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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce 5 6

Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac

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William J. McDonald a, Lynn N. Thomas a, Samir Koirala a, Catherine K.L. Too a,b,⇑ a b

Department of Biochemistry & Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada Department of Obstetrics & Gynaecology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada

a r t i c l e

i n f o

Article history: Received 16 July 2013 Received in revised form 20 September 2013 Accepted 26 September 2013 Available online xxxx Keywords: a4 Phosphoprotein EDD E3 Ubiquitin ligase PP2Ac Progesterone Prolactin 17b-Estradiol

a b s t r a c t Mammalian a4 phosphoprotein binds to the protein phosphatase 2A catalytic subunit (PP2Ac) to regulate PP2A activity, and to poly(A)-binding protein (PABP) and progestin-inducible EDD E3 ubiquitin ligase. This study showed induction of the EDD protein by progesterone, 17b-estradiol and prolactin in breast cancer cells. Co-immunoprecipitation analyses, using lysates of COS-1 cells transfected with a4-deletion constructs, showed the a4 N-terminus binding to endogenous PP2Ac and PABP, and the C-terminus to EDD. Monoubiquitinated a4 in MCF-7 cells was unaffected by EDD-targeting siRNA (siEDD) nor by non-targetting siNT, thus, EDD does not ubiquitinate a4. PP2Ac is polyubiquitinated, and 36-kDa PP2Ac only was detected in siEDD- or siNT-transfected cells. However, treatment with proteasomal inhibitor MG132 showed polyubiquitinated-PP2Ac molecules (65–250 kDa) abundantly in siNT controls but low in siEDD-transfectants, implicating PP2Ac as an EDD substrate. Finally, progesterone induction of EDD in MCF-7 cells correlated with decreased PP2Ac levels, further implicating hormone-inducible EDD in PP2Ac turnover. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

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Mammalian a4 phosphoprotein, the homolog of yeast Tap42, is an essential component of the mammalian target-of-rapamycin (mTOR) pathway that responds to nutrients and growth factors for the regulation of ribosome biosynthesis, the initiation of translation, and cell cycle progression (Schmelzle and Hall, 2000; Gingras et al., 2001; Raught et al., 2001). The a4 protein associates with the catalytic subunits of serine/threonine protein phosphatase 2A (PP2Ac), and the PP2A-related PP4 and PP6 (Inui et al., 1995; Chen et al., 1998). PP2A is a regulator of numerous signaling pathways, and it dephosphorylates many proteins in vitro, and specifically protein kinases and transcription factors in vivo (Zolnierowicz, 2000). Using a4 as the bait in yeast two-hybrid analysis, we have previously identified PP2Ac and an E3 ubiquitin ligase called EDD (E3 identified by differential display) as binding partners of a4 (McDonald et al., 2010). The EDD protein contains a domain designated PABC, that is also found in the poly(A)binding protein (PABP) C terminus. Recognizing this common PABC

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⇑ Corresponding author. Address: Department of Biochemistry & Molecular Biology, Sir Charles Tupper Medical Building, Room 9D-1, Faculty of Medicine, Dalhousie University, P.O. Box 15000, 5850 College Street, Halifax, Nova Scotia B3H 4R2, Canada. Tel.: +1 (902) 494 1108; fax: +1 (902) 494 1355. E-mail address: [email protected] (C.K.L. Too).

domain in EDD and PABP, we showed that a4 binds to PABP, using co-immunoprecipitation analyses (McDonald et al., 2010). The murine a4 has 340 amino acids and the PP2Ac-binding site is located in the N-terminal residues 94–202 (Inui et al., 1998). a4 and PP2Ac are mono- and poly-ubiquitinated proteins, respectively (McConnell et al., 2010). We reported that EDD binds to the C-terminal region of a4 in the absence of the PP2Ac-binding site (McDonald et al., 2010). Our identification of EDD E3 ubiquitin ligase as a protein partner of a4, suggests that a4 and/or PP2Ac could be ubiquitination targets of EDD. EDD, initially reported as a progestin-inducible gene in human T47D breast cancer cells (Callaghan et al., 1998; Henderson et al., 2002), is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development (Mansfield et al., 1994). EDD binds the progesterone receptor and potentiates progestin-mediated gene transactivation (Henderson et al., 2002). EDD also binds tumour suppressor p53 to block ataxia telangiectasia mutated (ATM) phosphorylation of p53, ensuring G1/S progression (Ling and Lin, 2011). EDD is overexpressed in some cancers (Clancy et al., 2003; Fuja et al., 2004; O’Brien et al., 2008), and is associated with the DNA damage response (Henderson et al., 2006; Munoz et al., 2007), and cisplatin resistance in vitro (O’Brien et al., 2008). In this study, we determined that progesterone and other hormones induce EDD gene expression in breast cancer cells. We further characterized the a4 binding sites for EDD, PABP and

0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.09.033

Please cite this article in press as: McDonald, W.J., et al. Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac. Molecular and Cellular Endocrinology (2013), http://dx.doi.org/10.1016/j.mce.2013.09.033

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PP2Ac. Lastly, we determined that polyubiquitinated PP2Ac is an EDD substrate, implicating an indirect regulation of PP2Ac turnover by hormones in breast cancer cells.

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2. Materials and methods

resolved proteins were transferred onto Biotrace™ NT nitrocellulose membranes (Pall Life Sciences, Pensacola, FL). Immun-Star™ WesternC™ Chemiluminescence reagents (1:1 mixture; Bio-Rad) were spread over the blots for 5 min before detection of chemiluminescence.

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2.1. Antibodies

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2.2. Cell culture

Similar to a4 deletion mutants D4 and D5 (McDonald et al., 2010), D1, D2 and D3 were generated by polymerase chain reaction (PCR) using as template the full-length pcDNA3-a4 construct (Boudreau et al., 2002). PCR products were excised from 1% agarose gels, purified using UltraClean 15 DNA Purification Kit (Medicorp Inc., Montreal, QC, Canada), subcloned into pcDNA3 or pcDNA4/ HisMax TOPO (Invitrogen) and sequenced. As shown in Fig. 3, D1, D2 and D3 contained the N-terminus of a4 and were all N-terminally Xpress-tagged. D3 also contained the PP2Ac-binding site. D4 lacked the PP2Ac-binding site but contained the a4 C-terminus, including the epitope that was used to raise polyclonal anti-a4 antibodies (Boudreau et al., 2002). D5 was N-terminal Xpresstagged and also had the C-terminal epitope.

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Primary antibodies were purchased and used at the indicated concentrations: rabbit anti-EDD (1:500) (Abcam Inc., Cambridge, MA); rabbit anti-PP2Ac (1:2000) and mouse anti-ubiquitin (1:1000) (Cell Signalling, Danvers, MA); mouse anti-Xpress (1:1000) (Invitrogen, Burlington, ON, Canada); rabbit anti-actin (1:5000) (Sigma–Aldrich, Oakville, ON, Canada). The secondary horseradish peroxidase (HRP)-conjugated antibodies used were goat anti-rabbit IgG (Sigma–Aldrich), goat anti-mouse IgG (GBioSciences, Maryland Heights, MO), and rat anti-mouse kappa-light chain (Abcam Inc.). Our custom rabbit anti-a4 antibodies have been described previously (Boudreau et al., 2002).

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2.7. Reverse transcription-plymerase chain reaction (RT-PCR)

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2.3. Total cell lysates

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Cells were harvested in RIPA cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitor cocktail P8340 (Sigma– Aldrich) and 1 mM phenymethylsulfonylfluoride. Cell lysates were produced by passaging cells through a 21-gauge needle, incubated on ice for 30 min, and centrifuged at 13,000 rpm for 20 min at 4 °C to remove cellular debris. The supernatant (total cell lysate) was used immediately or kept frozen at -20 °C until further analysis. In some immunoprecipitation experiments denatured cell lysates were prepared as described (Gordon et al., 2011), that is, cell lysates prepared in RIPA buffer above were precleared, then 0.5% SDS and 5 mM b-mercaptoethanol were added. The lysates were boiled for 5 min before addition of 1 volume of ice-cold 4x RIPA cell lysis buffer, followed by immunoprecipitation.

Total RNA was extracted from cells using GenElute™ Mammalian Total RNA Prep Kit (Sigma–Aldrich) and treated with DNase I (Fermentas, Burlington, ON, Canada). Synthesis of cDNA was performed in a reaction mixture containing 100 U of Maloney murine leukemia virus reverse transcriptase (MMLV-RT), 0.04 nM pdN6 random primers, 0.2 lM of deoxynucleotide triphosphates (dNTPs), 10 U of RNaseOUT™ and 10 mM dithiothreitol. Synthesized cDNA was amplified by PCR in a reaction mixture containing 12.5 lL of GoTaq™ Green Master Mix (Promega, San Luis Obispo, CA), 0.5 lL of forward primers, 0.5 lL of reverse primers, and 3 lL of synthesized cDNA, in a total volume of 25 lL. PCR was performed as follows: 94 °C for 75 s, 25 or 35 cycles of (94 °C for 45 s, 57 °C for 45 s, 72 °C for 60 s), and 72 °C for 10 min. PCR products were visualized in 1.2% (w/v) agarose gels in Tris–acetate

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Human MCF-7 breast cancer cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS), and supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 50 U/mL of penicillin/streptomycin. T47D breast cancer cells were maintained in DMEM containing 10% heat-inactivated FBS, 5 mM HEPES, 2 mM L-glutamine and penicillin/streptomycin. MCF-7 and T47D cells were made quiescent in DMEM containing 1% charcoal-stripped FBS for 48 h prior to treatment with hormones. SV40-transformed COS-1 cells were maintained in DMEM containing 5% heat-inactivated FBS and 2 mM L-glutamine.

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2.4. Protein assay

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Protein concentrations were determined using DC™ Protein Assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) following the manufacturer’s instructions. Absorbances were measured at 655 nm, using bovine serum albumin for standard curves.

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2.5. Immunoprecipitation and immunoblotting (IP/IB)

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Total cell lysates were used for IP, immunocomplexes were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 10–40 lg protein/lane, and followed by IB, as previously described (McDonald et al., 2010). For IB analysis, the

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Fig. 1. Hormones upregulate EDD in breast cancer cells. (A) T47D and (B) MCF-7 cells were made quiescent as described in Section 2, then treated with 17b-estradiol (E2; 10 nM), prolactin (PRL; 10 ng/ml) and progesterone (10 nM) for the indicated times. The control cells did not receive any hormone and were harvested at 0 h. Total cell lysates were prepared for Western analysis. Actin was used as a loading control. (A and B) Each is a representative of 2–3 separate experiments.

Please cite this article in press as: McDonald, W.J., et al. Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac. Molecular and Cellular Endocrinology (2013), http://dx.doi.org/10.1016/j.mce.2013.09.033

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B IP: EDD

EDD IgG HC α4

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IgG LC Fig. 2. Interactions between protein partners of a4. Total cell lysates were prepared from actively growing MCF-7 cells. Co-IP/IB analyses were performed using 1 lg each of anti-EDD, anti-PP2Ac antibodies or preimmune IgG as indicated. Each is a representative of at least 2 separate experiments.

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EDTA buffer (0.04 M Tris–acetate, pH 8.0, 1 mM EDTA) containing 0.005% ethidium bromide. Primers used were: human b-actin, 50 -AAA CTG GAA CGG TGA AGG TG-30 (forward) and 50 -AGA GAA GTG GGG TGG CTT TT-30 (reverse; 172-bp amplicon); MID1 (Harvard PrimerBank), 50 -TCC TAG TAT CAC ACT GTG CCA-30 (forward) and 50 -GAT GTT CTG TAG GGT GAC GTT G-30 (reverse; 137-bp amplicon).

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2.8. Transfection of a4 deletion constructs and siRNAs

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COS-1 and MCF-7 cells in growth medium containing 5% and 10% FBS, respectively, were seeded at a density of 2–3  106 cells per 10-cm dish. After 24 h, the medium was removed, cells were washed with 5 mL of PBS, and reincubated in 5 mL of serum-free DMEM prior to DNA transfection of a4 deletion constructs. Trypsinzied MCF-7 cells were resuspended in Opti-MEM medium and incubated with 10 nM siRNA in the presence of RNAiMax reagent (Invitrogen) for 20 min prior to seeding in 6-well plates at a density of 3  105 cells/ml. Deletion constructs pcDNA3-D5 (4 lg) or pcDNA3-D3 (8 lg) were incubated with 20 lL of PLUS™ reagent (Invitrogen) in 750 lL of serum-free DMEM for 15 min at room temperature. Subsequently, 30-lL of LipofectAMINE™ (Invitrogen) in 750 lL serum-free DMEM was added to the DNA mixture and incubated at room temperature for 15 min. The complete transfection mixture was added to COS-1 cells in 5 mL of serum-free DMEM. After 24 h at 37 °C, cells were given 5 mL of complete growth medium. Cells lysates were harvested after 48–72 h for IP/IB analyses. Predesigned SilencerÒ Select siRNAs and SilencerÒ Select Negative Control #1 were purchased from Applied Biosystems (Streetville, ON, Canada). The siRNAs targeting EDD were siEDD.1

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α4 epitope

PP2Ac binding site

full-length alpha4 cDNA

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1023 bp

D1 (272 bp; ~15 kDa) D2 (480 bp; ~22 kDa) D3 (656 bp; 27 kDa) D4 (558 bp; 33 kDa) D5 (435 bp; 23 kDa) Xpress-tag

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α4

PP2Ac PABP

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EDD α4 epitope

Fig. 3. N-terminus of a4 (D3) binds PABP and PP2Ac but not EDD. (A) Diagrammatic representation of a4 deletion mutants (cDNA and protein sizes are as indicated). The PP2Ac binding site is shown. The N-termini of D1, D2, D3 and D5 were Xpress-tagged (arrows). Like the full-length a4, the C-terminus of D5 contained the a4 epitope (arrowhead) used to raise polyclonal anti-a4 antibodies. (B–E) COS-1 cells were transfected with pcDNA3 plasmids containing D3 (8 lg) or D5 (4 lg). Cell lysates were prepared for IP with 1 lg each of anti-EDD (B), anti-PP2Ac (C) or anti-PABP (D, E) antibodies, or 1 lg preimmune IgG (B–E). Immunocomplexes or 5% lysates were resolved in 12% SDS–PAGE gels, and IB was performed with anti-Xpress antibodies. Each is a representative of at least 2 independent experiments. (F) Diagrammatic representation of the a4 protein complex.

Please cite this article in press as: McDonald, W.J., et al. Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac. Molecular and Cellular Endocrinology (2013), http://dx.doi.org/10.1016/j.mce.2013.09.033

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IP: IgG D D siE

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EDD (300 kDa)

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We determined that progesterone is not the only hormone that regulates EDD gene expression. Western analysis showed that EDD protein levels were rapidly upregulated within 3 h, following treatment of quiescent T47D and MCF-7 breast cancer cells with progesterone, 17b-estradiol or prolactin (Fig. 1).

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3.1. Several hormones upregulate EDD in breast cancer cells

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(50 -AGA CAA AUC UCG GAC UUG Att-30 ) and siEDD.2 (50 -GCG UGA ACG UGA AUC CGU Utt-30 ); targeting MID1: siMID1 (50 -GGC UGA UAG CUG GAU GAU Att-30 ). A final concentration of 10 nM of siRNA was incubated in 500 lL of Opti-MEM medium and 7.5 lL RNAiMAX (both from Invitrogen) for 20 min in 6-well plates. Following transfection, MCF-7 cells were incubated for 48 h at 37 °C prior to harvesting for co-IP/IB analyses.

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Co n

4

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3.2. Interactions between a4 and its protein partners

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3.4. Monoubiquitinated a4 is not an EDD substrate

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We have reported that EDD binds to the C-terminus of a4, as determined using a4 deletion mutants D4 and D5 in co-IP/IB analysis (McDonald et al., 2010) (see Fig. 3A). To further characterize protein-binding sites on a4, a new construct designated D3 that lacked the C-terminal region of a4 was generated. N-terminally Xpress-tagged D3 contained the PP2Ac-binding site whereas D5 lacked this site (Fig. 3A). Both D3 and D5 constructs were more readily transfected and expressed in COS-1 cells than in MCF-7 cells (data not shown), and the studies below were performed using COS-1 cells. In D3-transfected COS-1 cells, the endogenous EDD was immunoprecipitated with anti-EDD antibodies but the immunocomplex did not contain D3 (Fig. 3B), showing that EDD does not interact with the N-terminus of a4. In contrast, D3 interacted with PP2Ac (Fig. 3C). This was expected since D3 has the PP2Ac-binding site. PABP is another interacting partner of a4 (McDonald et al., 2010), and IP of PABP resulted in an immunocomplex that contained D3 (Fig. 3D) but not D5 (Fig. 3E). In summary, the C-terminus of a4 binds EDD whereas the N-terminus binds PP2Ac and PABP (Fig. 3F). The intensity of the D3-specific immunoreactive band was lighter in the D3-PP2Ac and D3-PABP immunocomplexes (Fig. 3C and D, respectively) as compared to the 5% cell lysates, implying that a fraction of D3 (i.e., a4) binds PP2Ac and PABP. Similarly, a fraction (5–10%) of cellular PP2Ac was reported to bind a4 (Murata et al., 1997). We also constructed two Xpress-tagged a4 deletion mutants, D1 and D2, to determine any overlap in the PP2Ac- and PABPbinding sites. However, D1 and D2 protein yields in transfected cells were consistently too low for co-IP analysis (data not shown), suggesting that perhaps the a4 protein is unstable.

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Using co-IP/IB analyses, we have shown that a4 interacts with PP2Ac, EDD and PABP in Nb2 rat lymphoma cells, COS-1 cells and/or MCF-7 cells (Boudreau et al., 2002; McDonald et al., 2010). The present study used MCF-7 cell lysates for co-IP analyses, which showed that the 300-kDa EDD interacted with a4 (Fig. 2A) but not with the a4 protein partners PP2Ac (Fig. 2B) nor PABP (data not shown).

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Con siNT siEDD1 siEDD2 kDa 5% IP 5% IP 5% IP 5% IP

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a4 is monoubiquitinated and is 50-kDa in western blotting

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(McConnell et al., 2010). This is in agreement with the predicted

Fig. 4. Monoubiquitinated a4 is not an EDD substrate. MCF-7 cells were transfected with siEDD.1, siEDD.2 or siNT for 48 h, or left untransfected (control). Cell lysates were prepared for (A) IB or (B) IP/IB analyses using rabbit anti-a4 antibodies or preimmune rabbit IgG. In (B), the 5% lysates and corresponding immunocomplexes were resolved in 12% gels. Immunoblottings were performed using HRP-conjugated goat anti-rabbit IgG for a4 and goat anti-mouse IgG for ubiquitin. Due to the strong immunoreactivity of the heavy chain IgG, the immunoblots were reexposed to film a second time after a considerable delay to allow the signals to decay. Representatives of 2–3 independent experiments.

size of 47.6-kDa for the monoubiquitinated a4 since ubiquitin is 8.5 kDa and we have reported the predicted size of the a4 phosphoprotein to be about 39.1 kDa (Boudreau et al., 2002). In addition, the 39-kDa a4 can undergo post-translational glycosylation to produce a protein of 45 kDa (Boudreau et al., 2002). The 39-kDa and/or 45-kDa a4 have been reported in human cancer cell lines, including breast cancer cells (McConnell et al., 2010; McDonald et al., 2010). To determine whether a4 is an EDD substrate, MCF-7 cells were transfected with the siRNAs, two sets of siEDD (EDD-targetting) or siNT (non-targetting). EDD gene expression was effectively knocked down in the siEDD-transfected cells, (Fig. 4A). Lysates of transfected cells and untransfected controls were used for IP with rabbit anti-a4 antibodies or preimmune IgG. The 39-kDa a4 was strongly detected only in the immunoprecipitates of the anti-a4 treatment group, but not in the anti-IgG group, and was abundant in siEDD, siNT and control samples (Fig. 4B, lower panel). The 5% cell lysates yielded this 39-kDa a4 as well as an immunoreactive band of 45-kDa that could be the glycosylated a4. In the 5% cell lysates, the different intensities of the a4-immunoreactive bands in transfected and control cells could be due to protein loading. Immunoblotting with mouse anti-ubiquitin antibody detected an immunoreactive band that was present only in the immunoprecipitates of the anti-a4 treatment group, and which ran very closely below the 50-kDa heavy chain IgG, suggesting that it could be

Please cite this article in press as: McDonald, W.J., et al. Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac. Molecular and Cellular Endocrinology (2013), http://dx.doi.org/10.1016/j.mce.2013.09.033

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the 47.6-kDa monoubiquitinated a4 (Fig. 4B, upper panel). More importantly, the persistent presence of a4, whether 39-kDa or 47.6-kDa, in the absence of EDD implied that a4 is not an EDD substrate. Attempts to better resolve the monoubiquitinated a4 were made using light chain-specific secondary antibodies in IB following IP of a4. Unfortunately, the heavy chain was still detected since IgG itself is a ubiquitinated protein (data not shown). Another E3 ubiquitin ligase, called MID1, also binds to the C-terminus of a4 (Liu et al., 2001; Trockenbacher et al., 2001). Unlike EDD, MID1 was reported to monoubiquitinate a4 (Han et al., 2011) and polyubiquitinate PP2Ac (Trockenbacher et al., 2001).

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3.5. EDD polyubiquitinates PP2Ac

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As indicated above, PP2Ac is a substrate of MID1, a cytoplasmic E3 ligase (Trockenbacher et al., 2001; McConnell et al., 2010). However, we have shown that PP2Ac is also present in the cell nucleus (Boudreau et al., 2002), where it could be the substrate

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of an nuclear E3 ligase. EDD is a nuclear-localized E3 ubiquitin ligase (Henderson et al., 2002) known to associate with other nuclear proteins (Benavides et al., 2013). To determine whether EDD ubiquitinates PP2Ac, MCF-7 cells were transfected with siEDD or siNT. Knockdown of EDD gene expression was confirmed by Western analysis (Fig. 5A and C). In the absence of proteasomal inhibitor MG132, IP analysis showed immunoprecipitation of the 36-kDa PP2Ac protein in all treatment groups (Fig. 5B, lower panel) whereas polyubiquitinated PP2Ac molecules, known to be >65-kDa (McConnell et al., 2010), were completely undetectable (Fig. 5A, upper panel). In the presence of MG132, the PP2Ac protein was immunoprecipitated and again detected in all treatment groups (Fig. 5D, bottom panel) together with the appearance of polyubiquitinated PP2Ac molecules (Fig. 5D, upper panel). The latter appeared as a heavy smear ranging from 65–250 kDa in the siNT control lane, and as a faint smear in the siEDD lanes. Similarly, polyubiquitinated PP2Ac molecules in this size range of 65–250 kDa has been demonstrated by others (McConnell et al., 2010). Therefore, our results clearly showed that EDD played a role in the polyubiquitination of PP2Ac. In contrast,

A - MG132

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IB: PP2Ac Fig. 5. EDD polyubiquitinates PP2Ac. MCF-7 cells were transfected with siEDD.1, siEDD.2 or siNT for 48 h. Transfected cells received (A and B) no MG132 or (C–E) 10 lM MG132 for 2 h prior to the preparation of denatured cell lysates (see Section 2), which ensured dissociation of PP2Ac-associated proteins and that any ubiquitination signals in B and D were from PP2Ac. (A and C) Western blot analysis. Cell lysates were used for IP using (B and D) anti-PP2Ac antibodies or (E) IgG, followed by IB as indicated. Each is a representative of 2–3 independent experiments.

Please cite this article in press as: McDonald, W.J., et al. Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac. Molecular and Cellular Endocrinology (2013), http://dx.doi.org/10.1016/j.mce.2013.09.033

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The a4-PP2Ac complex is involved with the rapamycinsensitive mTOR signal transduction pathway that ultimately results in cell-cycle progression (Inui et al., 1998; Nanahoshi et al., 1998). Crystallographic and biochemical analyses of yeast Tap42, the homolog of mammalian a4, suggests that Tap42/a4 could act as a scaffold for its protein partners, whereby interaction of PP2Ac at the N-terminus promotes the dephosphorylation of potential PP2A substrates recruited to the C-terminus (Yang et al., 2007). The present study showed that the N-terminal region of a4 binds PP2Ac and PABP. The C-terminal region of a4 binds to two E3 ubiquitin ligases, progestin-induced EDD (McDonald et al., 2010) and microtubule-associated MID1 (Liu et al., 2001). As we and others now show, PP2Ac itself is a substrate of EDD (present study) and MID1 (Trockenbacher et al., 2001). EDD gene expression is upreg-

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MCF-7 cells were transfected with siEDD or siNT, and total cell lysates were used for Western analysis. In the absence of MG132, PP2Ac levels in siNT-transfectants or untransfected controls were low, but markedly increased in cells transfected with siEDD (Fig. 6A). In the presence of MG132, PP2Ac protein levels were equally high in all treatment groups (Fig. 6B). Our results implicate EDD in the degradation of PP2Ac that was inhibited by MG132. In comparison, alpha4 levels were unaffected by siEDD, with or without MG132. We also confirmed that PP2Ac levels were regulated by MID1 E3 ligase. MCF-7 cells transfected with siMID1 showed decreased levels of the MID1 mRNA, determined using semi-quantitative RTPCR analysis (Fig. 6C and D). We resorted to RT-PCR analysis when Western analysis did not show any immunoreactive signal using commercial anti-MID1 antibodies (data not shown). In the absence of MG132, the PP2Ac protein level was high in siMID1-transfected cells but low in siNT controls (Fig. 6C). In the presence of MG132, PP2Ac protein levels were equally high in both treatment groups (Fig. 6D). Therefore, PP2Ac is a substrate of MID1 as previously

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If PP2Ac is a true substrate of EDD, hormonal upregulation of EDD in MCF-7 cells (Fig. 1) would decrease PP2Ac levels. The effect of progesterone was re-examined over a longer time period. Western analysis showed that progesterone treatment increased EDD protein levels which was accompanied by a decrease in PP2Ac levels over a 24-h period (Fig. 7). The increase in EDD levels was nearly 3-fold after 9 h of progesterone treatment whereas the decrease in PP2Ac levels was 2-fold at 12 h and almost 3-fold by 24 h.

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reported (Trockenbacher et al., 2001; McConnell et al., 2010), and a substrate of EDD (Figs. 5 and 6A and B).

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immunoprecipitation with IgG did not produce PP2Ac nor the smear (Fig. 5E). In Fig. 5 (A and D bottom panels), we anticipated PP2Ac protein levels to be increased in the siEDD-transfected cells over siNT controls. Our co-IP of PP2Ac could have reached a saturation point, preventing detection of any alteration of PP2Ac levels. Therefore, we examined the effect of EDD on PP2Ac protein levels using another procedure (see below).

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Fig. 6. EDD regulates PP2Ac protein turnover. (A and B) MCF-7 cells were transfected with siEDD.1, siEDD.2 or siNT for 48 h, or left untranfected (control). Cells received (A) no MG132 or (B) 10 lM MG132 for 2 h prior to the preparation of total cell lysates. SDS–PAGE (40 lg protein/lane; 10% gels) and IB were performed as indicated. (C and D) MCF-7 cells were transfected with siMID1 or siNT for 48 h, treated ± MG132 for 2 h, and cell lysates were prepared. MID1 and actin transcripts were determined by RT-PCR (upper panels). SDS–PAGE (40 lg protein/lane; 12% gels) and IB were performed to determine protein levels of PP2Ac and actin (lower panels). Each is a representative of at least 2 independent experiments.

Please cite this article in press as: McDonald, W.J., et al. Progestin-inducible EDD E3 ubiquitin ligase binds to a4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac. Molecular and Cellular Endocrinology (2013), http://dx.doi.org/10.1016/j.mce.2013.09.033

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Fig. 7. Progesterone increases EDD but decreases PP2Ac. Quiescent MCF-7 cells were treated with 10 nM progesterone for the times indicated. In (A), cell lysates were prepared for Western blot analysis of EDD, PP2Ac and actin. In (B), EDD and PP2Ac protein levels were normalized against actin. Representative of 2 separate experiments.

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ulated by progesterone, 17b-estradiol and prolactin, hormones which are involved in the growth and development of both normal and malignant mammary cells (McGuire et al., 1982; Jordan and Morrow, 1999; Clevenger et al., 2003). There is a possibility that through the regulation of EDD, these hormones may indirectly regulate pathways associated with the a4-PP2Ac complex in breast cancer cells. Crystallographic analysis showed that the N-terminal region of Tap42/a4 has an ordered a-helical structure, and the basic residues found within the PP2Ac-binding site interact with acidic residues on PP2Ac. In contrast, the C-terminal region of Tap42/a4 has a flexible secondary structure for recruitment of PP2Ac substrates but is predicted to adopt distinct conformations upon substrate binding (Yang et al., 2007). X-ray crystallography also showed several differences between the N-terminal regions of a4 and Tap42. The a4 N-terminus has a more open and flexible conformation than that of Tap42 and, furthermore, contains an ubiquitin-interacting motif (UIM) that is absent in Tap42 (Lenoue-Newton et al., 2011). Like other UIM-containing proteins, a4 is monoubiquitinated. The a4 UIM is located in N-terminal residues 46–60 and binds a single ubiquitin monomer (McConnell et al., 2010). Deletion of the UIM abolishes the protective effect of the native a4 towards PP2Ac (McConnell et al., 2010). Therefore, although the N-terminal UIM of a4 suppresses PP2Ac polyubiquitination, our results show that a4 recruitment of EDD and/or MID1 to its C-terminal region leads to PP2Ac polyubiquitination and degradation. EDD has a preference for binding to monoubiquitinated proteins (Kozlov et al., 2007). Although we showed formation of an EDD-a4 complex, our study suggests that a4 is not an EDD substrate. In contrast, the a4-binding MID1 is purported to monoubiquitinate a 45-amino-acid peptide derived from the C-terminus of a4 (Han et al., 2011). Our present study also showed that EDD polyubiquitinates PP2Ac but an EDD-PP2Ac immunocomplex was not detected in MCF-7 cells. We have also previously reported formation of an EDD-a4, but not EDD-PP2Ac, immunocomplex in rat Nb2 lymphoma cells (McDonald et al., 2010). The lack of a

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direct interaction between EDD and its substrate PP2Ac may reflect the dual HECT and RING domain structure of EDD. EDD is the only known E3 ubiquitin ligase that contains both RING and HECT domains (Henderson et al., 2002). HECT domain-containing ubiquitin ligases bind directly to their substrates and their cognate E2 ubiquitin-conjugating enzyme whereas some RING domaincontaining E3 ubiquitin ligases use adaptor proteins to bind their substrates for the transfer of ubiquitin from the E2 enzyme to the E3 ligase (Jackson et al., 2000; Pickart, 2001), a role that may be fulfilled by a4. Our identification of PABP as a protein partner of a4 further suggests a role for the a4-PP2Ac-EDD-PABP ternary complex in mRNA processing. PABP is a highly conserved protein that acts as a scaffold to organize mRNA–protein complexes (Mangus et al., 2003). PABP recruits mRNA-processing factors to promote mRNA circularization and maturation (Kozlov et al., 2004). Thus, acting through this ternary complex, hormones may have an indirect effect on the mTOR pathway and the protein translational machinery in breast cancer cells. In the case of MID1/PP2Ac protein complex, perturbations in this complex disrupts mTOR Complex 1 (mTORC1) signaling, downstream ribosomal S6 kinase phosphorylation, and cap-dependent protein translation (Liu et al., 2011). The MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) to increase stability and translational efficiency of these mRNAs (Aranda-Orgilles et al., 2011). Mutations in MID1, as seen in the human X-linked Opitz syndrome, decrease polyubiquitination and proteasome-mediated degradation of PP2Ac, resulting in elevated PP2Ac levels and hypophosphorylation of microtubule-associated proteins. This congenital disorder presents with defects in the formation of ventral midline structures, such as cleft palates (Liu et al., 2001; Trockenbacher et al., 2001). EDD gene mutation and how it may affect PP2Ac levels in a human disease have not been identified. Immunohistochemical analysis showed that staining for EDD correlated with progesterone receptor status in mammary ductal carcinomas (Fuja et al., 2004). Correlations between EDD levels and the expression of estrogen and/or prolactin receptors in breast cancer cells are as yet unknown. These studies and the molecular basis underlying hormonal regulation of EDD gene expression will be investigated in the future. In summary, the present study showed that a4 acts as a scaffold for EDD, PP2Ac and PABP. Progestin-inducible EDD is also upregulated by estrogen and prolactin in breast cancer cells. EDD plays a role in the polyubiquitination and proteasomal degradation of PP2Ac.

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This study was funded by the Canadian Breast Cancer Foundation/Atlantic Chapter, and by the CIHR-Regional Partnership Programme (CIHR-RPP), Nova Scotia Health Research Foundation and Dalhousie Cancer Research Programme (to CKLT).

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Aranda-Orgilles, B., Rutschow, D., Zeller, R., Karagiannidis, A.I., Kohler, A., Chen, C., Wilson, T., Krause, S., Roepcke, S., Lilley, D., Schneider, R., Schweiger, S., 2011. Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequencedependent regulator of translation efficiency controlling 3-phosphoinositidedependent protein kinase-1 (PDPK-1). J. Biol. Chem. 286, 39945–39957. Benavides, M., Chow-Tsang, L.F., Zhang, J., Zhong, H., 2013. The novel interaction between microspherule protein Msp58 and ubiquitin E3 ligase EDD regulates cell cycle progression. Biochim. Biophys. Acta 1833, 21–32. Boudreau, R.T.M., Sangster, S.M., Johnson, L.M., Dauphinee, S., Li, A.W., Too, C.K.L., 2002. Implication ofa4 phosphoprotein and the rapamycin-sensitive mTOR pathway in prolactin receptor signalling. J. Endocrinol. 173, 493–506. Callaghan, M.J., Russell, A.J., Woollatt, E., Sutherland, G.R., Sutherland, R.L., Watts, C.K., 1998. Identification of a human HECT family protein with homology to the

465 466 467 468 469 470 471 472 473 474 475 476 477

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Drosophila tumor suppressor gene hyperplastic discs. Oncogene 17, 3479– 3491. Chen, J., Peterson, R.T., Schreiber, S.L., 1998. Alpha 4 associates with protein phosphatases 2A, 4, and 6. Biochem. Biophys. Res. Commun. 247, 827–832. Clancy, J.L., Henderson, M.J., Russell, A.J., Anderson, D.W., Bova, R.J., Campbell, I.G., Choong, D.Y., Macdonald, G.A., Mann, G.J., Nolan, T., Brady, G., Olopade, O.I., Woollatt, E., Davies, M.J., Segara, D., Hacker, N.F., Henshall, S.M., Sutherland, R.L., Watts, C.K., 2003. EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer. Oncogene 22, 5070– 5081. Clevenger, C.V., Furth, P.A., Hankinson, S.E., Schuler, L.A., 2003. The role of prolactin in mammary carcinoma. Endocri. Rev. 24, 1–27. Fuja, T.J., Lin, F., Osann, K.E., Bryant, P.J., 2004. Somatic mutations and altered expression of the candidate tumor suppressors CSNK1 epsilon, DLG1, and EDD/ hHYD in mammary ductal carcinoma. Cancer Res. 64, 942–951. Gingras, A.C., Raught, B., Sonenberg, N., 2001. Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15, 807–826. Gordon, J., Hwang, J., Carrier, K.J., Jones, C.A., Kern, Q.L., Moreno, C.S., Karas, R.H., Pallas, D.C., 2011. Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3. BMC Biochem. 12, 54. Han, X., Du, H., Massiah, M.A., 2011. Detection and Characterization of the In Vitro E3 Ligase Activity of the Human MID1 Protein. J. Mol. Biol. 407, 505–520. Henderson, M.J., Munoz, M.A., Saunders, D.N., Clancy, J.L., Russell, A.J., Williams, B., Pappin, D., Khanna, K.K., Jackson, S.P., Sutherland, R.L., Watts, C.K., 2006. EDD mediates DNA damage-induced activation of CHK2. J. Biol. Chem. 281, 39990– 40000. Henderson, M.J., Russell, A.J., Hird, S., Munoz, M., Clancy, J.L., Lehrbach, G.M., Calanni, S.T., Jans, D.A., Sutherland, R.L., Watts, C.K., 2002. EDD, the human hyperplastic discs protein, has a role in progesterone receptor coactivation and potential involvement in DNA damage response. J. Biol. Chem. 277, 26468– 26478. Inui, S., Kuwahara, K., Mizutani, J., Maeda, K., Kawai, T., Nakayasu, H., Sakaguchi, N., 1995. Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal transduction. J. Immunol. 154, 2714–2723. Inui, S., Sanjo, H., Maeda, K., Yamamoto, H., Miyamoto, E., Sakaguchi, N., 1998. Ig receptor binding protein 1 (alpha4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. Blood 92, 539–546. Jackson, P.K., Eldridge, A.G., Freed, E., Furstenthal, L., Hsu, J.Y., Kaiser, B.K., Reimann, J.D., 2000. The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol. 10, 429–439. Jordan, V.C., Morrow, M., 1999. Tamoxifen, raloxifene, and the prevention of breast cancer. Endocrinol. Rev. 20, 253–278. Kozlov, G., De Crescenzo, G., Lim, N.S., Siddiqui, N., Fantus, D., Kahvejian, A., Trempe, J.F., Elias, D., Ekiel, I., Sonenberg, N., O’Connor-McCourt, M., Gehring, K., 2004. Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase. Embo J. 23, 272–281. Kozlov, G., Nguyen, L., Lin, T., De Crescenzo, G., Park, M., Gehring, K., 2007. Structural basis of ubiquitin recognition by the ubiquitin-associated (UBA) domain of the ubiquitin ligase EDD. J. Biol. Chem. 282, 35787–35795. Lenoue-Newton, M., Watkins, G.R., Zou, P., Germane, K.L., McCorvey, L.R., Wadzinski, B.E., Spiller, B.W., 2011. The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation. J. Biol. Chem. 286, 17665– 17671.

Ling, S., Lin, W.C., 2011. EDD inhibits ATM-mediated phosphorylation of p53. J. Biol. Chem. 286, 14972–14982. Liu, E., Knutzen, C.A., Krauss, S., Schweiger, S., Chiang, G.G., 2011. Control of mTORC1 signaling by the Opitz syndrome protein MID1. Proc. Natl. Acad. Sci. USA 108, 8680–8685. Liu, J., Prickett, T.D., Elliott, E., Meroni, G., Brautigan, D.L., 2001. Phosphorylation and microtubule association of the Opitz syndrome protein Mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit alpha 4. Proc. Natl. Acad. Sci. USA 98, 6650–6655. Mangus, D.A., Evans, M.C., Jacobson, A., 2003. Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol. 4, 223. Mansfield, E., Hersperger, E., Biggs, J., Shearn, A., 1994. Genetic and molecular analysis of hyperplastic discs, a gene whose product is required for regulation of cell proliferation in Drosophila melanogaster imaginal discs and germ cells. Dev. Biol. 165, 507–526. McConnell, J.L., Watkins, G.R., Soss, S.E., Franz, H.S., McCorvey, L.R., Spiller, B.W., Chazin, W.J., Wadzinski, B.E., 2010. Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination. Biochemistry 49, 1713–1718. McDonald, W.J., Sangster, S.M., Moffat, L.D., Henderson, M.J., Too, C.K.L., 2010. Alpha4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)binding protein. J. Cell Biochem. 110, 1123–1129. McGuire, W.L., Osborne, C.K., Clark, G.M., Knight 3rd, W.A., 1982. Steroid hormone receptors and carcinoma of the breast. Am. J. Physiol. 243, E99-102. Munoz, M.A., Saunders, D.N., Henderson, M.J., Clancy, J.L., Russell, A.J., Lehrbach, G., Musgrove, E.A., Watts, C.K., Sutherland, R.L., 2007. The E3 ubiquitin ligase EDD regulates S-phase and G(2)/M DNA damage checkpoints. Cell Cycle 6, 3070– 3077. Murata, K., Wu, J., Brautigan, D.L., 1997. B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc. Natl. Acad. Sci. USA 94, 10624–10629. Nanahoshi, M., Nishiuma, T., Tsujishita, Y., Hara, K., Inui, S., Sakaguchi, N., Yonezawa, K., 1998. Regulation of protein phosphatase 2A catalytic activity by alpha4 protein and its yeast homolog Tap42. Biochem. Biophys. Res. Commun. 251, 520–526. O’Brien, P.M., Davies, M.J., Scurry, J.P., Smith, A.N., Barton, C.A., Henderson, M.J., Saunders, D.N., Gloss, B.S., Patterson, K.I., Clancy, J.L., Heinzelmann-Schwarz, V.A., Murali, R., Scolyer, R.A., Zeng, Y., Williams, E.D., Scurr, L., Defazio, A., Quinn, D.I., Watts, C.K., Hacker, N.F., Henshall, S.M., Sutherland, R.L., 2008. The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro. Br. J. Cancer 98, 1085–1093. Pickart, C.M., 2001. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533. Raught, B., Gingras, A.C., Sonenberg, N., 2001. The target of rapamycin (TOR) proteins. Proc. Natl. Acad. Sci. USA 98, 7037–7044. Schmelzle, T., Hall, M.N., 2000. TOR, a central controller of cell growth. Cell 103, 253–262. Trockenbacher, A., Suckow, V., Foerster, J., Winter, J., Krauss, S., Ropers, H.H., Schneider, R., Schweiger, S., 2001. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat. Genet. 29, 287–294. Yang, J., Roe, S.M., Prickett, T.D., Brautigan, D.L., Barford, D., 2007. The structure of Tap42/alpha4 reveals a tetratricopeptide repeat-like fold and provides insights into PP2A regulation. Biochemistry 46, 8807–8815. Zolnierowicz, S., 2000. Type 2A protein phosphatase, the complex regulator of numerous signaling pathways. Biochem. Pharmacol. 60, 1225–1235.

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