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J. Biol. Chem., Vol. 282, Issue 13, 9688-9695, March 30, 2007

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Endofin, a FYVE Domain Protein, Interacts with Smad4 and Facilitates Transforming Growth Factor- Signaling^*

Ye-Guang Chen¹, Zhi Wang, Jing Ma, Long Zhang, and Zhongxian Lu

From the State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China and the Department of Medicine and Biological Chemistry, College of Medicine, University of California, Irvine, California 92697

Received for publication, December 21, 2006 , and in revised form, February 1, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Transforming growth factor- (TGF-) signaling is facilitated by scaffold proteins such as SARA (Smad anchor for receptor activation). Endofin, a member of the FYVE domain protein family, has been suggested to regulate membrane trafficking. In this study, we report that endofin functions as a scaffold protein to facilitate TGF- signaling. Overexpression of endofin FYVE domain-deletion mutants inhibited TGF--induced expression of CAGA-luciferase. Knockdown of endogenous endofin expression by RNA interference specifically led to reduction of the transcriptional responses of TGF-, but had no effect on BMP- or Wnt1-induced reporter expression. Furthermore, in endofin small interfering RNA-expressing stable cells, TGF--mediated expression of plasminogen activator inhibitor-1 and p21^Cip1 was significantly reduced, and TGF--promoted apoptosis was also impaired. We further showed that endofin could interact with Smad4 and TGF- type I receptors. Reduction of endogenous endofin expression resulted in a decrease of TGF--induced Smad2 phosphorylation and Smad2-Smad4 complex formation. Together, our findings suggest that endofin facilitates TGF- signaling as a scaffold protein to promote the R-Smad-Smad4 complex formation by bringing Smad4 to the proximity of the receptor complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Members of the transforming growth factor (TGF-)² superfamily, such as TGF-, activin, bone morphogenetic protein (BMP), and Nodal, regulate a variety of cellular events including cell growth, differentiation, apoptosis, and migration. They signal through specific cell-surface transmembrane serine/threonine kinase receptors, known as type I and type II receptors. Ligand binding promotes the formation of the receptor complex where the type II receptor phosphorylates the type I receptor. The activated type I receptor in turn phosphorylates the downstream effectors, the receptor-regulated Smads (R-Smads), which then form a complex with Smad4. In the nucleus, the Smad complex regulates the expression of TGF--targeted genes (1–6).

Several scaffolding proteins have been suggested to mediate TGF- signal transduction through their interaction with the receptor complexes and/or the Smads (7). One example is SARA (Smad anchor for receptor activation), a FYVE domain-containing protein that interacts directly with non-activated Smad2/3 and the TGF- receptors complex, thus forming a bridge between the receptors and R-Smad and assisting the specific phosphorylation of Smad2/3 by the type I receptor (8–10). SARA has also been suggested to target the catalytic subunit of protein phosphatase 1 to receptor complexes and negatively modulate type I receptor activity (11). Interestingly, a recent study showed that SARA is required for targeting to the spindle machinery of a subtype of endosomes containing Dpp and its receptor Tkv to ensure the equal segregation of Dpp signaling to two daughter cells (12). Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) is another FYVE-domain-containing protein and can facilitate Smad2 activation in cooperation with SARA (13), whereas both SARA and Hrs are reported to attenuate TGF- signaling in T cells (14). The FYVE domain is a motif known to bind phosphatidylinositol 3-phosphate and can specifically recruit the FYVE-domain-containing proteins to early endosomes where they are enriched with phosphatidylinositol 3-phosphate (15). In addition, several other adaptor proteins are also implicated in facilitating TGF- signaling by linking the receptor complex with R-Smads, Disabled2 (16), Axin (17), TRAP1 and the related protein TLP (18, 19), and Dok-1 (20). These studies collectively suggest that the scaffolding protein-facilitated interaction between TGF- receptors and Smad proteins is an important regulatory mechanism in TGF- signaling and it might provide an explanation for the complicated cell context-dependent specificity of TGF- responses.

Endofin (endosome-associated FYVE-domain protein) is closely related to SARA and shares about 50% identity in the carboxyl-terminal 800-amino acid region related to SARA. It was shown that endofin is targeted to early endosomes by its FYVE domain and colocalized with SARA (21). Overexpression of endofin induced aggregation of endosomes, suggesting that endofin is involved in endosome trafficking regulation (21–23). In this study, we provide evidence that either mislocalization of endofin or knockdown of endofin expression resulted in reduction of transcription responses of TGF-. Endofin interacts with both the TGF- type I receptor TRI/ALK5 and Smad4 and facilitates TGF- signaling. Reduction of endogenous endofin expression attenuated TGF--induced apoptosis and impaired TGF--promoted association of Smad2 and Smad4. Therefore, these results suggest that endofin may act as a scaffold protein to promote the R-Smad-Smad4 heterocomplex formation by bringing Smad4 to the vicinity of the receptor complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture and Establishment of Stable Cell Lines—HepG2 and Hep3B cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum (Hyclone) and antibiotics in 5% CO₂ at 37 °C in a humidified atmosphere. HEK293T and HeLa cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. At 48 h after transfection with anti-endofin siRNA expressing plasmids, stable transfectants were selected with 0.3 µg/ml of puromycin (Sigma) for 14 days. Individual clones were picked and amplified with 0.2 µg/ml of puromycin and cells were collected for analysis of endogenous endofin mRNA levels.

Luciferase Reporter Assay—Cells were co-transfected with luciferase reporters and the indicated plasmids, as well as Renilla luciferase (20 ng) as an internal control. One day after transfection, cells were seeded into 24-well plates and treated with TGF-1 (100 pM unless otherwise indicated) in minimal essential medium containing 0.2% fetal bovine serum for 20 h. After washing with phosphate-buffered saline, cells were harvested, and the luciferase activity of cell lysates was determined using a luciferase assay system (Promega) as described by the manufacturer. Total light emission during the initial 20 s of the reaction was measured in a luminometer (Berthold Lumat LB 9501).

Immunoprecipitation and Immunoblotting—HEK293T cells and Hep3B cells were transfected with the indicated constructs or empty vector by using the calcium phosphate method or Lipofectamine (Roche). At 48 h post-transfection, the cells were lysed. Immunoprecipitation and immunoblotting were performed as described previously (24).

Immunofluorescence—Cells were grown on glass coverslips, fixed with 4% paraformaldehyde for 15 min and blocked with 10% bovine serum albumin in phosphate-buffered saline (pH 7.0) for 60 min. The cells were then incubated with primary antibodies for 3 h, followed by fluorescein isothiocyanate (FITC) or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for an additional 40 min. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (Sigma). Images were obtained with the confocal Olympus fluoview 500 microscope.

Reverse Transcription (RT)-Polymerase Chain Reaction—At 48 h post-transfection, total RNA was extracted using TRIzol reagent (Invitrogen). RNA was further treated with DNase I (Promega) according to the manufacturer's recommendations. Reverse transcription was carried out in 20-µl volumes containing 1.5 µg of total RNA using the reverse transcription system (Promega). The final cDNA volume was diluted to 100 µl and used in PCR amplification for endofin or -actin, respectively. 1.5–2 µl of cDNA were amplified in a 25-µl reaction volume, containing 0.4 mM dNTPs, 2.5 µl of 10x LA Taq buffer, 1.25 units of Taq DNA polymerase (Takara), and 0.3 pM primers. Following 94 °C incubation for 2 min, 32 cycles of amplification for endofin or 24 cycles of amplification for -actin was performed at 94 °C for 20 s, 56 °C for 20 s, and 72 °C for 19 s. The endofin primers were sense 5'-AATGGTACAAATAACTCCAGAG-3' and antisense 5'-CTTTAGAAAGTAGAACACCTCG-3'; -actin, sense 5'-CGAGAAGATGACCCAGATC-3' and antisense 5'-AGCTTCTCCTTAATGTCACG-3'; plasminogen activator inhibitor-1, sense 5'-GAGACAGGCAGCTCGGATTC-3' and antisense 5'-GGCCTCCCAAAGTGCATTAC-3'; p21, sense 5'-TGTCCGTCAGAACCCATG-3' and antisense 5'-TGGGAAGGTAGAGCTTGG-3'; Bax, sense 5'-ATGGACGGGTCCGGGGAGCAG-3' and antisense 5'CATGATGGTTCTGATCAGTT-3'; Bim, sense 5'-GCCTTCAACCACTATCTCA-3' and antisense 5'-ATCCAGCTCGGTGTCTTCT-3'; Bcl-X_L, sense 5'-CTGCGGTACCGGCGGGCATT-3' and antisense 5'-TCCAAGGCTCTAGGTGGTCA-3'; DAPK1, sense 5'-TCTCCTCAGCCAAGGGTGTT-3' and antisense 5'-GGATGAAGAGTCCTCGGTGC-3'.

Real Time PCR Analysis—In real time PCR, 3 µl of cDNA was amplified in a 25-µl reaction volume, containing 0.4 mM dNTPs, 2.5 µl of 10x LA Taq buffer, 1.25 µl of 20x SYBR Green I buffer (OPE, Shanghai, China), 1.25 units of Taq DNA polymerase (Takara), and 0.4 pM primers. Reaction was performed in Mx3000P^TM Real-time PCR system (Stratagene). PCR was performed as for RT-PCR. Each sample was run in triplicate from two biological repeats. Data were analyzed as described previously (25).

Apoptosis Analysis—Cells were treated with 200 pM TGF-1 for 48 h in medium containing 0.2% fetal bovine serum and then collected by trypsinization. Flow cytometric analysis was performed to monitor the green fluorescence of the FITC-conjugated annexin V (530 ± 30 nm) and the red fluorescence of DNA-bound propidium iodide (630 ± 22 nm) with FACSCaliber (BD Biosciences). All data were analyzed with Cell Quest.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The FYVE Domain of Endofin Is Important for Its Subcellular Localization and TGF- Signaling—SARA was suggested to promote TGF- signaling and mislocalization of SARA affects its ability for regulation of TGF- signaling. As endofin is closely related to SARA, we attempted to investigate whether endofin plays a role in TGF- signaling. To this end, we generated a FYVE-domain deletion mutant of endofin and examined its subcellular localization by immunofluorescence. As shown in Fig. 1A, wild-type endofin was distributed in the cytoplasm as punctate spots and co-localized with EEA1, an early endosome marker, and deletion of the FYVE domain (endofin-dFYVE) led to a diffuse distribution in the cytoplasm. These data are consistent with the early report that the FYVE domain is required for the early endosomal localization of endofin (21). Then, we asked whether endofin has any effect on TGF- signaling with Smad3-responsive reporter CAGA-luciferase (26). Although overexpression of wild-type endofin marginally increased TGF--induced expression of the CAGA-luciferase reporter in human hepatoma Hep3B cells, overexpression of endofin-dFYVE effectively suppressed the transcriptional response of TGF- (Fig. 1B). A similar result was obtained in human hepatoma HepG2 cells (data not shown). These results suggest that the FYVE domain of endofin is critical for its localization in early endosomes and the correct subcellular localization of endofin seems to be important for cellular responses to TGF-.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. The FYVE domain of endofin is important for its subcellular mislocalization and for TGF- signaling. A, deletion of the FYVE domain in endofin caused a subcellular mislocalization of endofin. HeLa cells were transiently transfected with Myc-tagged wild-type endofin or the FYVE domain-deleted mutant. At 48 h post-transfection, the cells were subjected to indirect immunofluorescence with rabbit anti-Myc or mouse anti-EEA1 antibodies and FITC anti-mouse or rhodamine anti-rabbit secondary antibodies. The nuclei were counterstained in blue with 4,6-diamidino-2-phenylindole (DAPI). Scale bar:10 µm. B, mislocalization of endofin attenuated cellular responses to TGF- mediated expression of CAGA-luciferase. HepG2 cells were co-transfected with CAGA-luciferase (0.5 µg/ml) and Renilla (20 ng) together with endofin, endofin-dFYVE, and empty vector pCMV5 (as a control). Transfected cells were with TGF-1 (100 pM) for 20 h and harvested for luciferase assay. Each experiment was performed in triplicate and the data represent the mean ± S.D. after normalized to Renilla activity.

Next, the effect of Sh-1 and Sh-2 on TGF- signaling was examined with the reporter CAGA-luciferase in Hep3B cells. The TGF--induced expression of CAGA-luciferase was attenuated by transient expression of Sh-1 or Sh-2 (Fig. 2D). A similar result was obtained in HepG2 cells (Fig. 2E). Furthermore, knockdown of endofin expression by Sh-2 also decreased the TGF--induced expression of the Smad2-responsive reporter ARE-luciferase (Fig. 2F).

To determine whether endofin is also required for BMP signaling, Hep3B cells were transfected with siRNA constructs and the Smad1-responsive reporter BRE-luciferase (27). Like the nonspecific siRNA (Sh-NS), anti-endofin Sh-2 had no effect on BMP-induced expression of BRE-luciferase (Fig. 2G). Similarly, Sh-2 did not influence BRE-luciferase expression induced by the constitutively active BMP type I receptor BMPRIB(QD) (data not shown). Moreover, Sh-2 had no effect on Wnt-mediated lymphoid enhancer factor-1-luciferase expression (Fig. 2H).

Therefore, Sh-1 and Sh-2 siRNA can reduce endofin expression, and reduction of endogenous endofin expression leads to decreased cellular responses to TGF- but has no effect on BMP or Wnt signaling, suggesting that endofin specifically modulates TGF- signaling.

Endofin Mediates TGF- Signaling in the Physiological Condition—To further confirm the regulatory role of endofin in TGF- signaling, we established cell lines derived from Hep3B cells that stably expressed nonspecific siRNA Sh-NS (Hep3B-Sh-NS) or anti-endofin siRNA Sh-2 (Hep3B-Sh-2). The quantitative real time PCR analysis revealed a dramatic reduction of endofin expression in Hep3B-Sh-2 cells (Fig. 3A). We next performed reporter assays in Hep3B-Sh-NS and Hep3B-Sh-2 cells to examine their responses to TGF-. Consistent with the above results, the TGF--induced expression of CAGA-luciferase in Hep3B-Sh-2 cells was much lower than in Hep3B-Sh-NS cells (Fig. 3B), indicating that endogenous endofin plays an important role in mediating effective TGF- signal transduction.

We next investigated whether endofin is important for the expression of TGF- target genes by quantitative real time PCR. Both plasminogen activator inhibitor-1 and p21_Cip1 are well known TGF- targets (28, 29). Reduction of endofin expression apparently attenuated TGF--induced expression of plasminogen activator inhibitor-1 and p21_Cip1 in Hep3B-Sh-2 cells (Fig. 3C), supporting the importance of endofin in TGF--activated transcription.

To study the role of endofin in a more physiologically relevant condition, we compared TGF--induced apoptosis in these two cell lines. After cells were treated with 200 pM TGF- for 48 h, the cells were harvested for apoptosis assay. As shown in Fig. 3D, TGF- treatment resulted in about 40% cells undergoing apoptosis in Hep3B-Sh-2, significantly lower than the ones in Hep3B-Sh-NS (50%). Furthermore, about one-half of the apoptotic cells in Hep3B-Sh-2 were in the early stage of apoptosis, whereas most of the apoptotic cells in Hep3B-Sh-NS were in the late stage of apoptosis (41 versus 17% in Hep3B-Sh-2), indicating a postponed response to TGF- in Hep3B-Sh-2 cells lacking endofin. We further examined several genes that regulate apoptosis. Pro-apoptotic Bim and anti-apoptotic Bcl-X_L, all of which are the Bcl-2 family members, have been implicated to mediate TGF--induced apoptosis as they are up-regulated by TGF- (30–32). Quantitative PCR analysis revealed that the TGF--induced expression of BIM or TGF--suppressed expression of Bcl-X_L were impaired in Hep3B-Sh-2 cells (Fig. 3E). We also found that the TGF--up-regulated expression of pro-apoptotic BAX was attenuated in Hep3B-Sh-2 cells. These data are in agreement that endofin mediates TGF--induced apoptosis. DAPK1 was also suggested to mediate TGF--promoted apoptosis (33), but we found no difference of its expression between Hep3B-Sh-NS and Hep3B-Sh-2 cells (Fig. 3E).

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Knockdown of endofin expression attenuates cellular responses to TGF-. A, endogenous mRNA levels of ENDOFIN in different cell lines were analyzed by RT-PCR. -ACTIN served as an internal control. B, endofin siRNA effectively reduced endogenous mRNA levels of ENDOFIN in 293T cells. The cells transfected with empty vector pSUPER or siRNA constructs as indicated were harvested for real time PCR analysis. The -fold change in endofin mRNA level was presented as 2–Ct normalized to -actin. The data from three experiments were presented as mean ± S.D. C, HEK293T cells were co-transfected with Myc-endofin (2 µg), Myc-Smad3 (2 µg), and siRNA constructs (2 or 4 µg) as indicated. The protein expression was confirmed by anti-Myc immunoblotting with total cell lysates. D and E, Hep3B (D) and HepG2 (E) cells were transiently transfected with CAGA-luciferase (0.5 µg/ml), Renilla (20 ng), and the indicated siRNA constructs (1 µg/ml for each). The transfected cells were treated with TGF-1 (100 pM) for 20 h and harvested for luciferase analysis. F, Hep3B cells were transfected with ARE-luciferase (0.5 µg/ml), FoxH2 (0.2 µg/ml), and Renilla (20 ng), and together with Sh-NS or Sh-2 (1 µg/ml each). The transfected cells were treated with TGF-1 (100 pM) for 20 h and harvested for luciferase analysis. G, Hep3B cells were transfected with BRE-luciferase (0.5 µg/ml) and Renilla (20 ng), and together with Sh-NS or Sh-2 (1 µg/ml each). The transfected cells were then treated with BMP2 (1 nM) for 20 h and harvested for luciferase analysis. H, HEK293T cells were transfected with lymphoid enhancer factor-luciferase (0.5 µg/ml) and Renilla (20 ng), and Wnt-1 as well as Sh-NS or Sh-2 (1 µg/ml each). For luciferase assay, each experiment was performed in triplicate and the data represent the mean ± S.D. after normalized to Renilla activity.

SARA interacts with Smad2 via its Smad-binding domain (SBD) in the middle of its sequence (8, 9). Sequence alignment revealed that endofin contains a less-conserved SBD region (data not shown). To investigate whether this putative SBD is involved in Smad4 binding, we co-expressed Myc-endofin (wild-type or dSBD lacking the putative SBD) together with FLAG-Smad constructs in HEK293T cells. As shown in Fig. 4C, Smad4 interacted with both endofin wild-type and dSBD, indicating this putative SBD region does not mediate Smad4 binding.

It was reported that SARA associates with the TRI receptor via its carboxyl terminus (8). Because endofin shares a high similarity with SARA in their carboxyl-terminal regions, we explored whether endofin interacts with TGF- receptors. HEK293T cells were transfected with FLAG-endofin either alone or together with HA-tagged type I receptors. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody. The results shows that endofin strongly interacted with TRI, BMP receptors BMPRIA and BMPRIB, activin receptor ActRIB, and TGF- receptor ALK1, but not to ALK2 (Fig. 4D). Similar results were obtained in a reverse immunoprecipitation-immunoblotting experiment (Fig. 4E). To further examine whether the carboxyl-terminal region is important for endofin to associate with receptors, the amino-terminal region (1–886) of endofin was tested for interaction with receptors. As shown in Fig. 4F, no apparent interaction was detected between endofin-(1–886) and TRI or BMPRIB.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Endofin siRNA-expressing stable Hep3B cells exhibit impaired responses to TGF- stimulation. A, the endogenous ENDOFIN mRNA levels in Hep3B-Sh-2 cells were analyzed by real time PCR analysis. The relative endofin mRNA level was normalized to -ACTIN. B, Hep3B-Sh-NS and Hep3B-Sh-2 cells were transfected with CAGA-luciferase (0.5 µg/ml) and Renilla (20 ng) for 48 h. Then the cells were treated with TGF-1 (100 pM) for 20 h and harvested for luciferase analysis. The experiment was performed in triplicate and the data represent the mean ± S.D. after normalized to Renilla activity. C, Hep3B-Sh-NS and Hep3B-Sh-2 cells were treated with TGF-1 (100 pM) for 20 h. Total RNA was isolated and subjected to real time PCR to determine the expression of plasminogen activator inhibitor-1 (PAI-1) or p21. The -fold change in mRNA level of genes was normalized to -actin. D, Hep3B-Sh-NS and Hep3B-Sh-2 cells were treated with TGF-1 (200 pM) for 48 h and subjected to apoptosis analysis. The cells positive for annexin V-FITC (low right square) or positive for both annexin V-FITC and propidium iodide (top right square) represented the early or late stages of apoptosis, respectively. Corresponding percentages of total apoptotic cells (TA) and apoptotic cells at the late stages (LA) were presented as histograms (right panel). E, Hep3B-Sh-NS and Hep3B-Sh-2 cells were treated with TGF- (100 pM) for 24 h. Total RNA was isolated and subjected to real time PCR to determine the expression of Bcl-XL, BIM, BAX, or DAPK. The -fold change in mRNA level of genes was normalized to -ACTIN. All quantitative data were from three independent experiments and expressed as mean ± S.D. The asterisk (*) indicates a statistically significant difference between Hep3B-Sh-2 and Hep3B-Sh-NS cells (p < 0.05).

The above data that endofin can associate with both Smad4 and TRI receptor suggests that endofin might bring Smad4 to the vicinity of the TGF- receptor complex to facilitate R-Smad-Smad4 complex formation. To test this hypothesis, we examined the TGF--induced formation of endogenous Smad2-Smad4 complex in Hep3B-Sh-2 cells. Cells were harvested for anti-Smad2 immunoprecipitation and anti-Smad4 immunoblotting after a 1-h TGF- treatment. As shown in Fig. 5B, TGF- treatment led to the association of Smad2 with Smad4 in Hep3B-Sh-NS cells, whereas Smad complex formation was hampered in Hep3B-Sh-2 cells. These data indicate that endofin plays an important role in promoting Smad heterocomplex formation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The essential components to transduce TGF- signaling are relatively limited and only the receptors and Smad proteins are required for the canonical TGF-/Smad pathway. However, this pathway is known to be modulated by many other proteins. Several scaffold or adaptor proteins have been suggested to facilitate Smad activation by the receptors. In this study, we characterized the FYVE domain-containing protein endofin in regulation of TGF- signal transduction. Our data showed that deletion of the phosphatidylinositol 3-phosphate-binding FYVE domain in endofin caused its subcellular mislocalization from early endosomes to a diffuse distribution in the cytoplasm and reduced the TGF--dependent transcriptional responses. Knockdown of endogenous endofin expression by RNA interference attenuated TGF--induced expression of CAGA-luciferase and apoptosis. Furthermore, we showed that endofin interacted with TRI and Smad4 and interference of endogenous endofin expression impaired Smad2 phosphorylation and Smad2-Smad4 complex formation. Our findings suggest that endofin might facilitate TGF- signaling through recruiting Smad4 to the TGF- receptor complex and thus assisting the association between Smad4 and the receptor-activated Smad2.

View larger version (78K): [in this window] [in a new window]   FIGURE 4. Endofin interacts with Smad4 and type I receptors. A, endofin interacts with Smad4. HEK293T cells were transiently transfected with Myc-tagged endofin and FLAG-tagged Smads as indicated. At 48 h post-transfection, the cells were harvested for anti-FLAG immunoprecipitation (IP) and then anti-Myc immunoblotting (IB)(first panel), or anti-Myc immunoprecipitation and anti-FLAG immunoblotting (second panel). The protein expression was confirmed by immunoblotting with total cell lysates (lower panels). B, endofin associates with endogenous Smad4. HEK293T cells were transfected with FLAG-endofin and HA-Smad4 as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Smad4 antibody (upper panel). The protein expression was confirmed by immunoblotting with total cell lysates (middle and lower panels). C, the putative SBD region of endofin is not required for Smad4 interaction. HEK293T cells were transiently transfected with FLAG-Smads and Myc-endofin wild-type or dSBD mutant, and the cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Myc antibody (upper panel). Protein expression was confirmed by immunoblotting with total cell lysates (middle and lower panels). D and E, endofin binds to TRI, BMPRIA, BMPRIB, ActRIB, and ALK1 in HEK293T cells. HEK293T cells were transiently transfected with FLAG-endofin and HA-tagged receptors as indicated and cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody (D, upper panel) or immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG antibody (E, upper panel). Protein expression was confirmed by immunoblotting with total cell lysates (middle and lower panels). F, the carboxyl-terminal region of endofin is important for the interaction with TRI and BMPRIB. HEK293T cells were transiently transfected with HA-tagged receptors and Myc-endofin wild-type or the truncated mutant (1–886). At 48 h post-transfection, the cells were harvested for anti-HA immunoprecipitation and anti-Myc immunoblotting. Protein expression was confirmed by immunoblotting with total cell lysates (middle and lower panels).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Knockdown of endogenous endofin expression impairs Smad2 phosphorylation and Smad2-Smad4 complex formation. A, knockdown of endofin expression reduces TGF--mediated phosphorylation of endogenous Smad2. Hep3B-Sh-NS, Hep3B-Sh-2, or wild-type Hep3B cells were treated with different concentrations of TGF-1 for 2 h. The cells were harvested for immunoblotting with anti-phosphorylated Smad2 (upper panel) or anti-Smad2 (lower panel) antibodies. B, TGF--induced association of Smad2 and Smad4 is impaired in Hep3B-Sh-2 cells. Hep3B-Sh-NS and Hep3B-Sh-2 cells were treated with TGF-1 (100 pM) for 1 h. Cell lysates were immunoprecipitated (IP) with anti-Smad2 antibody and immunoblotted (IB) with anti-Smad4 antibody. Protein expression was confirmed by immunoblotting with total cell lysates (middle and lower panels).

SARA specifically interacts with Smad2 and Smad3 and was proposed to facilitate Smad2/3 phosphorylation by bringing the R-Smads to the signaling receptor complex (8, 9). Although endofin shares a high homology with SARA at the carboxyl terminus and both of them are localized in early endosomes, unlike SARA, endofin binds to Smad4 but not to Smad2/3. However, endofin might fulfill scaffolding functions together with SARA to promote R-Smad-Smad4 complex formation. Consistent with this, knockdown of endofin expression attenuated Smad2-Smad4 complex formation. Although endofin does not interact with Smad2/3, knockdown of its expression mitigated TGF--induced Smad2 phosphorylation. This could be because endofin and SARA form a heterocomplex and together they coordinate R-Smad activation. Indeed, our immunoprecipitation-immunoblotting assay revealed that endofin is able to interact with SARA (data now shown). Endofin was implicated in regulation of membrane trafficking by recruiting TOM1 and clathrin to endosomes (22, 23). It waits to be determined whether endofin controls the activity or turnover of TGF- receptors and other cell surface receptors.

	FOOTNOTES

 
^* This work was supported in part by National Natural Science Foundation of China Grants 30125021 and 30430360, and 973 Program Grants 2004CB720002 and 2006CB910100. 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.

¹ Chueng Kong Scholar. To whom correspondence should be addressed. Tel.: 86-10-62795-184; Fax: 86-10-6279-4376; E-mail: ygchen{at}tsinghua.edu.cn.

² The abbreviations used are: TGF-, transforming growth factor-; BMP, bone morphogenetic protein; R-Smads, receptor-regulated Smads; endofin, endosome-associated FYVE-domain protein; SARA, Smad anchor for receptor activation; RT, reverse transcription; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; SBD, Smad-binding domain; HA, hemagglutinin.

	ACKNOWLEDGMENTS

 
We are grateful to the RIKEN Institute for endofin (KIAA0305) cDNA.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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