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. 2004 May 16:5:20.
doi: 10.1186/1471-2121-5-20.

Direct interaction between Smad3, APC10, CDH1 and HEF1 in proteasomal degradation of HEF1

Claire Nourry  1 , Lola Maksumova, Mona Pang, Xiaohong Liu, Tongwen Wang
Affiliations

Direct interaction between Smad3, APC10, CDH1 and HEF1 in proteasomal degradation of HEF1

Claire Nourry et al. BMC Cell Biol. .

Abstract

Background: The Transforming Growth Factor-beta (TGF-beta) regulates myriad cellular events by signaling through members of the Smad family signal transducers. As a key signal transducer of TGF-beta, Smad3 exhibits the property of receptor-activated transcriptional modulator and also the novel ability of regulating the proteasomal degradation of two Smad3 interacting proteins, SnoN and HEF1. It has been shown that Smad3 recruits two types of Ub E3 ligases, Smurf2 and the Anaphase Promoting Complex (APC), to mediate SnoN ubiquitination, thereby enhancing SnoN degradation. The molecular mechanisms underlying Smad3-regulated HEF1 degradation are not well understood. Furthermore, it is not clear how Smad3 recruits the APC complex.

Results: We detected physical interaction between Smad3 and an APC component APC10, as well as the interaction between HEF1 and CDH1, which is the substrate-interacting component within APC. Detailed domain mapping studies revealed distinct subdomains within the MH2 domain of Smad3 for binding to APC10 and HEF1 and suggests the formation of a complex of these four proteins (Smad3, HEF1, APC10 and CDH1). In addition, the protein levels of HEF1 are subjected to the regulation of overexpressed APC10 and CDH1.

Conclusions: Our data suggests that Smad3 may recruit the APC complex via a direct interaction with the APC subunit APC10 to regulate the ubiquitination and degradation of its interactor HEF1, which is recognized as an ubiquitination substrate by the CDH1 subunit of the APC complex.

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Figures

Figure 1
Figure 1
Smad3 interacts with APC10 specifically. (A) Co-immunoprecipitation of Smad3 and APC10 from mammalian overexpression system. 293 cells were transiently transfected with T7-APC10 and Smad proteins as indicated. The cell lysates were subjected to Western blot analyses directly or immunoprecipitation followed by Western blot as indicated. (B) Smad3 binds to APC10 in GST pull down assays. Flag-tagged Smad constructs were transfected into 293 cells and tested against GST-APC10 purified from E. Coli. Total input Smad protein levels (100 ug) were detected by immunoblotting of the cell lysates with anti-Flag antibody (top panel). Smad proteins bound to the GST-APC10 beads were eluted and analyzed by immunoblotting with anti-Flag antibody (middle panel). As a control, F-Smad proteins were incubated with the GST alone (bottom panel). (*) We noted that GST-APC10 was recognized by the anti-Flag antibody on Western blot. (C) Smad3 binds to HEF1 and APC10 in in vitro binding assay. In vitro translated 35S-labeled HEF1, 35S Smad3 and 35S APC10 were incubated with GST-Smad3, GST-APC10 or GST as indicated. The in vitro translated proteins before and after binding were detected by electrophoresis and autoradiography.
Figure 2
Figure 2
Domain mapping studies of the interaction between Smad3 and APC10 reveal distinct domains involved in binding. (A) The Smad3C (MH2 domain) is necessary and sufficient for binding to APC10. Top panel: a cartoon to illustrate the deletion constructs of Smad3. The APC10 binding activities of each truncated Smad3 as detected by assays in the bottom panels are summarized at the right side of the panel. Bottom left panel: MH2 domain is necessary and sufficient for Smad3 to bind APC10 in GST pull down assay. Flag-tagged full-length or truncated Smad3, as indicated, was transfected into 293 cells and tested against GST-APC10 and GST (negative control). Bottom right panel: MH2 domain is necessary and sufficient for Smad3 to bind APC10 in 293 cells. Different Smad3 truncations tagged with Flag were co-transfected with T7-APC10 into 293 cells. The expression of these proteins was detected by Western Blot using anti-Flag and the interaction between APC10 and Smad3 truncations was detected by immunoprecipitation of T7-APC10 followed by Western blot using anti-Flag. "L.C." represents the antibody light chain. (B) The C-terminal domain of APC10 is necessary for binding to Smad3. Top panel: a cartoon that illustrates the deletion mutants of APC10. The top three deletion constructs are amino-terminal deletion mutants. The number of amino acids deleted in each construct is indicated. For example, the D2 construct lacks the N-terminal 41 amino acids, thus it is also labeled as D41N. Bottom left panel: the six deletion constructs of APC10, each tagged with Flag, were transfected into 293 cells. The expression of these deletion mutants was detected by Western blot using anti-Flag. Stable protein expression was detected only in cells transfected with two C-terminal deletion constructs (D8 and D9). Bottom right panel: the C-terminal 66 amino acids of APC10 are required for Smad3 binding. T7-tagged APC10 and Flag-tagged APC10 D9 were transfected into 293 cells. Cell lysates were subjected to immunoblotting with anti-T7 (lane 1) and anti-Flag (lane 2) antibody. APC10 and APC10 D9 were tested against GST-Smad3 (lanes 3 & 4) and GST alone as a control (lanes 5 & 6).
Figure 3
Figure 3
Smad3 MH2 domain contains overlapping but distinct binding sites for HEF1 and APC10. (A) A Cartoon to illustrate the deletion constructs of Smad3 (Smad3d2, d4 and d6) as well as a hybrid protein S3/S1/S3. (B) GST pull-down assay to map APC10 binding site on Smad3C. Flag-tagged Smad3 deletion mutants were transfected into 293 cells and the cell lysates were tested against GST-APC10. Cell lysates (top panel) and proteins bound to the beads (middle and bottom panels) were analyzed by Western blot with anti-Smad1/2/3 from Santa Cruz. The percentage listed at the right side of the listed constructs is derived from dividing the signals (measured by ImageQuant) of bound proteins in the middle panel by the signals of lysate proteins in the corresponding lane of the top panel and then times 100 percent. (C) GST pull-down assay to map HEF1 binding site on Smad3C. Same as in (B), with GST-APC10 replaced by GST-HEF1. (*) endogenous Smad1 and Smad3 recognized by anti-Smad1/2/3 antibody. (D) A cartoon to illustrate the complex of Smad3, HEF1 and APC10 and their potential interaction with the APC ligase core complex.
Figure 4
Figure 4
CDH1 interacts with HEF1 at the HEF1 C-terminal M2 domain. (A) In vitro binding test. In vitro translated 35S labeled CDH1 was incubated with GST-HEF1 or GST alone as a control in modified lysis buffer. 35S labeled CDH1 was separated by electrophoresis and detected by autoradiography. (B) A cartoon that illustrates putative D boxes on HEF1. (C) A cartoon that illustrates the deletion constructs of HEF1 used in (D). (D) Co-immunoprecipitation of HEF1/HEF1 deletions with CDH1 in 293 cells. Full-length HEF1 and T7-tagged HEF1 deletions were co-transfected with myc-tagged CDH1 into 293 cells. Myc-CDH1 was immunoprecipitated with anti-myc antiboby and CDH1 bound HEF1 or HEF1 deletions were detected by immunoblot with either anti-p130Cas antibody (top panel, lanes 1 & 2) or anti-T7 antibody (top panel, lanes 3–5). The expression level of CDH1 was detected with anti-myc antibody (second panel). The amount of HEF1 (third panel) or T7-HEF1 deletions (bottom panel) expressed was detected by anti-p130Cas for HEF1 or anti-T7 antibody. (E) A cartoon that illustrate the putative complex of HEF1, Smad3, APC and CDH1.
Figure 5
Figure 5
TGF-β type I receptor activation enhances Smad3 interaction with APC10 but does not alter CDH1 interaction with HEF1. (A) Smad3 interaction with APC10 is positively regulated by the activation of TGF-β type I receptor. Flag-Smad3 and T7-APC10 were co-expressed in the presence or absence of a constitutively active TGF-β type I receptor mutant R4T204D (R4TD). The interaction between Smad3 and APC10 was detected by immunoprecipitation of Smad3 with an anti-Smad3 polyclonal antibody followed by Western blot with anti-T7 (Top panel, lanes 4–6). The immunoprecipitated Flag-Smad3 was detected by anti-Flag antibody (middle panel). The expression of R4TD was detected by anti-TGF-β RI polyclonal antibody from Santa Cruz (Bottom panel). R4TD-p represents a potential cleavage product of R4. (B) HEF1 interaction with CDH1 Is not regulated by TGF-β type I receptor activation. The 293 cells were transiently transfected with myc-CDH1 and HEF1 in the presence or absence of Smad3 and R4TD, as indicated. The interaction between HEF1 and CDH1 was detected by immunoprecipitation with anti-p130Cas followed by Western blot with anti-myc (Top panel, lanes 7–13). The expression of myc-CDH1 and Flag-Smad3 was detected by Western blot by anti-myc and anti-Flag (Top panel, lanes 1–6). The expression levels of HEF1 was detected by anti-HEF1 (middle panel) and the levels of R4TD detected by Western blot with anti-TGF-β RI antibody (bottom panel).
Figure 6
Figure 6
Both APC10 and CDH1 can alter the steady state protein levels of HEF1 (A) Overexpressed APC10 caused the reduction of the steady-state protein levels of HEF1 and such an activity is compromised in a deletion mutant of APC10 (APC10 D8) that is defective in binding to Smad3. HEF1 was co-transfected with ubiquitin, Flag-Smad3, T7-APC10 or T7-APC10 deletion mutant APC10 D8. The expression level of HEF1 was detected by Western blot using anti-p130Cas antibody, as shown in the top panel; the expression of Smad3, APC10 and APC10D8 were detected by Western blot using anti-Flag and anti-T7, as shown in the bottom panel. (B) CDH1 enhances Smad3-regulated HEF1 degradation. HEF1 was co-expressed in different combinations with myc-CDH1, T7-APC10, Flag-Smad3 and R4T204D (R4TD), a constitutively active type I receptor in 293 cells. The steady state levels of HEF1 were detected by anti-HEF1 antibody (top panel) and the expression levels of Smad3 and APC10 were detected with anti-Flag and anti-T7 antibodies, respectively (second and third panels), while the expression levels of myc-CDH1 were detected by anti-Myc (fourth panel). To detect R4-TD, cell lysates were blotted with anti-TGF-β RI antibody (bottom panel).
Figure 7
Figure 7
A cartoon for possible signaling pathways that involve Smad3 interaction with APC10. A cartoon that illustrates possible pathways for Smad3-induced HEF1 degradation involving APC10 and CDH1. Please see text for details.
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