-dependent manner.
ip t
Cdh1 regulates MEF2A transcriptional activity via SENP2 destruction To investigate the functional significance of APCCdh1-mediated SENP2 destruction, we test
cr
us
dramatically repressed MEF2A transcriptional activity (Fig.6A). Indeed, Cdh1 repressed MEF2A
an
transcriptional activity in a dose dependent manner (Fig.6B). Furthermore, we found that overexpression of Cdh1 also decreased KCl-induced MEF2A de-SUMOylation, decreased
M
KCL-induced MEF2A acetylation, and KCl-induced MEF2A activation (Fig.6C, 6D). Indeed, using SynGAP and Nur77 neuronal specific promoters, we found that overexpressing Cdh1
ed
significantly decreased SynGAP- and Nur77-luciferase reporter genes activity in SHSY5Y cells
pt
treated with KCl (Fig.6E, Fig.S8). Furthermore, silencing the expression of Cdh1 by siRNA or
ce
overexpression of DN-Cdh1 promotes the transcriptional activity of MEF2A and decreases MEF2A SUMOylation (Fig.6F, Fig.S9). Co-expression of SENP2 Mut but not SENP2 WT significantly restored the transcriptional activity of MEF2A repressed by Cdh1 (Fig.6G).
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
whether Cdh1 regulates MEF2A transcriptional activity. Overexpression of Cdh1 but not Cdc20
Previous studies have showed that membrane depolarization by KCl promotes the survival of neurons, and this process is dependent on MEF2 transactivation activity27, 28. Thus, we proposed that APC/Cdh1–Senp2 axis may play a role in activity-dependent neuronal survival. To this end, we found that overexpression of Cdh1 or silencing the expression of SENP2 could prevent activity dependent neuronal survival, whereas overexpression of SENP2 or DN-Cdh1 could 9
promote
activity
dependent
neuronal
survival
(Fig.6H,
Fig.6I),
suggesting
that
APC/Cdh1–Senp2-MEF2 axis at least plays a role in activity dependent neuronal survival. Discussion
ip t
By checking the protein level of SENP2 in response to activity-dependent stimuli, we have found that exogenous SENP2 was rapidly stabilized to facilitate MEF2A de-SUMOylation and
cr
us
stimuli. SENP2 was reported to be a nucleocytoplasmic shuttling protein and ubiquitinated in the
an
cytoplasm. We found that activity-dependent stimuli could increase the half-life of SENP2 by preventing SENP2 ubiquitination and degradation. Our earlier work revealed that
M
activity-dependent stimuli could significantly increase global SUMOylation and alter the subcellular distribution of SUMO conjugated proteins4. Ce´ line et al. also demonstrated that in hippocampal neurons, the SUMOylation enzyme UBC9 and
ed
primary cultured rat
pt
deSUMOylation enzymes SENP1 and SENP6 are differentially redistributed in and out of
ce
synapses upon activity-dependent stimulation29. We then asked whether neuronal depolarization could also alter the distribution of SENP2. Indeed, SENP2 was translocated from cytosol to nucleus in response to activity-dependent stimuli, suggesting that activity-dependent stimuli
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
activation. In this study, we mainly focused on the regulation of SENP2 by activity-dependent
prevent SENP2 ubiquitination and degradation by promoting its nuclear translocation. We next asked which E3 ligase is required for the degradation of SENP2. Bioinformational
analysis found that SENP2 contains a canonical D-box degron which is also presented in the well-known substrates of APCCdh1. This clue led us to investigate the relationship between APCCdh1 and SENP2. By using various biochemistry studies, we found that Cdh1 but not Cdc20, 10
another APC/C activator, physically interacted with SENP2 and targeted SENP2 for ubiquitin-dependent degradation. We also test the functional significance of APCCdh1-mediated SENP2 destruction. We found that Cdh1 could enhance MEF2A SUMOylation and decrease the
ip t
MEF2A transcriptional activity. Overexpression of stable SENP2 form (SENP2 Mut) could significantly restore Cdh1-repressed MEF2A activation. We also found that both Cdh1 and
cr
us
previous unknown Cdh1-SENP2-MEF2A axis that mediated activity-dependent stimuli-induced
an
MEF2A transcriptional activation and activity-dependent neuronal survival.
Cdh1 is required to initiate APC activity during late mitosis and G1 and is the master of 30, 31
. Recently, SENP2 was reported to play a role in the
M
G0/G1 phase in non-neuronal cells
regulation of cell cycle progress 32. However, whether SENP2 itself is regulated during cell cycle
ed
progress is largely unknown. Further studies are needed to investigate the functional relevance
pt
between Cdh1 with SENP2 in the cell cycle regulation in the future.
ce
MEF2A has been reported to be able to regulate postsynaptic dendritic morphogenesis 18. It will be interesting to test whether SENP2 or Cdh1 could play a role in postsynaptic dendritic morphogenesis. APCCdh1 complex is highly expressed in the post-mitotic neurons, which
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP2 were participated in activity-dependent neuronal survival. Thus, our data clarified a
restrains the intrinsic axon growth potential33. APCCdh1 complex is required for associative fear memory and long-term potentiation in the amygdala of adult mice34, 35. APCCdh1 complex targets both SnoN and Id2 for destruction to elevate the expression of growth inhibitory molecules results in neurite outgrowth36, 37. It will be reasonable to test whether Cdh1 plays a role in postsynaptic dendritic morphogenesis in the future. SENP2 was also reported to be highly 11
expressed in central neuron system13. However, the biology function of SENP2 is completely unknown in central neuron system due to the embryonic lethal of SENP2-/- mice. Indeed, several neuronal SUMOylation substrates have been identified while the de-SUMOylases specific for
ip t
these substrates were remain to be identified38. Systematic identification of the neuron specific SUMOylation substrates of SENP2 might also be required to clarify the neuronal function of
cr
us
the neuronal function of SENP2 in the future.
an
Material and methods Isolating Rat Neurons
M
All the reagents used for neurons were purchased form Invitrogen. The cortex was dissected from ten E-18 rat embryo brains and put in a conical tube containing Hibernate®-E
ed
supplemented with 2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I at 4°C.
pt
Enzymatically digest the tissue in 4 mL of Hibernate®-E medium without Ca2+ containing 2
ce
mg/mL papain for 30 minutes at 30°C. 6 mL complete Hibernate®-E medium was added to the tube and centrifuge for 5 minutes at 150 × g. The supernatant was removed and the cells were transferred to a new tube in Neurobasal® medium with 2% B-27® Serum-Free Supplement and
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP2. Neuron- or glial-specific knockout of SENP2 mice will be a powerful tool to investigate
0.5 mM GlutaMAX™-I for culturing. Cell culture and treatments Primary rat cortex neuron cells were cultured in poly-D-lysine coated 6-well plate and cultured with in Neurobasal® medium. SHSY5Y neuroblastoma cell in high passages was purchased from ATCC, and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) 12
containing 10 % fetal bovine serum (FBS), 2 mM glutamine and 1 % antibiotics (penicillin–streptomycin). Cells were maintained at 37°C in a humidified 5 % CO2 atmosphere. The cells were depolarized by KCl in the medium with addition of 31 % depolarization buffer
ip t
(170 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES) at a final concentration of 50 mM KCl for the indicated lengths of time. MG132 was purchased from Calbiochem, TAME
cr us
Plasmids and Transfection
an
The 3 X MRE-luc and Gal4-MEF2A expression plasmids were gifts from Dr. Michael Greenberg. The pCDNA3-MEF2A-Flag expression plasmid was a gift from Dr. Rhonda Bassel-Duby.
M
Flag-SENP2, HA-SUMO1, His-Ubiquitin, Myc-SUMO1 and luciferase renilla reporter constructs, have been described previously. Cdh1 and Cdc20 were amplified from 293T cells by
ed
PCR and cloned into the pcDNA 3.1 vector. SENP2 MUT, SENP2 mNLS, DN-Cdh1 and
pt
DN-Cdc20 were generated using QuickChange Site-Directed Mutagenesis Kit (Stratagene). All cDNAs were completely sequenced. Cells were transiently transfected using Lipofectamine 2000
ce
(Invitrogen), according to the manufacturer’s instructions. RNAi
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
hydrochloride was purchased from sigma.
siRNA against SENP2 (J-006033-05) and nonspecific siRNA (D-001810-01) were purchased from Dharmacon RNA Technologies as previously described. The siRNA oligonucleotides targeting
human
Cdh1
(GAAGGGUCUGUUCACGUAUTT)
and
(CGGCAGGACUCCGGGCCGATT) was chemically synthesized by Dharmacon.
13
Cdc20
Nuclear-cytoplasmic fraction The nuclear-cytoplasmic fraction of rat primary cortex neurons and SHSY5Y cells were conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher
ip t
Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. SUMOylation assays
cr
us
cotransfected with expression plasmids for Flag-MEF2A and HA-SUMO1 were lysed in RIPA
an
buffer (150mM NaCl, 10mM Na2HPO4, pH 7.2, 2mM EDTA, 50mM NaF, 1mM NaVO4, 1%NP-40, 0.1% SDS, 0.75% sodium deoxycholate, 1mMPMSF, 10mM N-ethylmaleimide). Five
M
percent of this starting material was retained for detection of input proteins, and the remainder was subjected to immunoprecipitation overnight at 4°C. Flag M2 antibody (sigma) was used
ed
together with protein A-Sepharose beads. Immune complexes were washed eight times with
pt
RIPA buffer at 4°C and resuspended in 2XSDS loading buffer. Immune complexes and input
ce
samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with HA or Flag antibodies.
Immunoprecipitation (IP) assay
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
In vivo SUMOylation assays were performed as described previously21. Briefly, cells
Immunoprecipitation experiments were performed as described previously with minor modifications21. Briefly, cells cotransfected with tagged plasmids were lysed in IP buffer (150 mM NaCl, 50 mM TrisHCl, pH 7.5, 1 mM EDTA, 50 mMNaF, 1mMNaVO4, 1%NP-40, 1mMPMSF, 10mM N-ethylmaleimide). Five percent of this starting material was retained for detection of input proteins, and the remainder was subjected to immunoprecipitation with 14
anti-FLAG antibodies overnight at 4°C. Immune complexes were bound to protein A-beads for 1 h at 4°C, washed twice with IP buffer, once with PBS (pH 7.4), and resuspended in 2XSDS loading buffer. Immune complexes and input samples were subjected to SDS-PAGE, transferred
ip t
to nitrocellulose membranes, and probed with appropriate antibodies GST pull-down assay
cr
us
GST-SENP2 WT or GST-SENP2 MUT were expressed in BL21 cells and purified using
an
glutathione-Sepharose beads (Amersham Pharmacia) in binding buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 100 μg/ml
M
phenylmethylsulfonyl fluoride). After 1 h at 4 °C, equal amounts of GST or GST fusion proteins were resuspended in reaction buffer (200 mM NaCl, 50 mM HEPES pH 7.5, 1 mM MgCl2, and
ed
0.2% Triton X-100) containing 0.2 mg/ml bovine serum albumin (BSA) (Fraction V,
pt
Sigma-Aldrich) and incubated for 2 h at 4 °C . Then, 1 mg HA-Cdh1-transfected cell lysate were
ce
added to each mixture followed by rotation at room temperature for 1 h, centrifugation, and three washes. The beads were boiled in sodium dodecyl sulfate (SDS) sample buffer to elute the bound proteins, which were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Interaction between SENP2 and Cdh1 were assessed by GST-pull down assay. Briefly, GST or
by Western blot analysis. Western blotting and antibodies Protein extracts were equally loaded on 12 % SDS–polyacrylamide gels and subsequently transferred to nitrocellulose membrane by electrophoresis. The membrane was blocked in Tris–buffered saline with 0.1 % Tween-20 (TBST) containing 5 % nonfat dried milk for 1 h at 15
room temperature. Primary antibodies were incubated overnight at 4°C and washed three times each in TBST. Horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG as secondary antibody was added to TBST and membrane was incubated for 1 h followed by three washes in
ip t
TBST (5 min each). The images were visualized by FUJIFILM LAS-4000 Luminescent image analyzer. Western blot was performed using antibodies as indicated: SENP2 (H-300, Santa Cruz),
cr
us
Abcam), Lamin B (C-20, Santa Cruz), Cyclin B1 Antibody (GNS1, Santa Cruz), Cdh1 (ab89535,
an
Abcam), Cdc20 (ab26483, Abcam), Flag (M2, Sigma), Myc (ab9106, Abcam), HA (Y-11, Santa Cruz) and β-Actin (4967L, Cell Signaling Technology).
M
Luciferase assays
Luciferase experiments were performed as described previously with minor modifications21.
ed
Cells were transfected with firefly luciferase reporter plasmids 3XMRE-luc, MEF2A and other
pt
plasmids as indicated. For all reporter gene experiments, firefly luciferase plasmids were
ce
cotransfected with the TK-pRL vector, which expresses renilla luciferase and allows for normalization. Fresh growth media were added within 24 h of transfection. Cells were lysed 48 h after transfection. Luciferase activity was determined using the dual luciferase assay system from
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SENP2 (ab3660, Abcam), SynGAP (3200s, Cell Signaling Technology), beta Tubulin (ab6046,
Promega.
Quantitative Real-Time PCR Assays Total RNA was isolated with use of TRIzol reagent (Invitrogen). Quantitative PCR was performed with Light Cycler 480 (Roche, Switzerland) and SYBR Green (Molecular Probes, Eugene, OR) used as a fluorescent probe. Target gene mRNA levels were normalized to that of 16
β-actin in the same sample. The following sets of oligonucleotides were used as primers: SENP2 5’-primer
(5’-
GATTCCCATTCCAGCTGACCAC-3’)
(5’-CACTCTGATCTTTGGATAGTCA-3’)
β-Actin: and
3’-primer
5’-primer
3’-primer
(5
(5’’
-
ip t
TCATGAAGTGTGACGTTGACATCC-3’)
and
CCTAGAAGCATTTGCGGTGCACGA-3’). Thermal cycling parameters were 2 min at 50℃,
cr
us
denaturation, 30 sec at 60℃ for anneal. For each sample, average threshold (Ct) value assays
an
were repeated in triplicate, and the ΔCt value was determined by subtracting the average β-actin Ct value from the average SENP2 Ct value.
M
Immunofluorescence analysis.
Cells transfected with indicated plasmids were grown on cover slips. After NaCl or KCl
ed
treatment, cells were washed with PBS, fixed with 4% formaldehyde in PBS for 10 min and
pt
permeabilized with 0.5% Triton X-100 in PBS for 10 min. Then cells were incubated with
ce
anti-Flag or HA antibodies overnight. After washing with PBS three times, cells were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:1000; Invitrogen, Carlsbad, CA) for 1 h. DNA was stained with DAPI. Images were captured on Nikon DS-Ri1-U2 Digital Camera.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
and 10 min at 95℃, for cDNA denaturation followed by 40 cycles of 15 sec at 95℃ for
Apoptosis assay
Apoptosis was measured by the Annexin V Fluos Apoptosis detection kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer’s instructions. Fluorescent intensities of probes were determined by flow cytometry.
17
Statistical analysis The results are presented as mean ± SEM. The Student’s t test was used to compare the difference between two different groups. A value of p < 0.05 was considered to be statistically
ip t
significant. Funding
cr
and Specialized Research Fund for Outstanding Young Teachers of Higher
Education, Shanghai (to Dr. Han Lu).
M
Conflict of interest statement
an
Dr. Buwei Yu)
us
Republic of China, grant No. 81102513 (to Dr. Han Lu), grant No. 81072703 ＆ 81373492 (to
ce
pt
ed
None declared.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
This work was supported by the National Natural Science Foundation, Beijing, People’s
18
References 1. Bertrand SS, Cazalets JR. Activity-dependent Synaptic Plasticity and Metaplasticity in Spinal
ip t
Motor Networks. Curr Pharm Des 2013; 19:4498-508. 2. Cerda O, Trimmer JS. Activity-dependent phosphorylation of neuronal Kv2.1 potassium
cr
us
3. Renthal W, Maze I, Krishnan V, Covington HE, 3rd, Xiao G, Kumar A, Russo SJ, Graham A,
an
Tsankova N, Kippin TE, et al. Histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli. Neuron 2007; 56:517-29.
M
4. Lu H, Liu B, You S, Xue Q, Zhang F, Cheng J, Yu B. The activity-dependent stimuli increase SUMO modification in SHSY5Y cells. Biochemical and biophysical research communications 2009;
ed
390:872-6.
pt
5. Carulli D, Foscarin S, Rossi F. Activity-dependent plasticity and gene expression modifications in the adult CNS. Front Mol Neurosci 2011; 4:50.
ce
6. Muller S, Hoege C, Pyrowolakis G, Jentsch S. SUMO, ubiquitin's mysterious cousin. Nature reviews Molecular cell biology 2001; 2:202-10.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
channels by CDK5. J Biol Chem 2011; 286:28738-48.
7. Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nature reviews Molecular cell biology 2007; 8:947-56. 8. Shin EJ, Shin HM, Nam E, Kim WS, Kim JH, Oh BH, Yun Y. DeSUMOylating isopeptidase: a second class of SUMO protease. EMBO reports 2012; 13:339-46. 9. Wang Y, Dasso M. SUMOylation and deSUMOylation at a glance. Journal of cell science 19
2009; 122:4249-52. 10. Cheng J, Wang D, Wang Z, Yeh ET. SENP1 enhances androgen receptor-dependent transcription through desumoylation of histone deacetylase 1. Molecular and cellular biology
ip t
2004; 24:6021-8. 11. Cheng J, Perkins ND, Yeh ET. Differential regulation of c-Jun-dependent transcription by
cr
us
12. Itahana Y, Yeh ET, Zhang Y. Nucleocytoplasmic shuttling modulates activity and
an
ubiquitination-dependent turnover of SUMO-specific protease 2. Mol Cell Biol 2006; 26:4675-89.
M
13. Kang X, Qi Y, Zuo Y, Wang Q, Zou Y, Schwartz RJ, Cheng J, Yeh ET. SUMO-specific protease 2 is essential for suppression of polycomb group protein-mediated gene silencing during embryonic
ed
development. Molecular cell 2010; 38:191-201.
pt
14. Chiu SY, Asai N, Costantini F, Hsu W. SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages. PLoS biology 2008;
ce
6:e310.
15. Gong L, Yeh ET. Characterization of a family of nucleolar SUMO-specific proteases with
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
SUMO-specific proteases. The Journal of biological chemistry 2005; 280:14492-8.
preference for SUMO-2 or SUMO-3. The Journal of biological chemistry 2006; 281:15869-77. 16. Bawa-Khalfe T, Yeh ET. SUMO Losing Balance: SUMO Proteases Disrupt SUMO Homeostasis to Facilitate Cancer Development and Progression. Genes & cancer 2010; 1:748-52. 17. Rashid AJ, Cole CJ, Josselyn SA. Emerging roles for MEF2 transcription factors in memory. Genes Brain Behav 2013. 20
18. Shalizi A, Gaudilliere B, Yuan Z, Stegmuller J, Shirogane T, Ge Q, Tan Y, Schulman B, Harper JW, Bonni A. A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation. Science 2006; 311:1012-7.
ip t
19. Flavell SW, Cowan CW, Kim TK, Greer PL, Lin Y, Paradis S, Griffith EC, Hu LS, Chen C,
us
20. Shalizi A, Bilimoria PM, Stegmuller J, Gaudilliere B, Yang Y, Shuai K, Bonni A. PIASx is a MEF2
an
SUMO E3 ligase that promotes postsynaptic dendritic morphogenesis. The Journal of neuroscience : the official journal of the Society for Neuroscience 2007; 27:10037-46.
M
21. Lu H, Liu B, You S, Chen L, Dongmei Q, Gu M, Lu Y, Chen Y, Zhang F, Yu B. SENP2 regulates MEF2A de-SUMOylation in an activity dependent manner. Molecular biology reports 2013;
ed
40:2485-90.
pt
22. Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drug resistance in cancer. Biochem Pharmacol 2012; 83:1021-32.
ce
23. Barford D. Structure, function and mechanism of the anaphase promoting complex (APC/C). Q Rev Biophys 2011; 44:153-90.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
excitatory synapse number. Science 2006; 311:1008-12.
cr
Greenberg ME. Activity-dependent regulation of MEF2 transcription factors suppresses
24. Almeida A. Regulation of APC/C-Cdh1 and its function in neuronal survival. Mol Neurobiol 2012; 46:547-54.
25. Zeng X, Sigoillot F, Gaur S, Choi S, Pfaff KL, Oh DC, Hathaway N, Dimova N, Cuny GD, King RW. Pharmacologic
inhibition
of
the
anaphase-promoting
complex
induces
a
spindle
checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell 2010; 21
18:382-95. 26. Zeng X, King RW. An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination. Nat Chem Biol 2012; 8:383-92.
ip t
27. Wang X, She H, Mao Z. Phosphorylation of neuronal survival factor MEF2D by glycogen synthase kinase 3beta in neuronal apoptosis. J Biol Chem 2009; 284:32619-26.
cr
us
survival mediated by transcription factor MEF2. Science 1999; 286:785-90.
an
29. Loriol C, Khayachi A, Poupon G, Gwizdek C, Martin S. Activity-dependent regulation of the sumoylation machinery in rat hippocampal neurons. Biology of the cell / under the auspices of
M
the European Cell Biology Organization 2013; 105:30-45.
30. Hu D, Qiao X, Wu G, Wan Y. The emerging role of APC/CCdh1 in development. Semin Cell
ed
Dev Biol 2011; 22:579-85.
pt
31. Manchado E, Eguren M, Malumbres M. The anaphase-promoting complex/cyclosome (APC/C): cell-cycle-dependent and -independent functions. Biochem Soc Trans 2010; 38:65-71.
ce
32. Zhang XD, Goeres J, Zhang H, Yen TJ, Porter AC, Matunis MJ. SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
28. Mao Z, Bonni A, Xia F, Nadal-Vicens M, Greenberg ME. Neuronal activity-dependent cell
Mol Cell 2008; 29:729-41. 33. Yang Y, Kim AH, Bonni A. The dynamic ubiquitin ligase duo: Cdh1-APC and Cdc20-APC regulate neuronal morphogenesis and connectivity. Curr Opin Neurobiol 2010; 20:92-9. 34. Pick JE, Malumbres M, Klann E. The E3 ligase APC/C-Cdh1 is required for associative fear memory and long-term potentiation in the amygdala of adult mice. Learn Mem 2013; 20:11-20. 22
35. Pick JE, Wang L, Mayfield JE, Klann E. Neuronal expression of the ubiquitin E3 ligase APC/C-Cdh1 during development is required for long-term potentiation, behavioral flexibility, and extinction. Neurobiol Learn Mem 2013; 100:25-31.
ip t
36. Wan Y, Liu X, Kirschner MW. The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction. Mol Cell 2001; 8:1027-39.
cr
us
L, Pagano M, Bonni A, Iavarone A. Degradation of Id2 by the anaphase-promoting complex
an
couples cell cycle exit and axonal growth. Nature 2006; 442:471-4.
38. Krumova P, Weishaupt JH. Sumoylation in neurodegenerative diseases. Cell Mol Life Sci
ce
pt
ed
M
2013; 70:2123-38.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
37. Lasorella A, Stegmuller J, Guardavaccaro D, Liu G, Carro MS, Rothschild G, de la Torre-Ubieta
23
ip t cr us an M
ed
ubiquitination and degradation
pt
A. Lysates of primary rat cortex neuron cells depolarized with 50 mM KCl for the indicated
ce
time were immunoblotted for SynGAP and SENP2. β-actin was used as loading control. The number on the bottom of Western blot lanes indicated signal intensity of SENP2 or SynGAP protein against β-actin. The signal intensity of SENP2 or SynGAP protein against β-actin at 0
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.1. Activity-dependent stimuli activate MEF2A activation by preventing SENP2
min was set as 1.
B. SHSY5Y cells pretreated with 50 mM KCl or 150 mM NaCl (corresponds to physiological conditions: 0.9% NaCl) for 1h and 20mM CHX was added for the indicated time. Lysates from these cells were immunoblotted for SENP2. Histograms represent the ratio of the signal intensity of SENP2 protein quantified against β-actin. 24
C. SHSY5Y cells were transfected with Flag-SENP2 and HA-ubiquitin plasmids for 36h. Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h. Cells were lysed and subjected to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted
ip t
with anti-HA antibody. D. SHSY5Y cells were transfected with indicated plasmids for 36h. Cells were then treated
cr
us
immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted
an
with anti-HA antibody.
E. SHSY5Y cells were transfected with non-specific siRNA or siRNA against SENP2 for 48h.
immunoblotted for SENP2.
SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with
ed
F.
M
Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h. The whole cell lysates were
pt
non-specific siRNA or siRNA against SENP2 for 36h. Cells were then treated with 50 mM KCl
ce
or 150 mM NaCl for 2h, the luciferase activity was measured. Transfection efficiency was normalized by Renilla luciferase expression, and the results are presented as activation over that for non-specific siRNA transfected group. The y axis represents normalized luciferase activity ±
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
with 50 mM KCl or 150 mM NaCl for 2h. Cells were lysed and subjected to
SEM (n = 3). **represents p < 0.01, *** represents p < 0.001.
25
ip t cr us an M ed
pt
A. SHSY5Y cells were treated with 50 mM KCl or 150 mM NaCl for 2h and the
ce
nuclear-cytoplasmic fraction of these cells was conducted. Lysates from each fraction were immunoblotted for SENP2. Tubulin and Lamin B were used to indicate cytoplasmic or nuclear fraction, respectively. The number on the bottom of Western blot lanes indicated signal intensity
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.2. Activity-dependent stimuli promote SENP2 nuclear translocation
of SENP2 protein against β-Tubulin or Lamin B. The signal intensity of SENP2 protein against β-Tubulin or Lamin B without KCl treatment was set as 1. B. Schematic diagrams of the NLS, NES and Protease domain of SENP2. C. SHSY5Y cells were transfected with Flag-SENP2 WT or Flag-SENP2 mNLS for 36h. Cells were treated with 50 mM KCl or 150 mM NaCl for 2h. The whole cell lysates were 26
immunoblotted with Flag antibody. D. SHSY5Y cells were transfected with indicated plasmids for 36h. Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h, the luciferase activity was measured. The y axis
ip t
represents normalized luciferase activity ± SEM (n = 3). **represents p < 0.01. E. SHSY5Y cells were transfected with Flag-SENP2 for 32h and treated with 10nM LMB or
cr
us
each fraction were immunoblotted for Flag-SENP2. Tubulin and Lamin B were used to indicate
F.
an
cytoplasmic or nuclear fraction, respectively.
SHSY5Y cells were transfected with Flag-SENP2 and His-ubiquitin plasmids for 36h. Cells
M
were then treated with 10nM LMB. Cells were lysed and subjected to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted with anti-His antibody.
ed
G. SHSY5Y cells were transfected with Flag-MEF2A and HA-SUMO1 for 36h. Cells were
pt
then treated with 10nM LMB for 2h. Cells were lysed and subjected to immunoprecipitation with
ce
Flag M2 beads. Immunoprecipitated proteins were immunoblotted with anti-HA and Flag antibodies. H.
SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids for 36h.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
DMSO for 2h and the nuclear-cytoplasmic fraction of these cells was conducted. Lysates from
Cells were then treated with 10nM LMB for 2h, the luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). **represents p < 0.01 I.
Lysates of primary rat cortex neuron cells treated with 10nM LMB or DMSO were
immunoblotted for SynGAP and SENP2.
27
ip t cr us an
M
A. Alignment of D boxes in Skp2, Cyclin B1, Securin and SENP2.
pt
Homo sapiens
ed
B. Alignment of D boxes in SENP2 from Mus musculus, Rattus norvegicus, Pongo abelii and
ce
C. SHSY5Y cells were treated with 10μM proTAME for 12h. The whole cell lysates were immunoblotted with SENP2 and Cyclin B1 antibodies. D. SHSY5Y cells were treated with 10μM proTAME for 12h. The whole cell lysates were
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.3. SENP2 contains a canonical D-box motif.
immunoblotted with SUMO1 antibody. E. SENP2 mRNA level from SHSY5Y cells treated with 10μM proTAME for 12h was detected by Realtime-PCR. The y axis represents mean ± SEM (n = 3).
28
ip t cr us an M
ed
A. 293T cells were transfected with indicated plasmids for 36h. Cells were lysed and subjected
pt
to immunoprecipitation with Flag M2 or HA beads. Immunoprecipitated proteins were
ce
immunoblotted with indicated antibodies. B. Lysates of SHSY5Y cells depolarized with 50 mM KCl for 2h subjected to immunoprecipitation with SENP2, Cdh1 or IgG antibodies.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.4. SENP2 is a substrate of APCCdh1
Immunoprecipitated proteins were
immunoblotted with indicated antibodies. C. Beads coated with bacterially expressed GST or GST-SENP2 were incubated with purified HA-Cdh1 protein. Beads were washed, and the bound proteins were analyzed by Western blotting with indicated antibodies. The number on the bottom of Western blot lanes indicated 29
signal intensity of Cdh1. The signal intensity of input Cdh1 protein was set as 1. D. SHSY5Y cells were transfected with indicated increased HA-Cdh1 plasmid for 36h. In MG132 group, 10μM was added 6h before cell harvested. The whole cell lysates were
ip t
immunoblotted with indicated antibodies. E. SHSY5Y cells transfected with empty vector or HA-Cdh1 plasmids were treated with 20 μM
cr
us
The number on the bottom of Western blot lanes indicated signal intensity of SENP2 protein
F.
an
against β-actin. The signal intensity of SENP2 protein against β-actin at 0 min was set as 1. SHSY5Y cells were transfected with non-specific siRNA or siRNAs targeting Cdh1 for 48h.
M
The whole cell lysates were immunoblotted with SENP2 and Cdh1 antibodies. G. SHSY5Y cells were transfected with empty vector or HA-DN-Cdh1 for 48h. The whole cell
ed
lysates were immunoblotted with SENP2 and HA antibodies.
pt
H. 293T cells were transfected with indicated plasmids for 36h. Cells were lysed and subjected
ce
to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted with indicated antibodies.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
CHX for the indicated time. The whole cell lysates were immunoblotted with SENP2 antibody.
30
us
cr
ip t B.
an
A. Schematic diagrams of SENP2 WT and Mut
SHSY5Y cells were transfected with indicated plasmids for 48h. The whole cell lysates
were immunoblotted with Flag and HA antibodies.
M
C. Beads coated with bacterially expressed GST-SENPWT or GST-SENP2Mut were incubated with purified HA-Cdh1 protein. Beads were washed, and the bound proteins were analyzed by
D
ed
western blotting with indicated antibodies.
SHSY5Y cells transfected with Flag-SENP2 WT or Flag-SENP2 Mut for 36h were treated
pt
with 20 μM CHX for the indicated time. In MG132 group, 10μM was added 6h before cell
ce
harvested. The whole cell lysates were immunoblotted with indicated antibodies. E. SHSY5Y cells transfected with Flag-SENP2 WT or Flag-SENP2 Mut for 36h were treated
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
Figure.5. D-box-dependent degradation of SENP2 by APCCdh1
with 10μM TAME for 12h. The whole cell lysates were immunoblotted with indicated antibodies. F.
293T cells were transfected with indicated plasmids for 36h. Cells were lysed and subjected
to immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted with indicated antibodies. 31
ip t cr us an M ed pt
Downloaded by [Northwestern University] at 03:58 20 December 2014
ce
Ac
Figure.6. Cdh1 regulates MEF2A transcriptional activity via SENP2 destruction A. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with Cdh1 or Cdc20 plasmids for 36h. The luciferase activity was measured. Transfection efficiency was normalized by Renilla luciferase expression, and the results are presented as activation over that for empty vector transfected group. The y axis represents normalized luciferase activity ± 32
SEM (n = 3). **represents p < 0.01 compared with Con group. B. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with indicated increased Cdh1 for 36h. The luciferase activity was measured. The y axis represents
ip t
normalized luciferase activity ± SEM (n = 3). C. SHSY5Y cells were transfected with indicated plasmids for 36h. Cells were then treated
cr
us
immunoprecipitation with Flag M2 beads. Immunoprecipitated proteins were immunoblotted
an
with indicated antibodies.
D. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with
M
indicated plasmids for 36h. Cells were then treated with 50 mM KCl or 150 mM NaCl for 2h. The luciferase activity was measured. The y axis represents normalized luciferase activity ±
ed
SEM (n = 3). *represents p < 0.05, **represents p < 0.01.
pt
E. SHSY5Y cells were transfected with MEF2A and SynGap reporter plasmids with indicated
ce
plasmids for 36h. Cells were then treated with 50 mM KCl for 2h. The luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). *represents p < 0.05, **represents p < 0.01.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
with 50 mM KCl or 150 mM NaCl for 2h. Cells were lysed and subjected to
F. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with siRNAs specific targeting Cdh1 for 36h. The luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). **represents p < 0.01 compared with Con-siRNA group. G. SHSY5Y cells were transfected with MEF2A and MEF-luciferase reporter plasmids with 33
indicated plasmids for 36h. The luciferase activity was measured. The y axis represents normalized luciferase activity ± SEM (n = 3). *** represents p < 0.001 CDH1+ SENP2 Mut compared with CDH1 group.
ip t
H. Rat Primary Cerebellar Granule Neurons (CGNs) cultured in medium containing serum and depolarizing concentrations of KCl (29mM) were transfected with indicated plasmids for 30h.
cr
us
triggered CGN apoptosis model. The percentage of apoptotic cells were determined by
an
Annexin-V/PI staining assay. The y axis represents normalized luciferase activity ± SEM (n = 3). * represents p < 0.05 compared with Con group.
M
I. Rat Primary Cerebellar Granule Neurons (CGNs) cultured in medium containing serum and depolarizing concentrations of KCl (29mM) were transfected with indicated siRNAs for 30h.
ed
CGNs were switched to medium containing 5 mM KCl for 8h to generate KCl withdrawal
pt
triggered CGN apoptosis model. The percentage of apoptotic cells were determined by
ce
Annexin-V/PI staining assay. The y axis represents normalized luciferase activity ± SEM (n = 3). *represents p < 0.05 compared with Con-siRNA group.
Ac
Downloaded by [Northwestern University] at 03:58 20 December 2014
CGNs were switched to medium containing 5 mM KCl for 8h to generate KCl withdrawal
34
