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First published online 13 March 2007
doi: 10.1242/jcs.03400

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Endofin acts as a Smad anchor for receptor activation in BMP signaling

¹ Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
² Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. Endofin interacts with Smad1. (A) Co-immunoprecipitation (co-IP) of endofin with Smads. Flag-tagged Smad1 or Smad2 was co-transfected with HA-tagged endofin into COS1 cells. The lysate was subjected to co-IP with anti-HA antibody and probed with anti-Flag antibody, or co-IP with anti-Flag and probed with anti-HA. (B) Confirmation of the interaction between Smad1 and endofin by protein-fragment complementation assays (PCA). The complementary fragments (YFP1 and YFP2) of YFP were fused with Smad1 and endofin, respectively, to create YFP1-Smad1 (a.a. 1-158 of YFP) and endofin-YFP2 (a.a. 159-239 of YFP), and then transfected into COS1 cells. The interaction between endofin and Smad1 is indicated here as yellow fluorescence. Transfection of pcDNA3, co-transfection of pcDNA3-YFP1 and pcDNA3-YFP2 and co-transfection of YFP1-Smad1 and Hoxa1-YFP2 serve as negative and positive controls, respectively (Li et al., 2006). Bar, 10 µm. (C) Colocalization of Smad1 and endofin. Upper panel: C2C12 cells were transfected with HA-Endofin and Flag-Smad1. After transfection for 36 hours, cells were processed for immunofluorescence using antibodies against HA (rabbit polyclonal) and Flag (mouse monoclonal). Lower panel: Endogenous protein co-localization. Naive C2C12 cells were subjected to immunofluorescent microscopy using rabbit polyclonal anti-endofin and mouse monoclonal anti-Smad1. Nuclei were counterstained with Hoechst 33342. Representative images are shown. (D) Endofin interacts with both Smad1 and PP1c. COS1 cells were transfected with Flag-Smad1, HA-tagged endofin and Myc-tagged PP1c. Cells were treated with rhBMP2 (200 ng/ml) for 2 hours before lysis. The lysate was subject to co-immunoprecipitation with antibody against HA, the presence of Smad1 and PP1c in the precipitate was detected with anti-Flag and anti-Myc antibodies, respectively. (E) Co-IP of endogenous endofin, Smad1 and PP1c. C2C12 cells were cultured until subconfluent, with or without treatment of rhBMP2 (200 ng/ml, 2 hours), cells were lysed, and the lysate was subjected to immunoprecipitation with antibody against either endofin or SARA. The presence of Smad1 and PP1c in the precipitate was detected by anti-Smad1 and anti-PP1c antibodies. Bar, 5 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Identification of the endofin interaction domain in Smad1 and PP1c. (A) Schematic representation of wild-type and mutant endofin. Three mutants were generated to disrupt these conserved domains: FYVE domain (C753S), Smad-binding domain (deletion of amino acids 814-860) and PP1c-binding domain (F872A). (B) Deletion of Smad-binding domain in endofin diminishes its interaction with Smad1. COS1 cells were transfected with Flag-Smad1, HA-tagged endofin and its mutants. Immunoblots in the two lower panels show the protein expression levels of the COS1 cell lysates. The upper panel shows anti-HA immunoprecipitates probed with anti-Flag antibody. (C) Co-IP of PP1c with endofin and the PBD(F872A) mutant. HA-tagged endofin and its mutant with disrupted PP1c-binding domain were co-transfected with PP1c into COS1 cells. The lysate was subject to immunoprecipitation with anti-HA. Immunoblots indicating the protein expression levels of the lysates of COS1 cells are shown in the two lower panels. The upper panel shows anti-HA immunoprecipitates probed with anti-Flag antibody.

View larger version (35K): [in this window] [in a new window]   Fig. 3. The BMP type I receptor is the substrate of PP1. (A) C2C12 cells transfected with different combinations of genes were labeled with [32P]orthophosphate in the presence or absence of BMP2 as indicated. ALK3-HA was immunoprecipitated from lysates of treated cells and separated by 8.5% SDS-PAGE. Gels were dried and exposed to Biomax Mr film (Eastman Kodak). PBD (F872A) is a mutant with the disrupted PP1c-binding domain. Phosphatase inhibitor-1 is an inhibitor for PP1. (B) BMP type I receptors interact with PP1 regulatory subunit GADD34. COS1 cells transfected with HA-tagged type I receptors (ALK3 and ALK6) and Flag-tagged GADD34. Lysates were subjected to co-immunoprecipitation with anti-Flag and the presence of ALK3 and ALK6 were probed with antibody against HA. (C) Unlike in the TGF signaling pathway, GADD34 interacts with the BMP type I receptor without the bridging of inhibitory Smads. The GADD34-ALK3/6 complex was further confirmed by a sequential immunoprecipitation. COS1 cells were first co-transfected with ALK3/6-HA with or without Flag-Smad6/7 and GADD34. The lysates were subjected to immunoprecipitation with HA antibody, and the resultant precipitates were eluted from protein-G-Sepharose beads by HA peptide competition and then subjected to a second immunoprecipitation with anti-Flag antibody. The final precipitates were immunoblotted with antibodies against all these components.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Endofin modulates Smad1 phosphorylation and nuclear translocation. (A) Silencing of endofin expression by RNA interference inhibited Smad1 phosphorylation. C2C12 cells were transfected with Flag-tagged Smad1 and either vector, construct for GFP or endofin siRNA. On the third day following 3-hour BMP2 treatment, Flag-tagged Smad1 was precipitated with anti-flag and its phosphorylation level was detected with anti-phospho-Smad1. (B) Endofin, but not SARA, regulates BMP-specific phosphorylation and intracellular translocation of Smad1. C2C12 cells were transfected with either HA-SARA or HA-Endofin, then stimulated with BMP (100 ng/ml) for 3 hours. After fixation with paraformaldehyde (PFA), cells were immunostained with either anti-HA (green) or anti-phospho-Smad1 (red). Nuclei were visualized using Hoechst 33342. Representative images are shown. Bar, 20 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 5. Endofin regulates expression of BMP downstream genes and mineralization in human MSCs. (A) Transcriptional response assay. C2C12 cells stably expressing GFP (control), or endofin or its mutants were transfected with BMP signaling reporter construct 9XSBE-luc. Transfected cells were incubated in the presence or absence of BMP2 (200 ng/ml). Luciferase activity was normalized and plotted as the mean ± s.d. of triplicates from a representative experiment. *P<0.05 compared with the second group. (B) Alkaline phosphatase activity assay. C2C12 cells stably expressing GFP (control) or endofin or its mutant PBD(F872A) were cultured in 24-well plates treated with or without BMP2 and harvested at day 5 for alkaline phosphatase activity assay. Relative alkaline phosphatase activity was normalized and plotted as the mean ± s.d. of triplicates from a representative experiment. *P<0.05 compared with second group. (C) Alkaline phosphatase staining. C2C12 were cultured and treated as in alkaline phosphatase activity assay and harvested at day 3. After fixation with 4% paraformaldehyde, cells were subjected to alkaline phosphatase staining. Bar, 20 µm. (D) Overexpression of mutated endofin with disrupted PP1c-binding domain enhanced mineralization activity of human MSCs (von Kossa Assay). Human MSCs were cultured in Dulbecco's Modified Eagle's Medium: low glucose, 1x penicillin-streptomycin, 10% fetal bovine serum (BioWhittaker, MSC serum), 10 mM -glycerol phosphate, 50 µM Ascorbic acid 2-phosphate (AsAP) and 200 ng/ml BMP2. Mineralization assay was performed at day 24. After silver nitrate was added, a calcium deposit was visible as a black structure or focal dot. i, GFP; ii, GFP+BMP2; iii, Endofin+BMP2; iv, Endofin-PBD(F872A)+BMP2. Bar, 30 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Regulation of BMP-dependent mesodermal induction by endofin in Xenopus embryos. Capped RNAs encoding endofin (wild type and mutants) and BMP4 were synthesized in vitro and injected alone or in combination into both animal poles of two-cell-stage embryos. The ectodermal explants (animal caps) from injected embryos were dissected at blastula stages (stage 8.5-9) and cultured until gastrula stages (stage 11) before total RNA was extracted. RT-PCR was performed using the primers for different mesodermal marker genes. EF1- serves as a loading control. Although mutations in the FYVE and SBD domains reduced expression of BMP-responsive genes, the mutation in the PBD domain enhanced BMP-induced marker gene expression.