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RESEARCH COMMUNICATION
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
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Abstract |
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(TGF-) and bone morphogenetic protein (BMP) pathways. However, the phosphatase(s) involved in dephosphorylating and hence inactivating Smads remained elusive. Through RNA interference (RNAi)-based screening of serine/threonine phosphatases in Drosophila S2 cells, we identified pyruvate dehydrogenase phosphatase (PDP) to be required for dephosphorylation of Mothers against Decapentaplegic (MAD), a Drosophila Smad. Biochemical and genetic evidence suggest that PDP directly dephosphorylates MAD and inhibits signal transduction of Decapentaplegic (DPP). We show that the mammalian PDPs are important in dephosphorylation of BMP-activated Smad1 but not TGF--activated Smad2 or Smad3. Thus, PDPs specifically inactivate Smads in the BMP/DPP pathway.
[Keywords: Bone morphogenetic protein; Decapentaplegic; Mothers against Decapentaplegic; Smad; pyruvate dehydrogenase phosphatase]
Received October 14, 2005; revised version accepted January 13, 2006.
(TGF-) cytokines share an evolutionarily conserved mechanism of signal transduction (Attisano and Wrana 2002
; Shi and Massagué 2003; ten Dijke and Hill 2004). Genetic analyses of the DPP (Decapentaplegic) pathway in Drosophila identified MAD (Mothers against Decapentaplegic) as a key signal transducer (Raftery et al. 1995; Sekelsky et al. 1995). The orthologs of MAD in mammals include Smad2 and Smad3 in the TGF- pathway, and Smad1, Smad5, and Smad8 in the bone morphogenetic protein (BMP) pathway (Derynck et al. 1998; Whitman 1998). These Smads (referred to as R-Smads) contain an SSXS motif at their C termini, and the last two serines are phosphorylated in response to TGF- or BMP (Macias-Silva et al. 1996; Kretzschmar et al. 1997; Souchelnytskyi et al. 1997). This phosphorylation is a prerequisite for the Smads to accumulate in the nucleus and to interact with transcription cofactors (Chen et al. 1996; Lagna et al. 1996; Abdollah et al. 1997).
The C-terminal phosphorylation state of R-Smads is controlled by protein kinases and phosphatases. When the TGF- receptor kinase activity was blocked, the amount of phospho-Smad2/3 rapidly decreased with concomitant return of Smad2/3 to the cytoplasm (Inman et al. 2002; Xu et al. 2002). While degradation may account for part of the loss of phospho-Smad2/3, dephosphorylation plays a major role in inactivating R-Smads (Lo and Massagué 1999; Inman et al. 2002). However, the identity of Ser/Thr phosphatase(s) involved in dephosphorylation of R-Smads was unknown.
In this study, we uncovered pyruvate dehydrogenase phosphatase (PDP) to be a phosphatase for MAD. We found that genetic lesions in the pdp gene resulted in ectopic accumulation of C-terminally phosphorylated MAD in Drosophila embryos. Furthermore, we demonstrate that PDP directly dephosphorylates MAD and inhibits DPP-induced gene expression. Moreover, knockdown of mammalian orthologs of PDP (PDP1 and PDP2) by small interfering RNA (siRNA) led to reduced dephosphorylation of Smad1, but not Smad2 or Smad3, suggesting that PDPs specifically regulate BMP signaling in mammalian cells.
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Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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In Drosophila S2 cells, the level of DPP-induced phosphorylation of MAD remained elevated in the continuous presence of DPP (Fig. 1A). However, upon removal of DPP, the amount of phospho-MAD decreased rapidly while the total protein level of MAD was unchanged (Fig. 1B). Thus, we reason that phospho-MAD is dephosphorylated once the DPP signal subsides.
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; Lum et al. 2003). The dsRNA against CG12151 (CG12151-A, targeting the Drosophila PDP) consistently impeded dephosphorylation of MAD (Fig. 1C,D). A similar observation was made with a second dsRNA for PDP (CG12151-B) targeting a different region (Fig. 1C,D). We noticed that eventually (i.e., 2 h after removal of DPP), the level of phospho-MAD decreased substantially even when PDP was knocked down (Fig. 1C,D). This could reflect activities from residual PDP or other phosphatases that can compensate partially. Another possibility is that if phospho-MAD constitutes a very small fraction of the total MAD, then degradation of phospho-MAD specifically may also result in reduction of phospho-MAD without grossly changing the level of total MAD. In parallel to the decrease in phospho-MAD, the expression of Daughters against Decapentaplegic (DAD), a DPP-dependent target gene, dropped substantially from the peak DPP-induced level 2 h after DPP removal (Fig. 1E). dsRNA against PDP significantly reduced such decrease in DAD expression, consistent with its ability to delay dephosphorylation of MAD (Fig. 1E).
Loss of PDP results in ectopic phosphorylation of MAD in Drosophila embryos
To further determine the importance of PDP in regulating the C-terminal phosphorylation state of MAD, we examined Drosophila strains carrying genetic lesions that affect the PDP locus on the X chromosome (division 7B7). The strain PBacRBCG12151e02351 (PBacCG12151) harbors a piggyback transposon 7 base pairs upstream of the first exon of the PDP gene (Thibault et al. 2004). Another strain, Df(1)ct4b1 (uncovers 7B2–7C4), carries a chromosome deletion that includes the PDP gene. We stained whole embryos with the PS1 antisera that specifically recognize phospho-MAD in Drosophila embryos (Persson et al. 1998; Tanimoto et al. 2000; Sutherland et al. 2003).
Similar to previous reports, in wild-type blastoderm-stage embryos, phospho-MAD is distributed only in the dorsal-most cells and predominantly nuclear (Fig. 2A,B; Sutherland et al. 2003). In the PBacCG12151 and Df(1)ct4b1 embryos, in addition to the dorsal nuclei, we observed punctate staining for phospho-MAD throughout the embryo (Fig. 2A, C–E). The ectopic PS1 staining was mostly outside the nucleus (Fig. 2C–E), and was detected in embryos at most, if not all, developmental stages (Fig. 2A). The ectopic accumulation of phospho-MAD was not observed in the Df(1)RF19 (uncovers 7A4–7B3) strain, with a chromosome deletion adjacent to but not encompassing the PDP locus (data not shown). In control experiments, no abnormal staining patterns for Dorsal (data not shown) and Toll were observed in either PBacCG12151 or Df(1)ct4b1 embryos (Fig. 2A). In cleavage-stage embryos, which undergo rapid mitosis, we detected aggregated signals for phospho-MAD in PBacCG12151 and Df(1)ct4b1, but no PS1 signal in wild-type embryos (Fig. 2A,E). Double staining with DAPI showed that in these mutant embryos, phospho-MAD accumulated on both sides of the condensed chromosomes (Fig. 2E).
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PDP directly dephosphorylates DPP-activated MAD
Thickveins (TKV) and Punt are the receptor kinases upstream of MAD that are themselves activated by phosphorylation (Raftery and Sutherland 1999). Conceivably, phosphatases toward either TKV or Punt could also impact the level of phospho-MAD. We therefore determined if PDP directly dephosphorylates MAD as its substrate.
Recombinant GST-PDP effectively dephosphorylated phospho-MAD (Fig. 3A), at as low as 0.3 µM (Fig. 3B). Removal of the GST moiety by thrombin did not affect the efficiency of dephosphorylation (Fig. 3A). Asp93 in PDP is highly conserved and critical for metal ion chelating (Das et al. 1996; Kusuda et al. 1998). When Asp93 was mutated to Ala, the phosphatase activity toward MAD was largely abolished (Fig. 3A,B). As controls for specificity in the in vitro phosphatase assay, PDP was inactive against phospho-Jun kinase 1 (JNK1 phosphorylated on Thr185) under the same conditions (Fig. 3C, right); and phospho-MAD was not dephosphorylated by protein phosphatase 1 (PP1), which exhibited strong activity toward phospho-JNK1 (Fig. 3C).
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). PDPs belong to the PPM family whose catalytic domains are similar to that of PP2C, and pyruvate dehydrogenase was the only known substrate of PDPs (Lawson et al. 1993; Simonot et al. 1997; Huang et al. 1998). We have thus identified a novel substrate and function for PDP.
PDP inhibits DPP signaling
The C-terminal phosphorylation is critical for MAD to regulate gene transcription. We therefore tested if PDP could inhibit MAD-mediated transcriptional activation. In the Drosophila S2R+ cells, 2xUbx-lacZ, a reporter controlled by a DPP response element from the Ultrabithorax promoter, was activated upon expression of MAD, Medea (MED), Punt, and TKV, as previously reported (Fig. 3D; Kirkpatrick et al. 2001). The expression of this reporter was significantly repressed when PDP was overexpressed in a dose-dependent manner (Fig. 3D). Moreover, dsRNA against PDP, but not dsRNAs against PP1-like (CG8822) or PP4-like (CG11597) Ser/Thr phosphatases, enhanced the expression of 2xUbx-lacZ upon activation of MAD (Fig. 3E). Either overexpression or knockdown of PDP had little effect on the basal 2xUbx-lacZ expression (Fig. 3D [right], E). Thus, the level of PDP is an important determinant of the strength of DPP signaling.
Interaction between PDP and MAD
After DPP stimulation, endogenous phospho-MAD was readily coimmunoprecipitated with V5-tagged PDP (Fig. 4A). In S2 cells transfected with Flag-MAD and PDP-V5, anti-Flag immunoprecipitation brought down PDP-V5, demonstrating that the coimmunoprecipitation of MAD and PDP works in reciprocal order (Fig. 4B). Anti-V5 immunoblotting revealed two V5-containing proteins in cells transfected with PDP-V5, suggesting the presence of different forms of PDP (input in Fig. 4A,B).
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3% of phospho-MAD was bound by GST-PDP; while <1% of unphosphorylated MAD was bound (Fig. 4D). Thus, while not being required for interaction, the C-terminal phosphorylation substantially enhanced MAD interaction with PDP.
Subcellular localization of PDP
In live S2 cells, PDP-GFP not only distributed throughout the cell, but also exhibited a punctate pattern that overlapped with mitochondria as revealed by MitoTracker (Fig. 4E). Such distribution of PDP was not changed upon activation of the DPP pathway (Fig. 4F). Overexpression of PDP-GFP did not apparently prevent nuclear accumulation of MAD, possibly because the receptor activation overrode PDP activity under the condition or because changes in the kinetics of MAD nuclear import/export require more sensitive and quantitative methods to measure (Fig. 4F). Our observations suggest that PDP has broad subcellular localization and can therefore gain access to substrates in mitochondria, cytoplasm, and nucleus. Indeed, in both the cytosolic and nuclear fractions, coimmunoprecipitation of phospho-MAD and PDP could be detected (Fig. 4G). Less phospho-MAD was coimmunoprecipitated with PDP in the nuclear fraction, which could be due to the fact that less PDP was present in the nucleus and that the buffer used to extract nuclei contained a higher salt and detergent concentration, which is more stringent for protein–protein interaction (Fig. 4G).
Mammalian PDPs are involved in dephosphorylation of BMP-activated Smad1
We next investigated if mammalian PDPs had similar functions to their Drosophila counterpart. Two orthologs of Drosophila PDP, PDP1 and PDP2, have been identified in the human genome, and both share 40% identity with the Drosophila PDP in amino acid sequences (Lawson et al. 1993; Simonot et al. 1997; Huang et al. 1998). Coimmunoprecipitation between PDP2 and Smad1 was detected in 293T cells, suggesting that the functional interaction between PDP and Smad is conserved in mammalian cells (Fig. 5A).
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receptor kinases can be inhibited by the compound SB431542 (Inman et al. 2002), while SB202190 was shown to block BMP receptor kinase activity toward Smad1 (Nishihara et al. 2003). In HeLa cells, TGF-- and BMP2-induced phosphorylation of Smad2/3 and Smad1 decreased considerably after SB431542 or SB202190 treatment, respectively, reflecting dephosphorylation of these Smads (Fig. 5C–E). Therefore, we used these two kinase inhibitors to monitor phosphatase activities toward Smad1, Smad2, and Smad3. Compared with the control, siRNA against PDP1 or PDP2 resulted in reduced Smad1 dephosphorylation (Fig. 5C). However, the same siRNA treatment did not significantly change the dephosphorylation of Smad2 and Smad3 (data not shown; but see Fig. 5E). Even when we combined siRNAs against PDP1 and PDP2, dephosphorylation of Smad2 and Smad3 was largely unaffected (Fig. 5D,E). Moreover, the expression of one BMP target gene, Smad6, was enhanced in cells transfected with siRNA against PDP1 and PDP2, both at the basal state and after BMP2 stimulation (Fig. 5F). The increase in Smad6 expression without added BMP2 could reflect enhancement of low-level autocrine BMP signaling.
These observations suggest that PDP1 and PDP2 are important for dephosphorylation of Smad1. Therefore, the mechanism of Smad dephosphorylation in vertebrates is similar to that in Drosophila. BMP is more closely related to DPP, and Smad1 is more similar to MAD than Smad2/3 are. Interestingly PDP1 and PDP2 did not appear to be rate-limiting in dephosphorylation of Smad2 and Smad3. This raised the possibility that different phosphatases are used to inactivate different R-Smads.
The activation state of various signal transduction pathways is often dictated by phosphorylation–dephosphorylation control of key signaling molecules. We report here that PDPs are phosphatases for R-Smads in the DPP pathway in Drosophila and the BMP pathways in mammals. PDP can act to reduce the concentration of phospho-R-Smads in the nucleus and consequently weaken the transcriptional responses to BMP. Moreover, our findings also raised the possibility that MAD may be phosphorylated by kinases outside of the DPP pathway, and the role of PDP is to remove such aberrant phosphorylation and prevent ectopic DPP signaling.
TGF-/BMP cytokines control the biology of many cell types in different physiological contexts (Massagu|$$|Aae et al. 2000). In addition to the receptors and Smads, other factors must participate to modify and fine-tune the signaling. The identification of phosphatases that inactivate R-Smads provides us with a new angle to study how TGF-/BMP signaling is modulated.
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Materials and methods |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Drosophila S2 and S2R+ cells were cultured at 26°C in Schneider’s medium (Invitrogen) with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. Cells were transfected using Effectene (Qiagen). For mitochondria staining, cells were cultured with 100 nM MitroTracker Deep Red 633 (Invitrogen) for 1 h, washed twice, and then observed directly using confocal microscopy (Leica). Drosophila embryo fixation and immunostaining were carried out as previously described (Hemavathy et al. 2004). The PS1 antisera for phospho-MAD (1:1000) were a gift from Dr. P. ten Dijke (The Netherlands Cancer Institute, Amsterdam, The Netherlands).
In reporter assays, transfected cells were induced with 0.5 mM CuSO4 to express MAD, PDP, Punt, and TKV as indicated. A TK promoter-driven luciferase reporter was used as a normalization standard. Cell lysates were assayed for both luciferase and -galactosidase activities (Promega).
Cell-based assay for phosphatase activity against MAD and Smad
A stable S2 cell line was generated expressing Flag-MAD under the control of the metallothionine promoter. The cells were induced with 0.5 mM CuSO4 overnight and then treated with 10–9 M DPP (R&D Systems) for 2 h. After three washes with PBS, the cells were cultured in the absence of DPP. At indicated time points, cells were harvested and analyzed by immunoblotting using anti-phospho-Smad1 (Cell Signaling).
HeLa cells were starved in 1% FBS/DMEM media for 3 h before being treated with 100 ng/mL BMP2 (R&D Systems) or 100 pM TGF- (R&D Systems) for 1 h. The cells were then treated with 10 µM SB431542 (Tocris) or 20 µM SB202190 (Calbiochem) for 30 min before being harvested and analyzed by immunoblotting.
RNAi and quantitative real-time PCR
For RNAi in Drosophila cells, PCR amplicons of targeted cDNA were transcribed in vitro using the MEGAscript kit (Ambion) to generate dsRNAs, which were introduced into S2 cells as described previously (Clemens et al. 2000). siRNA duplexes against PDP1 and PDP2 (SiPDP1-a, CAAGTTGGTGATCCTAATT; siPDP1-b, CAGTCACGCTGTCTA ATGA; siPDP2-a, GAAGCATTAATGTACTCCT; and siPDP2-b, CA ATCGGTGCATATTACAA) were provided by Dr. Biliang Zhang (Ribobio Co. Ltd, Guangzhou, China). siRNAs (25–40 nM final) were transfected using HiperFect (Qiagen). After 72 h, the transfection was repeated, and another 48 h later, cells were harvested for analyses.
Real-time PCR was carried out using the iQ SYBR Green Supermix (Bio-Rad). Total RNA was extracted using the RNeasy kit (Qiagen), and cDNA was synthesized with the iScript cDNA Synthesis Kit (Bio-Rad). The level of test cDNA relative to that of 18s rRNA or ribosomal protein rp49 was calculated by the 2–
CT method (Livak and Schmittgen 2001).
In vitro phosphatase assay
S2 cells were transfected with Flag-MAD and treated with 10–9 M DPP for 2 h to generate phospho-MAD. 293T cells transfected with Flag-JNK1 were exposed to UV light (60 J/m2) to induce phosphorylation of JNK1. Cells were lysed in 20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.5% NP-40, and Flag-MAD or Flag-JNK1 was immunoprecipitated with anti-Flag (Sigma). After washing in the lysis buffer, the beads were suspended in 50 mM Tris-Cl (pH 7.4) and 5 mM MnCl2, and mixed with GST-PDP or PP1 (Calbiochem). The reaction was carried out at 30°C for 30 min.
Protein–protein interaction
For GST pull-down, cell lysate was prepared by sonication in 20 mM Tris (pH 7.4), 175 mM NaCl, 0.5% Triton X-100, and 2 mM DTT with protease inhibitors. Such a whole-cell extract was incubated with GST-PDP on ice for 1 h before adding glutathione-conjugated agarose beads. After 1 h at 4°C, the beads were washed three times in the lysis buffer and analyzed by immunoblotting. For coimmunoprecipitation, cells were lysed in 20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.5% NP-40, and 20 mM DTT and immunoprecipitated with anti-Flag (Sigma) or anti-V5 agarose beads (Invitrogen) for 5 h at 4°C. The beads were washed three times in the lysis buffer.
Subcellular fractionation was done using the NE-PER nuclear and cytoplasmic extraction reagents following the manufacturer’s protocol (Pierce).
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Acknowledgments |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Footnotes |
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E-MAIL lan.xu{at}umassmed.edu; FAX (508) 856-6662. 
Article published online ahead of print. Article and publication date are at http://www.genesdev.org/cgi/doi/10.1101/gad.1384706
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-MAD-P). (B) The same S2 cells as in A were first stimulated by DPP for 2 h and washed by PBS to remove DPP, and continued to be cultured in regular media. At indicated time points, phospho-MAD and total Flag-MAD were detected by the indicated antibodies. (C) The same S2 cell line was treated with the indicated dsRNAs for 72 h before being analyzed as in B. (D) Quantitation of phospho-MAD signal in C. The signal of phospho-MAD was quantitated using NIH Image, normalized to that of total Flag-MAD, and plotted. The phospho-MAD level at the 0 time point was arbitrarily set as 100%. (E) The DAD mRNA level was measured by quantitative real-time PCR at the peak DPP-induced level (10–9 M for 2 h) (white bars) and 2 h after DPP removal (black bars). The peak level was arbitrarily set as 100%. The mRNA level of the ribosomal protein rp49 was used as the quantitation standard. The presented values are based on four independent experiments.
