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Originally published In Press as doi:10.1074/jbc.M508365200 on June 15, 2006

J. Biol. Chem., Vol. 281, Issue 33, 23748-23756, August 18, 2006
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The Novel PIAS-like Protein hZimp10 Enhances Smad Transcriptional Activity*

Xiaomeng Li, Gregory Thyssen, Jason Beliakoff, and Zijie Sun1

From the Departments of Urology and Genetics, Stanford University School of Medicine, Stanford, California 94303-5118

Received for publication, July 29, 2005 , and in revised form, June 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor beta (TGF-beta) plays critical roles in the control of cell proliferation, differentiation, and apoptosis. Smad proteins are substrates of the TGF-beta type I receptor and are responsible for transducing receptor signals to target genes in the nucleus. The PIAS (protein inhibitor of activated STAT) proteins were originally identified as transcriptional co-regulators of the JAK-STAT pathway. Subsequently, cross-talk between the PIAS proteins and other signaling pathways has been shown to be involved in various cellular processes. Importantly, PIAS proteins modulate TGF-beta signaling by regulating the transcriptional activity of Smad3. In this study we tested whether hZimp10, a novel PIAS-like protein, acts as other PIAS proteins to regulate Smad3-mediated transcription. We show that expression of exogenous hZimp10 enhances the transcriptional activity of Smad3, which appears to be Smad4-dependent and responsive to TGF-beta induction. Furthermore, knockdown of endogenous hZimp10 reduced the transcriptional activity of Smad3. A protein-protein interaction between Smad3 and Smad4 with hZimp10 was identified in glutathione S-transferase-pulldown and co-immunoprecipitation assays. The Miz domain of hZimp10 and the MH2 domains of Smad3 and Smad4 were mapped as the regions responsible for binding. Results from immunostaining assays further demonstrated that Smad3, Smad4, and hZimp10 co-localize within cell nuclei. Finally, we demonstrated that Smad3/4-mediated transcription is significantly impaired in response to TGF-beta induction in Zimp10 null (zimp10-/-) embryonic fibroblasts. Taken together, these results provide the first line of evidence to demonstrate a role for Zimp10 in regulating the TGF-beta/Smad signaling pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transforming growth factor-beta (TGF-beta)2 family comprises a large number of structurally related polypeptide growth factors that play critical roles in cell proliferation, differentiation, motility, adhesion, and death (1). TGF-beta and related factors activate signaling by binding and bringing together members of two subfamilies of transmembrane protein serine/threonine kinases, the type I (TbetaR-I) and type II receptors (TbetaR-II). Smad proteins are the substrates of TGF-beta type I receptor and play a central role in transducing receptor signals to target genes in the nucleus (2). The Smads can be loosely grouped into three categories. Smad2 and Smad3 are substrates and mediators of the related TGF-beta and activin receptors, whereas Smad4 acts as a cofactor for the receptor-regulated Smads. Smad6 and 7, termed anti-Smads, inhibit the signaling function of the other two groups (3).

Recent studies have shown that Smad proteins can modulate transcription through interactions with other transcriptional co-activators or co-repressors (4). For instance, Smad3 and Smad4 interact with multiple members of the AP1 family (5, 6). The interaction between Smads, p300/CBP, and p300/CBP-associated factor may dictate promoter specificity and mediate signal integration (7, 8). Smads also associate with other transcription factors including SP1 and leukemia inhibitory factor and allow for a higher level of promoter specificity and transcription activity (9, 10). Smad3 is responsible for TGF-beta-mediated transcriptional repression of c-myc (11). Smad2, Smad3, and Smad4 can interact with the nuclear oncoproteins SnoN and Ski to repress transcription (12, 13). Smad2/Smad4 complexes can recruit histone deacetylase to promoters through association with the homeodomain protein, 5' TG 3' interacting factor, and Sin3A (14, 15). Recently, several lines of evidence have shown that Smad3 can be regulated directly or indirectly by phosphatidylinositol 3-kinase and AKT signaling pathways (16, 17).

The PIAS (protein inhibitor of activated STAT) proteins were first identified as transcriptional co-regulators of the JAK-STAT pathway (18). PIAS1 and PIAS3 have been shown to inhibit the activity of STAT1 and STAT3, respectively (19-21). However, recent studies have suggested that the PIAS proteins may play a more general role in regulating chromatin structure (22). An increased interest has been focused on the role of PIAS proteins in sumoylation (23). Sequence analysis has shown that the SUMO E3 ligase RING domain shares significant homology with the Miz domain of PIAS proteins (24). Moreover, PIASx{alpha}, -xbeta, -1, and -3 have been found to interact with SUMO-1 and Ubc9 and mediate the sumoylation of p53 and steroid hormone receptors (25-31).

Recent studies have shown that PIAS proteins interact with the TGFbeta/Smad pathway. PIASy was reported to repress the transcriptional activity of Smad3, and this repressive effect was due to enhanced recruitment of HDAC1 (32). In contrast, PIAS3 showed an opposite effect, enhancing Smad3-mediated transcription (33). The RING domain of PIAS3 can interact with the transcriptional co-activator p300/CBP and form a ternary complex with Smad3. Moreover, the SUMO-conjugating enzyme Ubc9 and PIAS proteins have been shown to enhance the sumoylation of Smad4 (34). The sumoylation of Smad4 by PIAS proteins is regulated by the p38 mitogen-activated protein kinase pathway (35).

hZimp10 is a novel PIAS-like protein (36). It shares a ring finger domain, termed Miz (msx-interacting zinc finger), with other PIAS proteins (37), which appears to be important for protein-protein interactions. A novel Drosophila gene, termed tonalli (tna), was identified recently and is the ortholog of hZimp10 (38). The protein encoded by tna genetically interacts with the chromatin remodeling complexes SWI2/SNF2 and the Mediator complex, suggesting that it may play a role in transcription. In this study, we tested whether hZimp10 affects Smad3-mediated transcription in a manner similar to that of the PIAS proteins. Using several in vivo and in vitro approaches, we demonstrated that Zimp10 interacts with Smad3/4 proteins and augments Smad-mediated transcription, which provides the first line of evidence that Zimp10 plays a critical role in the regulation of the TGF-beta/Smad signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The pcDNA3-FLAG-Smad3 and pcMV5-FLAG-Smad4 expression vectors were described previously (39, 40). The HA-tagged Smad4 expression plasmid was constructed by inserting the full-length Smad4 cDNA into pcDNA3 with an N-terminal HA epitope tag (Invitrogen). Various deletion mutants of Smad3 and Smad4 were cloned into pGEX vectors (Amersham Biosciences). The pGEX4T1-Smad4 and 3TP-Luc were kindly provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York). pSV-beta-gal, an SV40 driven beta-galactosidase reporter plasmid (Promega, Madison, WI), was used in this study as an internal control. The pcDNA3-hZimp10, pcDNA3-FLAG-hZimp10, and pcDNA3-FLAG-hZimp7 were generated as described previously (36, 41). The fragments of hZimp10, including the N terminus (amino acids 1-333), Miz domain (amino acids 728-809), and C terminus (amino acids 932-1064), were generated by PCR with appropriate primers and subcloned in-frame to the pGEX4T3 for making GST fusion proteins. The hZimp10 mutants containing double point mutations (C755G/H757A and C760G/H762A) within the Miz domain were generated by a PCR-based site-directed mutagenesis approach in the pcDNA3-FLAG vector. The pGEX4T3-PIASx{alpha}/ARIP3 plasmid was kindly provided by Dr. J Palvimo (Helsinki, Finland).

Cell Cultures and Transient Transfections—A monkey kidney cell line, CV-1, a human prostate cancer cell line, PC3, a human colon cancer cell line, SW480.7, and a human embryonic kidney cell line, HEK293, were maintained in Dulbecco's modified Eagle's medium supplemented with 5 or 10% fetal bovine serum (HyClone, Denver, CO). Transient transfections were carried out using a LipofectAMINE2000 kit (Invitrogen). Approximately 1.5 x 104 cells were seeded into a 48-well plate 16 h before transfection. 300 ng of total plasmid DNA and 0.5 µl of Lipofectamine2000 per well were used in the transfection. The total amount of plasmid per well was equalized by the addition of pcDNA3 or pBluescript empty vector. Approximately 48 h after transfection, luciferase activity was measured as relative light units in a Monolight 3010 luminometer (Pharmingen) according to the manufacturer's protocol. The relative light units from individual transfections were normalized by beta-galactosidase activity in the same samples. Individual transfection experiments were done in triplicate, and the results are reported as mean relative light units/beta-galactosidase (±S.D.) from representative experiments.

GST Pulldown Assay—Expression and purification of GST fusion proteins were performed as described previously (42). The full-length Smad3, Smad4, and hZimp10 proteins were generated and labeled in vitro by the TNT-coupled reticulocyte lysate system (Promega). Equal amounts of GST fusion proteins coupled to glutathione-Sepharose beads were incubated with the radiolabeled proteins at 4 °C for 2 h in a modified binding buffer (20 mM Tris-HCl (pH 7.8), 180 mM KCl, 0.5 mM EDTA, 5 mM MgCl2, 50 µM ZnCl2, 10% glycerol, 0.1% Nonidet P-40, 0.05% dry nonfat milk, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). Beads were carefully washed 3 times with 500 µl of binding buffer and then analyzed by SDS-PAGE followed by autoradiography.

Immunoprecipitation and Western Blotting—The HA-tagged pcDNA3-hZimp10 expression plasmid, alone or with a FLAG-tagged pcDNA3-Smad3 and/or FLAG-tagged pCMV5-Smad4 expression plasmids, was transfected into CV-1 cells. Transfected cells were cultured for 48 h and then harvested in a buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 2 mM MgCl2, 50 mM HEPES-KOH (pH 7.4), 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 25 mM NaF. Lysates were clarified by incubation on ice and centrifugation for 5 min. Four hundred µl of clarified lysate from each sample was precleared for 20 min with 10 µl of protein-A-Sepharose beads bound to 1 µg of normal mouse IgG (Pharmacia). Precleared lysates were then incubated with pre-equilibrated protein-A-Sepharose beads with either normal mouse IgG or FLAG monoclonal antibody (Sigma) at 4 °C for 3 h. The beads were washed 3 times in 500 µl of lysis buffer and eluted by boiling in SDS-PAGE sample buffer. After SDS-PAGE, proteins were transferred to nitrocellulose (Schleicher and Schuell) and blocked overnight at 4 °C in TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.08% Tween 20) with 5% lowfat milk. Membranes were probed with HA, FLAG, Smad3, Smad4, or the hZimp10 antibody at the appropriate dilutions. Anti-rabbit, mouse, or chicken IgG conjugated to horseradish peroxidase were used as secondary antibodies (Promega). Detection was performed with ECL reagents according to the manufacturer's protocol using ECL Hyperfilm (Amersham Biosciences).

Immunostaining—CV-1 or PC3 cells were co-transfected with pcDNA3-hZimp10, FLAG-tagged pcDNA3-Smad3, and HA-tagged pcDNA3-Smad4 in the presence or absence of TGF-beta 1 growth factor (R&D Systems, Minneapolis, MN). Specific primary antibodies and Fluorophore-conjugated secondary antibodies were used (Molecular Probes, Eugene, OR). Images were acquired using a confocal microscope.

Mouse Embryonic Fibroblasts—Mice heterozygous for a neomycin-disrupted allele of the Zimp10 gene were mated, and females were sacrificed at 9.5 days post-coitus. Embryos were isolated in cold phosphate-buffered saline and then incubated in 250 µl of trypsin (0.05%) for 10 min at 37 °C with intermittent agitation. Embryos were disrupted by pipetting and then added to at least a 3x volume of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were directly plated into 6- or 48-well plates, allowed to adhere overnight, and used for assays. To determine the mouse embryo fibroblasts (MEF) genotype, embryo sacs isolated during the dissection were digested, genomic DNA was extracted, and the wild type or mutant Zimp10 alleles were PCR-amplified using specific primers.


Figure 1
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  		FIGURE 1.
hZimp10 augments Smad3-mediated transcription. A, CV-1 cells were transiently transfected in 48-well plates with 100 ng of 3TP-Luc, 25 ng of pSV40-beta-gal, 8 ng of pcDNA3-FLAG-Smad3, and where indicated, 20 or 60 ng of pcDNA3-FLAG-hZimp10, pcDNA3-FLAG-hZimp7, or pcDNA3-FLAG-PIASx{alpha}. The total amount of plasmid per well was normalized in all transfections by the addition of pcDNA3 empty vector. Luciferase activity is reported as relative light units and represented as the mean ± S.D. B, the transient transfection was repeated as described above but in the presence of 4 ng of pcDNA3-FLAG-Smad4. C, SW480.7 cells were transiently transfected in 48-well plates with 100 ng of 3TP-Luc, 25 ng of pSV40-beta-gal, and other plasmids as indicated in the figure. D, HEK293 cells were transfected with 100 ng of 3TP-Luc, 25 ng of pSV40-beta-gal, 8 ng of pcDNA3-FLAG-Smad3, and 4 ng of pcDNA3-FLAG-Smad4 as well as other plasmids as indicated and incubated with serum-free Dulbecco's modified Eagle's medium overnight. Then TGF-beta1 factor was added into cells, and whole cell lysates were prepared after 24 h for luciferase and beta-galactosidase assays.

 
RNA Isolation and Reverse Transcription (RT)-PCR Assay— Mouse embryo fibroblasts were established as described above and serum-starved overnight. TGF-beta1 was then added directly to the media to achieve a final concentration of 50 ng/ml. 5.5 h after stimulation, total RNA was isolated using RNABee (TEL-TEST, Inc., Friendswood, TX). The RT-PCR method was carried out as described previously (43). Briefly, cDNA was synthesized from 1-5 µg of total RNA with 9 units of avian myeloblastosis virus reverse transcriptase (Promega) using 0.1 µM oligo-dT primer in a total volume of 20 µl. One µl of cDNA was added to a standard PCR mix containing 1 µM concentrations of each primer. The PCR reaction was performed on a thermal cycler using 26-30 cycles of 45 s at 95 °C, 40 s at 58 °C, and 45 s at 72 °C for glyceraldehyde-3-phosphate dehydrogenase and 30 s at 95 °C, 30 s at 52 °C, and 50 s at 72 °C for PAI-1. The final polymerization step was extended an additional 10 min at 72 °C. Primers for PAI-1 (5'-TCATCAATGACTGGGTGGAA-3';5'-CTGCTCTTGGTCGGAAAGAC-3') and glyceraldehyde-3-phosphate dehydrogenase (5'-CCATGGAGAAGGCTGGGG-3'; 5'-CAAAGTTGTCATGGATGACC-3') were synthesized and used in the PCR reactions.


Figure 2
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  		FIGURE 2.
Knockdown of hZimp10 expression reduces Smad3/4-mediated transcription. A, CV-1 cells were transfected with 1 µg of pcDNA3-FLAG-hZimp10 (F-hZimp10) and 200 ng of different pBluescript (pBS)/U6-hZimp10-shRNA constructs (Z1, Z2, and Z3) or pBS/U6 vector only in 6-well plates. Whole cell lysates were prepared after 48 h of transfection and analyzed by Western blotting with either FLAG or tubulin antibody. B, different hZimp10 shRNA constructs or pBS/U6 vector were transfected into HEK293 cells. Cells were harvested, and cell lysates were analyzed as described in A. C, CV1 cells were transfected with different amounts of plasmids as indicated in the figure. Luciferase and beta-galactosidase activities were measured in whole cell lysates as described above. The asterisk indicates that the data are significantly different by t test (p < 0.05). D, HEK293 cells were transiently transfected in 48-well plates with 100 ng of 3TP-Luc, 25 ng of pSV40-beta-gal, 8 ng of pcDNA3-FLAG-Smad3, 4 ng of pCMV5-FLAG-Smad4, and 15 or 45 ng of hZimp10 shRNA. The shRNA vector backbone was used as a control. The data showing statistical significance is marked with an asterisk.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hZimp10 Augments Smad3-mediated Transcription—PIAS proteins have been shown to regulate TGF-beta/Smad3 activity. Here, we investigated a possible role for hZimp10 (36) and hZimp7 (41), novel PIAS-like proteins, in regulating Smad3-mediated transcription. A plasmid containing the TGF-beta-inducible luciferase reporter (3TP-Luc) was co-transfected into CV-1 cells with plasmids expressing Smad3, hZimp7, hZimp10, or PIASx{alpha}.An ~5-fold induction of Smad3-mediated transcriptional activity was observed when cells were transfected with Smad3 (Fig. 1A). Smad3 activity was enhanced ~2-fold in the presence of 60 ng of hZimp10, and this enhancement was dose-dependent. In contrast, co-transfection of hZimp7 and PIASx{alpha} showed no significant effect (Fig. 1A). There was no effect when only hZimp7, hZimp10, or PIASx{alpha} was transfected alone with the reporter plasmid (data not shown). These results indicate that hZimp10, but not hZimp7 or PIASx{alpha}, augments Smad3-mediated transcription.

Previous studies have shown that Smad4 can form a heterodimer with Smad3, which can then translocate into the nucleus to activate the transcriptional response (44, 45). To test whether the enhancement of hZimp10 is mediated through the transcriptionally active Smad3/Smad4 complex, we repeated the transient transfection assays presented in Fig. 1A in the presence of a Smad4 expression vector. As shown in Fig. 1B, Smad4 increases Smad3-mediated transcription by nearly 30%, and hZimp10 further enhances Smad3/4-mediated transcription to ~0.6-1.2-fold. Again, no enhancement was observed with hZimp7 or PIASx{alpha}. To further confirm that hZimp10 enhances the activity of the Smad3/Smad4 transcriptional complex, we repeated the above experiments in the Smad4-negative cell line SW480.7. As expected, overexpression of Smad3 showed no significant transcriptional activity on 3TP-Luc in this human colon cancer cell line (Fig. 1C). There was also no significant effect of hZimp10 on Smad3-mediated transcription. However, expression of exogenous Smad4 resulted in a dosage-dependent enhancement of Smad3-mediated transcription. In the presence of Smad4, hZimp10 further increased the activity of 3TP-luc in a dosage-dependent manner. Taken together, these data indicate that hZimp10 can enhance the activity of the Smad3/Smad4 transcriptionally active complex.

Previous studies have demonstrated that TGF-beta signals are transmitted through Smad proteins (2, 46). To determine whether enhancement of Smad3/4 by hZimp10 is induced by TGF-beta, we repeated the transfection experiments with serum-free medium with or without TGF-beta1 in HEK293 cells, which respond to TGF-beta induction. As shown in Fig. 1D, there was only a slight increase in luciferase activity in cells transfected with Smad3 and Smad4 expression vectors in the absence of TGF-beta1. However, co-transfection of hZimp10 with Smad3 and 4 resulted in 20-50% increased luciferase activity in cells treated with TGF-beta1 (Fig. 1D). These results suggest that hZimp10 affects TGF-beta-induced Smad3/4-mediated transcription.

Next, we investigated the involvement of endogenous hZimp10 in regulating the transcriptional activity of the Smad3/Smad4 complex. We first generated three short hairpin RNA (shRNA) constructs for hZimp10 (47) and tested their knockdown effects on ectopically expressed hZimp10 in CV-1 cells (Fig. 2A). All three hZimp10 shRNA constructs reduced the expression of FLAG-tagged hZimp10 protein. There was no change in tubulin expression, confirming the specificity of the hZimp10 shRNAs. Particularly, the hZimp10 shRNA construct 2 appeared most effective in this knockdown experiment. In addition, this construct also diminished hZimp10 enhancement of Smad3/Smad4-mediated transcription (Fig. 2C). A t test showed that the hZimp10 shRNA-mediated knockdown effect is significant (p < 0.05). Using this construct, we further tested the role of endogenous hZimp10 on Smad3/4-mediated transcription in HEK293 cells. As shown in Fig. 2B, the hZimp10 shRNA2 significantly reduced the expression of the endogenous protein. This knockdown effect resulted in an ~35 or 50% reduction in Smad3/4-mediated transcription at 15 or 45 ng of the shRNA2 construct, respectively (Fig. 2D). Taken together, the above data indicate an important role for endogenous hZimp10 in augmenting the activity of the Smad3/Smad4 transcriptional complex.

The Miz Domain of hZimp10 Is Involved in the Interaction with Smad3 and Smad4 Proteins—Previous reports suggest that the Miz domain plays a role in interacting with target proteins (37). Particularly, it has been shown that the Miz domain of PIAS3 and PIASy is responsible for interacting with the Smad3 and Smad4 proteins (32, 33). To directly assess the involvement of the hZimp10 Miz domain in the interaction with Smad3 and Smad4, we performed in vitro GST-pulldown assays. [35S]Methionine-labeled full-length Smad3 or Smad4 bound to different GST-hZimp10 fusion proteins or GST protein alone was analyzed by SDS-PAGE and detected by autoradiography. As shown in Fig. 3A, Smad3 and Smad4 proteins bound to GST-PIASx{alpha}/ARIP3, which was used as a positive control. Importantly, a weak interaction was observed in samples containing GST-hZimp10-Miz (amino acids 728-809) but not with GST-hZimp10-N' (amino acids 1-333), GST-hZimp10-C' (amino acids 932-1064), or GST beads alone. Next, we used two hZimp10 Miz domain mutants that contain double point mutations, Mut1 (C755G/H757A) and Mut2 (C760G/H762A), to further assess the importance of the Miz domain in the interaction. Either the wild type hZimp10 or the mutants of hZimp10 proteins were synthesized and tested in GST-pulldown experiments (Fig. 3C). As shown in Fig. 3B, a specific interaction was observed between the full-length GST-hZimp10 protein and Smad3 or Smad4. However, with equal amounts of inputs, the Smad proteins showed no interaction with the two hZimp10 Miz domain mutants (Fig. 3C). These results not only provide a line of evidence demonstrating an interaction between Smad3 and Smad4 with hZimp10 in vitro but also show that the Miz domain of hZimp10 is required for the interaction with the Smad proteins.

The MH2 Domains of Smad3 and Smad4 Interact with hZimp10—Smad3 and Smad4 proteins contain a number of functional domains, including MH1, MH2, and the linker region (2). It appears that the MH2 domain is involved in many biological processes through interaction with other regulatory proteins (45, 48). Previous studies have shown that the MH2 domain is involved in the interaction with PIAS3 and PIASy (32, 33). Here, we used in vitro GST-pulldown experiments to test which regions of Smad3 and Smad4 are required for the interaction with hZimp10. GST fusion proteins containing either the full-length Smad3 or Smad4 or the truncated mutants containing the MH1, MH2, or the linker regions were produced and isolated (Fig. 3E). The full-length hZimp10 protein was translated in vitro and incubated with equal amounts of the various Smad3 or Smad4 GST fusion proteins. The full-length Smad3 or Smad4 and their MH2 domains showed an interaction with [35S]methionine-labeled full-length hZimp10 protein (Fig. 3D). In contrast, there is no retention in the samples with GST fusion proteins of Smad3 and Smad4 MH1 domains or Smad4 linker region. These data demonstrate that the MH2 domains of Smad3 and Smad4 are involved in the interaction with hZimp10.


Figure 3
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  		FIGURE 3.
The Miz domain of hZimp10 and the MH2 domains of Smad3 and Smad4 are involved in the Zimp10-Smad3/4 interaction. A, equal amounts of GST-hZimp10 N-terminal (1-333 amino acids), MIZ domain (728-809 amino acids), or C-terminal (932-1064 amino acids) fusion proteins were used to pull down in vitro translated [35S]methionine-labeled Smad3 or Smad4. GST-ARIP3 fusion protein was used as a positive control, and GST protein alone was used as a negative control. B, GST-Smad3 and Smad4 fulllength fusion protein as well as GST alone were isolated, purified, and subjected to binding assays. Equal amounts of the above GST proteins were analyzed on SDS-PAGE. C, hZimp10 fusion proteins (728-809 amino acids) containing either Miz domain wild type (WT) or mutants (Mut1 (C755G/H757A) and Mut2 (C760G/H762A)) were translated and labeled with [35S]methionine-labeled in vitro and bound to glutathione-Sepharose containing the indicated Smad3 or Smad4 fusion proteins. GST protein alone was used as a negative control. Materials bound to GST columns were subjected to SDS-PAGE and autoradiography. D, GST-Smad4 full-length (FL), MH1 domain (MH1, 1-146 amino acids), linker domain (L, 147-308 amino acids), and MH2 domain (MH2, 309-553 amino acids) and GST-Smad3 full-length (FL), MH1 (1-220 amino acids), and MH2 domain (221-423 amino acids) fusion proteins were isolated and bound to in vitro translated [35S]methionine-labeled hZimp10. E, equal amounts of different GST-Smad4 and GST-Smad3 proteins were isolated, analyzed on SDS-PAGE, and subjected to in vitro binding assays.

 
hZimp10 Interacts with Smad3 and Smad4 Proteins—To confirm that hZimp10 interacts with Smad3 or Smad4 in intact cells, co-immunoprecipitation assays were carried out to detect possible protein complexes. Initially, we co-transfected FLAG-tagged Smad3 and Smad4 together with HA-tagged hZimp10 in CV1 cells (Fig. 4A). Whole cell lysates containing FLAG-Smad3, FLAG-Smad4, and HA-hZimp10 proteins were immunoprecipitated with normal mouse IgG or an anti-FLAG monoclonal antibody. As shown in Fig. 4B, FLAG-Smad3 and FLAG-Smad4 proteins were detected in the anti-FLAG immunoprecipitates from cells co-transfected with HA-tagged hZimp10. Intriguingly, HA-tagged hZimp10 proteins were detected in the FLAG immunoprecipitates by an HA antibody or by a specific antibody against hZimp10 (36). These data indicate that Smad3 and Smad4 can form a protein complex with hZimp10 in intact cells.


Figure 4
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  		FIGURE 4.
Physical interaction between hZimp10 and the Smad3/Smad4 complex in intact cells. A, CV-1 cells were transfected with pcDNA3-FLAG-tagged Smad3 (0.5 µg) and pCMV-FLAG-Smad4 (0.5 µg) together with HA-hZimp10 (1 µg). Cell lysates were then immunoprecipitated with anti-FLAG antibody or normal mouse IgG. 5% input of the cell lysates was probed with anti-FLAG antibody or anti-HA antibody. IB, immunoblot. B, FLAG and IgG immunoprecipitates (IP) were probed by FLAG, HA, and hZimp10 antibodies. C and D, CV-1 cells were transfected with pcDNA3-FLAG-Smad3 (1 µg) or pCMV-FLAG-Smad4 (1 µg) together with pcDNA3-HA-hZimp10 (1 µg). Cell lysates were then immunoprecipitated with anti-FLAG antibody and normal IgG. 5% of the input lysate and the immunoprecipitation elutions were detected by anti-FLAG antibody or anti-HA antibody. E, whole cell lysates were prepared from HEK293 cells and used for Western blotting. Expression of hZimp10, Smad3, and Smad4 was detected by each of the specific antibodies as indicated in the F-H. Whole cell lysates were immunoprecipitated with anti-hZimp10 (36), anti-Smad3 (Santa Cruz Biotechnology, SC-6202), or anti-Smad4 antibody (Santa Cruz Biotechnology, SC-7966), respectively. The immunoprecipitation elutions from each antibody or normal rabbit IgG were analyzed by Western blotting with the hZimp10, Smad3, or Smad4 antibody.

 
Previous studies have shown that Smad4 can form a heterodimer with Smad3, which can then translocate into the nucleus to activate the transcriptional response (45, 49). Results from the in vitro GST pulldown experiments have shown that hZimp10 can interact with Smad3 or Smad4 individually. To further verify if Smad3 or Smad4 can interact with hZimp10 in intact cells, we repeated immunoprecipitation experiments with cell lysates co-transfected with HA-hZimp10 and FLAG-Smad3 or -Smad4 expression vector, respectively (Fig. 4C). As shown in Fig. 4D, HA-hZimp10 was detected in both the FLAG-Smad3 and FLAG-Smad4 immunoprecipitates. These data are consistent with the above immunoprecipitation assay and further demonstrate that Smad3 and Smad4 can interact with hZimp10 in an intact cell context.

Next we examined endogenous protein complexes of hZimp10 and Smad3 and -4 proteins. The expression of these three proteins was detected in HEK293 cells (Fig. 4E). Using the specific antibodies against hZimp10, Smad3, and Smad4, we immunoprecipitated endogenous hZimp10, Smad3, and Smad4 proteins, respectively, from whole cell lysates of HEK293 cells (Fig. 4, F, top panel and G and H, bottom panels). Intriguingly, both Smad3 and Smad4 were detected in the immunoprecipitates pulled down by the hZimp10 antibody (Fig. 4F, middle and bottom panels). Moreover, hZimp10 was also co-immunoprecipitated with either the Smad3 or Smad4 antibody (Fig. 4, G and H, top panels). The above data further support our conclusion that the interaction between hZimp10 with Smad3 and Smad4 is a biologically relevant event.

Smad3 and Smad4 Co-localize with hZimp10 in the Nucleus—Next we examined whether a dynamic interaction between Smad3, Smad4, and hZimp10 exists in cells. Expression vectors containing FLAG-tagged Smad4, HA-tagged Smad3, and the full-length hZimp10 were co-transfected into CV-1 and PC3 cells. Transfected cells were incubated with normal medium containing 5% fetal bovine serum. As shown in Fig. 5, all three proteins display a nuclear distribution in both CV-1 and PC3 cells, which is consistent with previous reports (7, 36). Intriguingly, a significant amount of overlay between Smad3, Smad4, and hZimp10 was observed in these cells (Fig. 5). Based on these observations, we conclude that hZimp10 can co-localize with Smad3 and Smad4 in the nucleus, in which hZimp10 may form a ternary transcriptional complex with Smad3 and Smad4.

Loss of Smad3/Smad4-mediated Transcription in Zimp10 Null Cells—To investigate the biological role of Zimp10 in vivo, we have recently generated mice in which the Zimp10 gene locus has been disrupted by replacing the second and third exons with a neomycin resistance cassette. The consequence of this disruption is embryonic lethality at approximately E10.5. 3 To determine whether endogenous Zimp10 regulates Smad3/4-mediated transcription, we generated MEFs from E9.5 day embryos and transfected them with the Smad3/4-responsive 3TP-luciferase reporter with increasing concentrations of FLAG-Smad3 and FLAG-Smad4. As shown in Fig. 6A, an induction of Smad3/4-mediated transcription on the 3TP promoter/reporter was observed in MEFs prepared from Zimp10 heterozygous embryos (+/-). In contrast, no activity was observed in MEFs where both Zimp10 alleles were disrupted (-/-). To further characterize the physiological role of Zimp10 in Smad3/4-mediated transcription, we assessed the expression of PAI-1, a downstream target gene of Smad3/4 in the MEFs derived from Zimp10(+/-) or -(-/-) mice by RT-PCR assay (44, 50). Although low basal levels of PAI-1 expression appear in both +/- and -/- MEFs, a significant induction of PAI-1 expression in response to TGF-beta treatment is only observed in the Zimp10-positive cells (zimp10(+/-)) (Fig. 6B). These data provide a solid line of evidence that demonstrates a physiological role for endogenous Zimp10 in the regulation of Smad3/4-mediated transcription. In addition, we also examined the expression of PAI-1 in Zimp10 mouse embryos. The results from RT-PCR approaches showed that the expression of PAI-1 was much lower in Zimp10(-/-) embryos than in Zimp 10(+/+) and -(+/-) embryos (data not shown).


Figure 5
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  		FIGURE 5.
Smad3 and Smad4 co-localize with hZimp10 in the nucleus. CV-1 (top) and PC3 (bottom) cells were transfected with pcDNA3-hZimp10 (100 ng), pcDNA3-FLAG-Smad3 (100 ng), and pcDNA3-HA-Smad4 (100 ng) together. Six hours after transfection cells were cultured with 10 ng/ml TGF-beta1. Twenty-four hours after transfection, cells were fixed, incubated with a chicken anti-hZimp10 (1:375 dilution), an anti-FLAG (1:750 dilution) monoclonal antibody and an anti-HA (1:500) polyclonal antibody, and then visualized with Fluorophore-conjugated secondary antibodies goat-anti-chicken-488, goat-anti-mouse-350, goat-anti-rabbit 594 at a dilution of 1:500, respectively. Merge (right panel) of different stains indicates areas of co-localization. Ab, antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have shown that hZimp10, a novel PIAS-like protein, acts as a transcriptional co-activator to augment Smad3-mediated transcription. These findings are consistent with previous reports of an interaction between PIAS proteins and Smad3 (32, 33) and provide an additional line of evidence demonstrating the cross-talk between PIAS proteins and the TGF-beta/Smad pathway. We show that expression of exogenous hZimp10 or knockdown of endogenous hZimp10 affects Smad3-mediated transcription. Using a Smad4-negative cell line, we further demonstrate that the enhancement of Smad3 by hZimp10 depends upon the presence of Smad4, suggesting that hZimp10 may mediate Smad3 activity by interacting with the Smad3/Smad4 transcriptionally active complex. Sequence analysis showed that unlike other PIAS proteins, hZimp10 contains a strong intrinsic transactivation domain in the C-terminal proline-rich region (36). It appears that through this domain hZimp10 can act as a transcriptional co-activator to augment androgen receptor-mediated transcription. The finding that hZimp10 enhances Smad3-mediated transcription is consistent with these previous observations, suggesting that hZimp10 may play an important role in transcriptional regulation.

In this study we determined that hZimp10 interacts with Smad3 and Smad4. Using both in vitro GST-pulldown and co-immunoprecipitation experiments, we have shown that Smad3 and Smad4 can interact with hZimp10 individually. The Miz domain of hZimp10 and MH2 domains of Smad3 and Smad4 were shown to be required for the interaction. The Miz domain of hZimp10 shares high sequence similarity with other PIAS proteins, and this domain has been suggested to mediate the interactions of PIASy and PIAS3 with Smad3 (32, 33). Our finding that the Miz zinc finger domain of hZimp10 binds to Smad3 and Smad4 further supports the biological importance of this region in regulating various pathways through protein-protein interactions. Because PIASy and PIAS3 have been shown to negatively affect Smad3-mediated transcription through binding to the protein, it will be very interesting to examine the mechanisms by which the different Miz-containing PIAS proteins cooperatively regulate Smad3 activity in response to different cell signals in a biologically relevant context.

The MH2 domain of Smad3 and Smad4 were shown to be required for hZimp10 binding. These data are consistent with previous reports on Smad protein structure-function, which showed that the MH2 domain is involved in many biological processes through interaction with regulatory proteins (2, 14). The MH2 domain mediates both homomeric and receptor-induced heteromeric interactions between Smad4 and receptor-regulated Smads (45, 51). In this study we have shown that the MH2 domains of Smad3 and Smad4 interact with hZimp10, which is not surprising since Smad3 and Smad4 MH2 domains share a high degree of sequence similarity. The biological activity of the MH2 domain may be modulated by interaction with the MH1 domain when the protein is not phosphorylated. Upon receptor-mediated phosphorylation, this interaction may be altered, and each domain may form the DNA and protein interactions required for the proper activity of the transcriptional complex (52). It will be interesting to investigate whether posttranslational modification of the Smad3/Smad 4 complex affects its association with hZimp10 in the nucleus.


Figure 6
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  		FIGURE 6.
Loss of Zimp10 significantly reduces Smad3/4-mediated transcription. A, mouse embryo fibroblasts were isolated from either +/- or -/- animals as described under "Materials and Methods." Cells were transfected with 3TP-luc (100 ng), CMV-beta-galactosidase (25 ng), and increasing concentrations of FLAG-Smad3 and FLAG-Smad4. 48 h after transfection luciferase and beta-galactosidase values were measured, and relative luciferase/beta-galactosidase units were calculated. Bars represent the mean and S.D. of triplicate determinations. Similar results were obtained from two independent MEF isolations. B, transcript levels of the Smad target gene PAI-1 was assessed by RT-PCR using RNA samples isolated from Zimp10 knock-out (-/-) or heterozygous (+/-) mouse embryos. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is included as a control. C, densitometry of the gels was performed, and the PAI-1 relative fold induction rates were reported (induced/uninduced).

 
We have shown that hZimp10 co-localizes with newly synthesized DNA at replication foci throughout S phase (36). These data suggest that hZimp10 may play an important role in both chromatin assembly and maintenance of chromatin. Several lines of evidence also demonstrate that hZimp10 may act as a transcriptional co-regulator to initiate and mediate formation of an active transcriptional complex. A homologue of hZimp10, termed tonalli (tna) was identified recently in Drosophila (38). Intriguingly, the protein encoded by tna was shown to interact with SWI2/SNF2 and the Mediator complex, suggesting a potential role for hZimp10 in chromatin modification. Our current finding that hZimp10 interacts with Smad3 and Smad4 and enhances Smad3-mediated transcription corroborates our previous observation that hZimp10 plays an important role in transcriptional regulation. Early reports have shown that PIAS3 recruits transcriptional co-activators, such as p300/CBP, to Smad3-containing transcriptional complexes and augments Smad3-mediated transcription (33). In this study we have not investigated the involvement of p300/CBP in hZimp10-mediated enhancement of Smad3 transcriptional activity. However, given the observations that hZimp10 contains an intrinsic transcriptional activation domain and its orthologue interacts with SWI2/SNF2 and the Mediator complex, it appears that hZimp10 may regulate Smad3-mediated transcription through a different mechanism than PIAS3. Further investigation into the role of hZimp10 in human SWI/SNF BAF complexes is required to understand the precise mechanism by which hZimp10 modulates the activity of Smad3 and other transcription factors.

PIAS proteins have been found to interact with SUMO-1 and Ubc9 and to mediate sumoylation of nuclear hormone receptors and other transcription factors (27, 31). Furthermore, it has been shown that PIAS1 and PIASxbeta act as E3 ligases to mediate the sumoylation of Smad4 (35). Our previous studies have shown that hZimp10 also co-localizes with SUMO-1 at replication foci and is involved in the sumoylation of the androgen receptor (36). Although it is currently unclear whether the modulation of Smad3 activity by PIASy and PIAS3 is through sumoylation, we did not observe a significant effect of hZimp10 on modulating the sumoylation of Smad3 and Smad4 in our experiments. Therefore, the sumoylation of Smad4 by PIAS proteins must be further explored to fully understand the biological consequences and molecular mechanisms of this modification in the TGF-beta/Smad network.

Our recent data shows that disruption of the Zimp10 gene in mice results in embryonic lethality at approximately E10.5. This result implies a critical role for Zimp10 in normal development. Using MEFs generated from these mice, we demonstrated that the disruption of Zimp10 inhibits Smad3-mediated transcription. In MEFs with an intact wild type Zimp10 allele, a clear dose-dependent induction of Smad3 transcriptional activity was observed in cells transfected with increasing amounts of Smad3 and Smad4. In contrast, no enhancement was observed in cells where both Zimp10 alleles were disrupted. Moreover, we also observed that the expression of PAI-1 in MEFs isolated from Zimp10(-/-) knockouts shows no response to TGF-beta induction. In contrast, a significant induction of PAI-1 expression to TGF-beta was observed in Zimp10(+/-) MEFs. These data provide an intriguing line of evidence that Zimp10 plays an important role in Smad-mediated transcription in vivo.

In conclusion, this study demonstrates for the first time that hZimp10, a novel PIAS-like protein, augments the transcriptional activity of the Smad3/Smad4 protein complex. The interaction between hZimp10, Smad3, and Smad4 provide an additional line of evidence demonstrating cross-talk between the TGF-beta pathway and PIAS proteins. The data also indicate that hZimp10 functions as a transcriptional co-regulator to modify the transcriptional activity of Smad3. Further studies of the molecular mechanisms by which hZimp10 and other PIAS proteins regulate Smad3-mediated transcription may provide new insight into the biological role of PIAS and PIAS-like proteins in transcriptional regulation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA070297, CA087767, DK061002, and 5T32 CA09302-27 and Dept. of Army Prostate Cancer Grant DAMD17-03-1-0090. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom requests for reprints should be addressed: Depts. of Urology and Genetics, Stanford University, 300 Pasteur Dr. Grant Bldg. S287, Stanford, CA 94305-5118. E-mail: zsun{at}stanford.edu.

2 The abbreviations used are: TGF, transforming growth factor; STAT, signal transducers and activators of transcription; CBP, cAMP-response element-binding protein (CREB)-binding protein; GST, glutathione S-transferase; HEK cells, human embryonic kidney cells; HA, hemagglutinin; MEF, mouse embryo fibroblast; RT, reverse transcription; shRNA, short hairpin RNA; PAI, plasminogen activator inhibitor. Back

3 J. Beliakoff, J. Lee, and Z. Sun, unpublished observations. Back



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J. Beliakoff, J. Lee, H. Ueno, A. Aiyer, I. L. Weissman, G. S. Barsh, R. D. Cardiff, and Z. Sun
The PIAS-Like Protein Zimp10 Is Essential for Embryonic Viability and Proper Vascular Development
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