Overexpression of the mTOR alpha4 phosphoprotein activates protein phosphatase 2A and increases Stat1α binding to PIAS1

Molecular and Cellular Endocrinology 263 (2007) 10–17

Overexpression of the mTOR alpha4 phosphoprotein activates protein phosphatase 2A and increases Stat1␣ binding to PIAS1 Wei Lun Nien a,1 , Shauna M. Dauphinee a,1 , Lori D. Moffat a , Catherine K.L. Too a,b,∗ a

Department of Biochemistry & Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada b Department of Obstetrics & Gynecology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada Received 12 May 2006; received in revised form 8 August 2006; accepted 14 August 2006

Abstract Alpha4 phosphoprotein in the mTOR pathway is a prolactin (PRL)-downregulated gene product that interacts with the catalytic subunit of serine/threonine protein phosphatase 2A (PP2Ac) in rat Nb2 lymphoma cells. Transient overexpression of alpha4 in COS-1 cells inhibited PRLinducible interferon-regulatory-1 (IRF-1) promoter activity, but the mechanism underlying this inhibition was not known. The present study showed a stable alpha4–PP2Ac complex that was not dissociated by rapamycin in COS-1 cells. Transient overexpression of alpha4 in COS-1 cells had no effect on endogenous PP2Ac protein levels but significantly increased PP2Ac carboxymethylation and PP2A activity as compared to controls. The increased PP2A activity was accompanied by decreased phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP1) but had no effect on Stat phosphorylation. However, overexpressed alpha4 decreased arginine methylation of Stat1␣ and increased Stat1␣ binding to the Stat1␣-specific inhibitor, PIAS1. In summary, ectopic alpha4 increased PP2A activity in COS-1 cells and this was accompanied by Stat1␣ hypomethylation and increased Stat1␣–PIAS1 association. These events would inhibit Stat action and ultimately inhibit PRL-inducible IRF-1 promoter activity. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: mTOR alpha4 phosphoprotein; PRL; PP2A; Stat1␣; PIAS1

1. Introduction Alpha4 phosphoprotein is the mammalian homologue of yeast Tap42, an essential component of the target-of-rapamycin (TOR) kinase pathway that controls translation initiation and cell survival in yeast (Di Como and Arndt, 1996). Yeast Tap42 associates with the type 2A phosphatases, Sit4 and Pph21/22, to regulate their activities (Duvel et al., 2003). Similarly, the conserved mammalian TOR (mTOR) pathway responds to growth factors and nutrients to stimulate translation initiation and cell growth (Thomas and Hall, 1997; Cutler et al., 1999; Bjornsti and Houghton, 2004). The mammalian alpha4 phosphoprotein associates with serine/threonine protein phosphatases PP2A, PP4 and PP6 (Chen et al., 1998). The physical interaction of ∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Sir Charles Tupper Medical Building, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada. Tel.: +1 902 494 1108; fax: +1 902 494 1355. E-mail address: [email protected] (C.K.L. Too). 1 These authors contributed equally to this work.

0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.08.015

alpha4 with the catalytic subunit of PP2A (PP2Ac) is well documented in human, murine and rat cell lines (Murata et al., 1997; Inui et al., 1998; Boudreau et al., 2002). mTOR is believed to control translation through the action of the alpha4–PP2Ac complex on p70S6 kinase (p70S6K) and/or the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (Nanahoshi et al., 1998; Peterson et al., 1999). The mTOR pathway is sensitive to the immunosuppressive drugs, rapamycin and FK506, which bind to the cytosolic family of FK506-binding proteins (FKBP). The rapamycin–FKBP complex acts as an intracellular toxin that disrupts the alpha4–PPAc interaction. However, the stability of the alpha4–PP2AC complex varies with cell types and appears to depend on the rapamycin-sensitivity of the cell lines (Inui et al., 1998). PP2A is a multiprotein enzyme complex that plays a key role in the regulation of many cellular processes, including transcription, translation, cell cycle progression and apoptosis (Lechward et al., 2001). The core enzyme is a dimer, consisting of a 36 kDa catalytic subunit (PP2Ac) and a 65 kDa regulatory subunit (PP2Aa), which associates with one of multiple B-subunits (PP2Ab, 54–130 kDa) (Janssens and Goris,

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2001). PP2A activity is regulated by post-translational modification of PP2Ac (e.g., by reversible phosphorylation and carboxymethylation), association of PP2Ac with regulatory proteins (e.g., alpha4) and targeting of the PP2A holoenzymes to specific subcellular structures. Carboxymethylation of the Cterminal L309 residue of PP2Ac is essential for interaction with the regulatory B␣ subunit (Bryant et al., 1999). Alpha4 is also recognized as a novel regulator of PP2A activity but there is no consensus on its action (Nanahoshi et al., 1999). For example, alpha4 inhibits the phosphatase activites of PP2A, PP4 and PP6 (Nanahoshi et al., 1999) but positively regulates PP2A activity on the Opitz syndrome protein, MID1 and the related MID2 protein (Short et al., 2002). In addition, whereas the alpha4–PP2Ac interaction inhibits PP2A-mediated dephosphorylation of 4E-BP1 (Nanahoshi et al., 1998), it augments PP2A-mediated dephosphorylation of myelin basic protein, histone H1 (Inui et al., 1998) and elongation factor 2 (Chung et al., 1999). We have previously identified alpha4 phosphoprotein as a prolactin (PRL)-downregulated gene in PRL-dependent rat Nb2 lymphoma cells and showed that it co-immunoprecipitated with PP2Ac and several unidentified proteins (Boudreau et al., 2002). We also showed that transient overexpression of alpha4 in COS1 cells inhibited PRL-inducible interferon-regulatory-1 (IRF1) promoter activity (Boudreau et al., 2002), but the mechanism underlying this inhibition was not known. IRF-1 is an immediate-early gene under transcriptional regulation by PRL in Nb2 cells (Yu-Lee et al., 1990), thus implicating cross-talk between the mTOR and PRL receptor (PRLr) signaling cascades. Recently, we have shown that PRL rapidly stimulated phosphorylation of mTOR, which preceded the phosphorylation of downstream p70S6K and 4E-BP1 in Nb2 cells (Bishop et al., 2006). A transient p70S6K–PP2Ac complex was detected upon PRL stimulation for 1–2 h, whereas a PP2Ac–4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells, both suggesting that p70S6K and 4E-BP1 were substrates of PP2A (Bishop et al., 2006). The PRL receptor (PRLr) signals primarily through the Jak2/Stat pathway. Ligand binding results in receptor dimerization, Jak2 tyrosine kinase activation and PRLr phosphorylation, leading to the recruitment and activation of the cytoplasmic Stat proteins (see review (Clevenger et al., 1998)). The PRL-activated Stats form homo/heterodimers, translocate to the nucleus and bind to the interferon␥-activated sequence (GAS) of PRLresponsive genes, such as IRF-1 (Stevens et al., 1995). Tyrosine phosphorylation on a conserved C-terminal carboxyl-terminal residue is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Stat1, -3 and -5 also undergo phosphorylation on a specific serine residue for maximal transactivation potential (see review (Heinrich et al., 2003)). Recently, Stat1 methylation on Arg31 was found to be functionally necessary as it suppressed the interaction of Stat1 with the negative regulator protein PIAS1 (Mowen et al., 2001; Duong et al., 2004). The Stats are substrates of PP2A and PP2Amediated serine dephosphorylation of Stat1 (Eilers et al., 1995) or Stat3 and Stat6 (Woetmann et al., 1999, 2003) decreased Stat binding to DNA.

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In the present study, we examined the mechanism underlying the inhibitory action of ectopic alpha4 on PRLr signaling. We showed that transient overexpression of alpha4 in COS-1 cells increased PP2Ac methylation and PP2A activity. These events were associated with decreased phosphorylation of 4EBP1, hypomethylation of Stat1␣ and increased Stat1␣-binding to PIAS1. 2. Materials and methods 2.1. Antibodies Rabbit anti-alpha4 antibodies, raised against a synthetic peptide corresponding to 20 C-terminus amino acids of the deduced alpha4 protein, was purified as previously described (Boudreau et al., 2002). Commercial antibodies were from the following sources and used at the indicated concentrations: mouse anti-PP2A catalytic subunit (PP2Ac; 1:2500) and anti-methyl-PP2Ac (C-terminal L309; 1:100), rabbit anti-phospho-Stat1␣ (Ser727; 1:1000), antip70S6K (1.5 ␮g/ml) and anti-phospho-p70S6K (0.5 ␮g/ml) were all purchased from Upstate Biotechnology (Lake Placid, NY); mouse anti-Stat1␣ p91 (C-111; 1 ␮g/ml) and rabbit anti-4E-BP1 (1:1000) were from Cell Signaling Technology, New England Biolabs. Ltd. (Mississauga, Ontario, Canada). Mouse anti-PRLr (clone U5; 1 ␮g/ml) was from ABR-Affinity Bioreagents (Golden, CO). Monoclonal antibody to mono/dimethyl arginine (ab412, clone 7E6; 1:1000) was from Abcam Inc. (Cambridge, MA). Rabbit anti-TFIIB (1:200) was from Santa Cruz Biotechnology, Santa Cruz, CA. Secondary antibodies goat anti-mouse IgG-horse radish peroxidase (HRP) conjugate and donkey anti-rabbit IgGHRP conjugate were from BioRad Laboratories (Mississauga, Ontario, Canada) and Amersham Pharmacia Biotechnology (Baie d’Urfe, Quebec, Canada), respectively.

2.2. Cell culture and lysates SV40-transformed African green monkey COS-1 kidney cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS). In some experiments, COS-1 cells were cultured in DMEM containing 5% horse-serum (HS), tested to be free of PRL and interleukin-2 (Too et al., 1987), prior to treatment of cells with 100 ng/ml human PRL (a gift from Dr. Robert Shiu, Department of Physiology, University of Manitoba). Rat Nb2 lymphoma cells were cultured as previously described (Too et al., 1987). For subcellular fractionation, COS-1 cells were washed in phosphatebuffered saline (PBS), pH 7.5, and homogenized in cold, detergent-free RIPA buffer, pH 7.5 (150 mM NaCl, 50 mM sodium phosphate, 1 mM sodium fluoride, 1 mM vanadate, 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylene glycol bis-(␤-aminoethyl ether) N,N,N ,N -tetraacetic acid (EGTA)) containing freshly added protease inhibitors (1 mM phenylmethylsulphonyl fluoride and 10 ␮g/ml each of antipain, leupeptin and pepstatin). The 700 × g cell pellet (nuclear fraction) and supernatant (cytosol fraction) were prepared and then a final concentration of 0.1% Triton X-100 was added. For immunoprecipitation studies, PBS-washed COS-1 and Nb2 cells (∼20 × 106 cells/treatment) were lyzed in 1 ml RIPA buffer containing 0.25% sodium deoxycholate and 0.1% NP-40. PBS-washed COS-1 cell pellets were resuspended in the appropriate buffer for PP2A assays.

2.3. Immunoprecipitation, electrophoresis and western analyses Immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western analysis were performed as previously described (Dodd et al., 2000). In fractionation studies, SDS-PAGE was performed with 10–30 ␮g protein per lane, representing about 15% of the total protein in the nuclear or cytosolic fraction, followed by western analysis. Purified rabbit anti-alpha4 antibodies (7.34 ␮g/␮l IgG) was used for coimmunoprecipitation (1 ␮g/sample) or diluted 1:300 for western blotting. Commercial antibodies were used at concentrations indicated in Section 2.1. The appropriate HRP-conjugated secondary antibodies (1:2000–1:5000 donkey anti-

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rabbit IgG or 1:1250 goat anti-mouse IgG) were used to detect immunoreactive signals with SuperSignal ULTRA (Pierce, Rockford, IL).

2.4. DNA transfection COS-1 cells were transiently transfected as previously described (Boudreau et al., 2002). Briefly, 2 × 105 cells/well in 6-well dishes were cultured in DMEM5% FBS for 24 h and then washed with serum-free DMEM. To each well was added a LipofectAMINE-DNA mixture containing 5 ␮l LipofectAMINETM reagent (Invitrogen) and 1 ␮g pcDNA3.1-␣4 or 1 ␮g pcDNA3.1 in a total volume of 1 ml DMEM. In some experiments, a PRLr isoform (Nb2-PRLr) was tranfected, using 1 ␮g pcDNA3.1-Nb2-PRLr. After 5 h at 37 ◦ C, the LipofectAMINE-DNA mixture was replaced with DMEM-1% HS for 24–48 h, and in some cases PRL (100 ng/ml) was added during this period. Cell lysates were prepared for various studies.

2.6. Chloramphenicol acetyltransferase (CAT) assay CAT assays were performed as previously described (Boudreau et al., 2002). Briefly, COS-1 cells were transiently transfected with 1 ␮g pcDNA3Nb2-PRLr, 0.2 ␮g 1.7 kb IRF-1-CAT, 1 ␮g pcDNA3-Stat1␣ (all generous gifts of Dr. Li-yuan Yu-Lee, Baylor College of Medicine, Houston, TX) and 1 ␮g pcDNA3-␣4. Cell extracts were prepared and equal amounts of protein were used to measure CAT activity, in a reaction mixture containing 0.5 M Tris–HCl, pH 7.8, 0.2 mCi [3 H]-acetyl-coenzymeA and 1.75 mM chloramphenicol. The data were analyzed by regression analysis and expressed as c.p.m./␮g protein.

2.7. Statistical analysis ANOVA and Scheffe’s F-test were performed using Statview (Abacus Concepts Inc., Berkeley, CA). Differences of p < 0.05 were taken as significant.

2.5. Protein phosphatase 2A assay The Serine/Threonine Phosphatase Assay System (Promega, Madison, Wisconsin) was used following the manufacturer’s instructions. In this assay, PP2A activity was selectively measured using the phosphopeptide substrate, RRA(pT)VA, in a PP2A-specific reaction buffer. Briefly, transfected COS-1 cells were resuspended in phosphate-free lysis buffer containing 25 mM Tris–HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA and 2 mM ␤-mercaptoethanol plus protease inhibitors. Cell lysates were prepared and endogenous free phosphate, which could interfere with the assay, was removed from the sample by centrifugation through the supplied Sephadex G-25 Spin columns. Assays were performed in 96-well plates in 1 × PP2A-specific reaction buffer containing 50 mM imidazole, pH 7.2, 0.2 mM EGTA, 0.02% ␤-mercaptoethanol, 0.1 mg/ml BSA and 0.1 mM of the phosphopeptide substrate. Negative controls also contained 5 ␮M okadaic acid, as recommended by the manufacturer to abrogate PP2A-specific activity. After a 3 min preincubation at 30 ◦ C, the phosphatase reaction was initiated by addition of 15 ␮l phosphate-free cell lysate to each well. After a 30 min incubation, the reaction was terminated by addition of 50 ␮l molybdate dye solution. Absorbance at 600 nm was measured. The amount of free phosphate released was determined against a standard curve of free phosphate ranging from 200 to 3000 pmol. Additional controls included reactions without cell lysate (i.e., no enzyme) or complete reactions (substrate plus cell lysate) terminated at time zero.

3. Results 3.1. Nuclear alpha4 interacts with PP2Ac in COS-1 cells In COS-1 cells, the alpha4 phosphoprotein was a nuclear protein (Fig. 1A) that also co-immunoprecipitated with PP2Ac (Fig. 1B). In Fig. 1A, autoradiography of the western blot was carried out for as long as 60 min and clearly demonstrated nuclear but not cytosolic distribution of alpha4. PP2Ac was predominantly nuclear but was also detected in the cytosol (Fig. 1A). The alpha4–PP2Ac complex can be dissociated by 100 nM rapamycin in some (Murata et al., 1997; Inui et al., 1998) but not all cells (Nanahoshi et al., 1998; Kloeker et al., 2003). Our study showed that alpha4 did not dissociate from PP2Ac after treatment of COS-1 cells with rapamycin (100 nM) for 30 min (Fig. 1C), nor after 24 h (data not shown), indicating that the complex was relatively stable in these cells.

Fig. 1. Nuclear alpha4 interacts with PP2Ac in COS-1 cells: (A) COS-1 nuclear (Nuc) and cytosolic (Cyt) fractions were prepared for SDS-PAGE and western analysis as described in Section 2. Transcription factor TFIIB was used as a nuclear marker. (B) Precleared COS-1 cell lysates were equally divided and used for immunoprecipitation (IP) of alpha4 (␣4), followed by SDS-PAGE and immunoblotting (IB) as indicated. IP/IB of alpha4 showed successful pull-down of alpha4 before IB for PP2Ac. Molecular weight markers (in kD) are indicated. Representatives of at least two experiments each. (C) COS-1 cells were treated with rapamycin (RAP; 100 nM) for 30 min or left untreated. Precleared cell lysates were used for IP/IB as indicated. Undiminished association of PP2Ac with alpha4 was seen in at least three separate experiments.

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Fig. 2. PP2Ac levels and distribution are not affected by alpha4 overexpression. COS-1 cells (equal number of dishes) were transfected with the pcDNA3 vector or pcDNA3-␣4 construct. Nuclear (Nuc) and cytosolic (Cyt) fractions were used for western analysis as in Fig. 1. TFIIB served as a nuclear marker and input control. Representative of two separate transfections.

3.2. Overexpression of alpha4 increases PP2Ac methylation and phosphatase activity We next examined the effects of transient overexpression of alpha4 on its partner PP2Ac. COS-1 cells were transfected with the alpha4 expression construct or the vector alone. Western analysis showed an abundance of the alpha4 protein in the transfectants that received the alpha4 construct as compared to the vector controls (Fig. 2). The overexpressed alpha4 distributed in the cell nucleus and in the cytoplasm whereas the endogenous alpha4 was detected in the nucleus only (Figs. 1 and 2). Overexpression of alpha4 had little, if any, effect on endogenous PP2Ac protein levels and distribution (Fig. 2). Transient overexpression of alpha4 resulted in a significant increase in the carboxymethylation of PP2Ac by more than twofold (Fig. 3) and this was accompanied by an increase in PP2A activity by at least 2.5-fold (Fig. 4), each compared to the vector controls. Since carboxymethylation of PP2AC was reported to be essential for its interaction with the regulatory B␣ subunit (Bryant et al., 1999), our results suggested that ectopic alpha4 might enhance PP2A holoenzyme assembly to increase its enzymatic activity.

Fig. 4. Overexpression of alpha4 increases PP2A activity. COS-1 cells were left untransfected (Con, lane 1) or transfected with pcDNA3 (lane 2) or pcDNA3-␣4 (lane 3). Cell lysates were used for: (A) western analysis (20 ␮g protein/lane) of alpha4 or TFIIB (as loading control), and (B) PP2A enzyme assay. In (B), each phosphatase assay was performed in duplicate. Data shown are mean ± S.D. of three separate transfections. (*) Significantly increased over samples with vector alone, P ≤ 0.05 (n = 3). Absence of error bar indicates small S.D.

3.3. PP2Ac interacts with Stat1α Since PRL activation of the IRF-1 promoter is regulated positively by Stat1␣ (Wang and Yu-Lee, 1996; Yu-Lee, 2001), we examined if Stat1␣ was a PP2A substrate and if the two proteins interacted physically. Fig. 5A showed that PP2Ac co-immunoprecipitated with Stat1␣ in COS-1 cells. Unlike COS-1 cells, rat Nb2 lymphoma cells have a robust Jak/Stat machinery, therefore, we examined if the PP2Ac–Stat1␣ complex was present in Nb2 cells. Fig. 5B showed that PP2Ac also co-immunoprecipitated strongly with Stat1␣ but not with

Fig. 3. Overexpression of alpha4 increases PP2Ac methylation. COS-1 cells (equal number of dishes) were transfected with pcDNA3 or pcDNA3-␣4: (A) whole cell lysates (20 ␮g protein/lane) were used for western analysis. After IB with anti-methyl-PP2Ac (m-PP2Ac) antibodies, the blots were stripped and re-immunoblotted for the endogenous PP2Ac (also served as a loading control). Representative of five separate transfections. (B) Densitometric scanning showing the ratio of mPP2Ac/PP2Ac. Mean ± standard deviation (S.D.; n = 5). (*) Significantly higher than controls, P < 0.05.

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Fig. 5. PP2Ac associates with Stat1␣. Precleared lysates prepared from: (A) COS-1 or (B) Nb2 cells (20 × 106 cells/treatment) were used for IP/IB as indicated. In (B), Nb2 cell homogenate (H) served as a positive control for PP2Ac. For each cell line, the data shown is a representative of at least two separate experiments.

ERK kinase in Nb2 cells, demonstrating specificity in this protein–protein interaction. 3.4. Overexpression of alpha4: Stat1α phosphorylation is unchanged but 4E-BP1 is hypophosphorylated Since ectopic alpha4 increased PP2A activity (Fig. 4), its effect on Stat1␣ phosphorylation was examined. The endogenous Stat1␣ in COS-1 cells was serine phosphorylated and ectopic alpha4 had no significant effect on this status (Fig. 6A and B). COS-1 cells do not express the PRLr and PRL may be required to modulate alpha4 action on Stat phosphorylation. To test this possibility, COS-1 cells were co-transfected with

expression constructs of alpha4 and the PRLr or transfected with the vector alone (Fig. 6C–F). Increased protein levels of alpha4 and expression of the PRLr were confirmed in western analysis (Fig. 6C). A functional PRLr was demonstrated by PRL activation of the IRF-1-promoter whose activity was increased by two to three-fold over controls which did not receive PRL (Fig. 6D, lanes 2 and 3). The endogenous Stat1␣ in all treatment groups was serine phosphorylated (Fig. 6E) but ectopic alpha4, with or without the addition of PRL, did not significantly alter the phosphorylation of Stat1␣ (Fig. 6F; upper left, lanes 2 and 3). Overexpression of alpha4 also had no effect on tyrosine phosphorylation of Stat1␣ on Y701 nor serine phosphorylation of Stat5a/b on S726/731 (data not shown). Recently, we reported that PP2Ac could be induced to associate with p70S6K and 4E-BP1 (Bishop et al., 2006). The present study showed that ectopic alpha4 had no effect on the phosphorylation of p70S6K in COS-1 transfectants, regardless of PRL addition (Fig. 6E and F, lower left). In western analysis, the ␣-, ␤- and ␥-forms of 4E-BP1 were readily detected in COS-1 cells and they represented the hypo-(␣) to hyperphosphorylated (␥) 4E-BP1 protein (Fig. 6E). Overexpression of alpha4, with or without PRL, increased the intensity of the ␣-band of 4E-BP1 (Fig. 6E and F, right panel), suggesting that alpha4 stimulation of PP2A activity decreased phosphorylation of 4E-BP1. 3.5. Overexpression of alpha4 decreases methylation of Stat1α but increases Stat1α binding to PIAS1 We next examined the effects of ectopic alpha4 on Stat1 methylation. COS-1 transfectants, either overexpressing alpha4 or with the vector alone, were used to immunoprepitate the

Fig. 6. Ectopic alpha4 has no effect on Stat phosphorylation but 4EBP-1 is hypophosphorylated. (A) COS-1 cells were transfected with pcDNA3 or pcDNA3-␣4. Cells were maintained in DMEM-1% HS (free of PRL to reduce Stat activation) for 48 h and whole cell lysates were prepared for (A) western analysis (20 ␮g protein/lane) followed by (B) densitometric scanning of phospho-Stat1␣/Stat1␣ (mean ± S.D., n = 8). Stat1␣ also served as a loading control. (C–F) COS-1 cells were transfected with the pcDNA3 vector alone (lane 1) or with pcDNA3-␣4 and pcDNA3-Nb2-PRLr together (lanes 2 and 3). Cells were maintained in DMEM-5% HS for up to 48 h and then given PRL (100 ng/ml) for 1 h (+; lane 3) or left untreated (−; lane 2). Cells were harvested and in (C, E) western analysis was performed. In (D), CAT assay was performed to measure IRF-1 promoter activity (mean ± S.D., n = 3). In (F), densitometric scanning of phospho-Stat1␣/Stat1␣ (upper left) or phospho-p70S6K/p70S6K (lower left) (mean ± S.D., n = 3); or a representative scan of the ␣, ␤ and ␥ forms of 4EBP1 (right). Representatives of 8 (A) and 3 (C, E) separate transfections.

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Fig. 7. Ectopic alpha4 decreases Stat1␣ methylation but increases Stat1␣ binding to PIAS1. COS-1 cells were transfected with pcDNA3 or pcDNA3-␣4 and cell lysates were prepared. (A) An aliquot of each cell lysate (20 ␮g/lane) was used for western analysis of alpha4 (to confirm overexpression) or TFIIB (loading control). (B) Precleared cell lysates were used for IP of Stat1␣. IB was performed, first with anti-mono/dimethyl arginine antibodies. The blots were then stripped and reimmunoblotted with anti-Stat1␣ or with anti-PIAS1 antibodies. Representative of three separate experiments. (C) Densitometric scanning as indicated. Mean ± S.D. (n = 3). (*) significantly different from controls, P < 0.05.

endogenous Stat1␣. Western analysis and densitometric scanning showed that Stat1␣ was hypomethylated in cells overexpressing alpha4 as compared to controls and there was an increased association of the hypomethylated Stat1␣ to PIAS (Fig. 7). 4. Discussion PRL downregulation of the alpha4 phosphoprotein in Nb2 cells and ectopic alpha4 inhibition of the PRL-inducible IRF-1 reporter gene in COS-1 cells (Boudreau et al., 2002) both suggested that a decrease in alpha4 levels was essential for PRLr signal transduction. By overexpressing alpha4 in COS-1 cells, we attempted to elucidate the inhibitory action of alpha4 on PRLr signaling. Similar to our findings in Nb2 cells, the present study showed that the COS-1 alpha4 phosphoprotein was nuclear and it formed a relatively stable complex with PP2Ac. We also showed that transient overexpression of alpha4 in COS-1 cells increased carboxymethylation of PP2Ac and increased PP2A activity. Rapamycin has been shown to dissociate the alpha4–PP2Ac complexes in COS-7 and Jurkat cells (Murata et al., 1997; Inui et al., 1998) but has no effect on this complex in HEK293 and COS-M6 cells (Nanahoshi et al., 1998; Kloeker et al., 2003). The physical interaction between alpha4 and PP2Ac may also be disrupted by PP2A inhibitors such as okadaic acid and microcystin (Kloeker et al., 2003). Okadaic acid has been postulated to confer conformational changes near the PP2Ac binding site for alpha4, and potentially negating the positive regulation by alpha4 and abrogating PP2A activity (Kloeker et al., 2003). The association of PP2Ac with the PP2A B regulatory subunits, critically mediated by carboxymethylation of PP2Ac, was shown to increase PP2A phosphatase activity (Tolstykh et al., 2000; Wu et al., 2000). Thus, in our study, increased carboxymethylation of

PP2Ac could enhance its association with the B subunits, giving rise to increased PP2A activity. PRL activation of the IRF-1 promoter is regulated positively by Stat1␣ but negatively by Stat5a/b (Wang and YuLee, 1996; Yu-Lee, 2001). PP2A has been reported to inhibit IFN␥-activated serine phosphorylation of Stat1 and to reduce DNA binding of a transcriptional complex consisting of a Stat1 homodimer and the IFN-␥-activated GAF transcription factor (Eilers et al., 1995). Inhibition of PP2A has also been shown to increase serine phosphorylation of Stat3 and Stat6, resulting in decreased Stat-DNA binding (Woetmann et al., 1999, 2003). In our study, overexpression of alpha4, by increasing PP2A activity, might be expected to decrease serine phosphorylation of the Stat proteins to modulate IRF-1 promoter activity in COS-1 transfectants. However, we showed that the upregulation of PP2A activity by ectopic alpha4 was accompanied by hypomethylation of Stat1␣, without affecting Stat1 phosphorylation, and an increased Stat1␣–PIAS binding. Similar to our observations, hepatitis C virus inhibition of IFN␣ signaling was reported to occur through upregulation of PP2A, hypomethylation of Stat1 without a change in Stat1 phosphorylation, and an increased Stat1–PIAS interaction (Duong et al., 2004). PIAS1 is a Stat1specific inhibitor, which blocks Stat1 binding to DNA to inhibit gene activation (Liu et al., 1998). Although our study showed no effect on Stat phosphorylation, upregulation of PP2A activity by ectopic alpha4 was accompanied by decreased phosphorylation of 4E-BP1 in COS-1 cells. We and others have shown that PP2A dephosphorylates and/or interacts with 4E-BP1 in Jurkat1 and Nb2 lymphoma cells (Peterson et al., 1999; Bishop et al., 2006). Since dephosphorylation of 4E-BP1 increases its repressor activity and its sequestration of eukaryotic initiation factor 4E (Clemens, 2001), our study suggests that ectopic alpha4 may activate PP2A to inhibit translation.

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N-terminal arginine methylation of Stat1 is catalyzed by arginine methyltransferase and inhibition of this enzyme by methylthioadenosine was shown to increase PIAS1 association with phosphorylated Stat1 dimers, resulting in impaired Stat1-DNA binding and inhibition of Stat1-mediated IFN responses (Mowen et al., 2001). Inhibition of Stat1 arginine methylation was also shown to prolong the half-life of Stat1 tyrosine phosphorylation, an effect that was mediated by increased PIAS1-Stat1 binding and decreased Stat1 association with TcPTP tyrosine phosphatase (Zhu et al., 2002). Recently, arginine methylation of Stat proteins has been disputed (Meissner et al., 2004; Komyod et al., 2005). In those studies, anti-methylarginine antibodies were used for immunoprecipitations. It has been suggested that certain Stat antibodies used for the subsequent immunoblotting might cross-react with proteins that were precipitated by the anti-methylarginine antibodies (Komyod et al., 2005). In our study, we first immunoprecipitated Stat1 and then immunoblotted with anti-methylarginine antibodies. Others studying hepatitis C virus infection have also consistently shown hypomethylation of Stat1 and increased Stat1–PIAS1 association leading to the impairment of IFN-activated Jak/Stat signaling (Duong et al., 2005, 2006). In summary, transient overexpression of alpha4 in COS-1 cells increased PP2Ac carboxymethylation and PP2A activity and was accompanied by Stat1␣ hypomethylation and increased Stat1␣–PIAS1 binding. The increase in PP2A activity was also accompanied by decreased phosphorylation of 4E-BP1. These events, resulting from alpha4 overexpression in COS-1 cells, would culminate in the inhibition of Stat-mediated responses and inhibition of translation. In contrast, PRL downregulation of alpha4 in the Nb2 cells may be expected to facilitate Statmediated responses and activation of translation. Acknowledgements The excellent technical assistance of Ms. Shirley Sangster is gratefully acknowledged. WLN was awarded two summer studentships from the Izaak–Walton–Killam, including the IWK Mendel Burstein Award, and a partial summer studentship from the Dalhousie Cancer Biology Research Group (CBRG). SMD was a recipient of a graduate studentship from Cancer Research and Education (CaRE) Nova Scotia. LDM received a partial summer studentship from CBRG. This work was supported by the Canadian Institutes of Health Research (to CKLT). References Bishop, J.D., Nien, W.L., Dauphinee, S.M., Too, C.K.L., 2006. Prolactin activates mTOR through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-BP1 in lymphoma cells. J. Endocrinol. 190, 307–312. Bjornsti, M.A., Houghton, P.J., 2004. The TOR pathway: a target for cancer therapy. Nat. Rev. Cancer 4, 335–348. Boudreau, R.T.M., Sangster, S.M., Johnson, L.M., Dauphinee, S., Li, A.W., Too, C.K.L., 2002. Implication of ␣4 phosphoprotein and the rapamycinsensitive mTOR pathway in prolactin receptor signalling. J. Endocrinol. 173, 493–506.

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