TI - Discussion . AB - HEF1 has been implicated in many different signaling pathways such as those mediated by integrin , TCR and BCR where it plays the role of an adaptor protein . Recent studies have shown that HEF1 degradation is regulated by Smad3 via the proteasomal degradation pathway and is further enhanced by TGF-beta stimulation [17] . These findings reveal a novel ability for Smad3 , which has been primarily considered as a DNA -binding transcriptional factor , and also suggest a novel cross-talk mechanism between TGF-beta/activin pathways and multiple HEF1-involved pathways . The molecular mechanisms underlying Smad3 -regulated proteasomal degradation are not clear . Our current studies revealed the ability of Smad3 to bind to APC10 , which is a regulatory component of the APC ligase core complex required for subSTRate interaction , as well as the ability of HEF1 to bind to CDH1 , which is a co-activator of APC ligase for specific SUBstrate recognition . The interaction between Smad3 and APC10 is subjected to the regulation by the TGF-beta type I receptor , while the interaction between CDH1 and HEF1 is constitutive . Both APC10 and CDH1 exhibit the ability to regulate the steady state levels of HEF1 upon co-expression with HEF1 . These data suggests a novel mechanism for Smad3 to regulate the proteasomal degradation of HEF1 via assisting the recognition of HEF1 by the APC E3 ligase . The interaction between APC10 and Smad3 was first observed in the yeast two hybrid system . This interaction was confirmed in mammalian over-expression system by co-immunoprecipitation , then by in vitro GST pull-down assay , and finally via in vitro binding assays to demonstrate a potential direct interaction between Smad3 and APC10 . Domain mapping studies showed that Smad3 MH2 domain is necessary and sufficient to bind APC10 , whose C-terminal domain is required for Smad3 binding , while the N-terminal domain for APC10 stability . These data strongly suggested a direct and domain -specific interaction between Smad3 and APC10 . Previously Smad3 has been shown to recruit APC complex to ubiquitinate its nuclear interactor SnoN , but it was not clear how Smad3 recruits APC complex [18,19] . The ability of Smad3 to bind directly to APC10 suggests that this interaction could be the missing link for Smad3 to recruit APC complex in the ubiquitination of SnoN . Future studies will be carried out to directly test this possibility . Our previous studies have shown that Smad3 interlocks with HEF1 , with the MH1 domain binds to the N-terminal domain of HEF1 , while the MH2 domain of Smad3 binds to the C-terminal domain of HEF1 [17] . Considering the involvement of the MH2 domain of Smad3 in binding to APC10 , we compared the ability of various deletion constructs of Smad3 MH2 domain in their ability to bind to APC10 and HEF1 . These studies showed that residues located within 237 to 362 on Smad3 are involved in Smad3 binding to APC10 , while a more localized region between residues 301 to 330 is involved in binding to HEF1 . These data suggests that Smad3 , APC10 and HEF1 can potentially co-exit in one single complex . Since APC10 is known to play a role in regulating SUBstrate recognition and ubiquitination by mammalian APC Hartmut C.et al. , Current Biology 13 : . . . . . . . 1459 , 2003 } the interaction between Smad3 , APC10 and HEF1 suggests that HEF1 and Smad3 are potential ubiquitination SUBstrates for APC ligase . Our finding of the direct interaction between HEF1 and the WD40 repeat protein CDH1 further qualifies HEF1 as a subSTRate for APC , since interaction with CDH1 or CDC20 has been shown to be a prerequisite for APC SUBstrates [37] . The role of APC10 and CDH1 in regulating HEF1 degradation was confirmed by the ability of overexpressed APC10 and CDH1 in enhancing Smad3 -regulated HEF1 degradation in 293 cells ( Figure 6 ) . The physical interaction between HEF1 and CDH1 , a regulator at late anaphase of cell cycle , could be functionally linked to a previous observation of the ability of a processed form of HEF1 , p55HEF1 , in interaction with mitotic spindles [22] . The exact role of HEF1 in cell cycle regulation and how Smad3 , via interacting with the co-activators of APC complex to regulate such a role is also an important subject for future studies . While Smad3 can regulate the proteasomal degradation of both HEF1 and SnoN , PHOSphorylation of Smad3 by the activated type I receptor is required only for SnoN degradation but not for HEF1 degradation [17-19] . The mechanism for such a difference warrants future investigation . One apparent difference between HEF1 and SnoN is their intracellular localization . While HEF1 is predominantly cytoplasmic , SnoN is primarily a nuclear protein . It has been shown that the inactive Smad3 is primarily cytoplasmic , and its nuclear translocation is triggered upon its PHOSphorylation by the type I receptor of TGF-beta at the C-terminal SSVS motif . Thus , the dependence of SnoN degradation on Smad3 phosphorylaTION at the SSVS motif could be atleast partially due to the dependence of Smad3 PHOSphorylation for Smad3 accumulation into the nucleus . However , a recent study indicated that the in vitro translated Smad3 can induce HEF1 degradation in an in vitro degradation assay , which does not involve the issue of nuclear localization , thus suggesting that additional component s involving PHOSphorylation -dependent structural changes of Smad3 may be involved in regulating SnoN ubiquitination and degradation . One such structural change has been recently revealed . Previously it was shown that inactive unphosphorylaTED Smad3 is bound to proteins such as SARA ( Smad anchor for receptor activation ) in the cytoplasm and that PHOSphorylation of Smad3 by the type I receptor decreases its affinity for SARA thus allowing Smad3 to interact with the Co-Smad Smad4 before entering the nucleus [38] . In a recent study , molecular details were revealed regarding the interaction between inactive Smad3 , SARA and the TGF-beta type I receptor . The inactive Smad3 exists in a monomer form in a complex with SARA and the TGF-beta type I receptor , at the cell membrane or at early endosome [39,40] . The PHOSphorylation of Smad3 releases it from SARA and allows Smad3 to adopt a different conformation that favors trimmer formation . Interestingly , the later conformation is preferred by the nuclear oncoprotein Ski [41] . Thus , SARA serves as a molecular guardian of Smad3 to prevent it from forming aberrant trimmers for constitutive activation . Therefore , one possible explanation for the dependence of Smad3 PHOSphorylation for SnoN degradation , is that SnoN , like Ski , only interacts with Smad3 in oligomers , which could be either Smad3 homo-trimmers or Smad3 (2) /Smad4 (1) hetero-trimmers , and that the ubiquitination of SnoN occurs in such a complex . For HEF1 , since neither the interaction nor the degradation is dependent upon Smad3 phosphorylaTION , we therefore consider it likely that Smad3 binds to HEF1 in monomeric form before its PHOSphorylation and that such a complex is sufficient for assisting HEF1 ubiquitination and degradation . It also remains a possibility that HEF1 could function like SARA to keep Smad3 in an inactive conformation , thereby assisting Smad3 recognition by the type I receptor kinase and directly regulating Smad3 trimmer formation . Our preliminary data have suggested such an inhibitory role of HEF1 [17] . Future studies of the crystal structures of the complex of Smad3 and HEF1 in the presence or absence of TGF-beta type I receptor cytoplasmic domain , as well as the structural studies of the complex of Smad3 , HEF1 APC10 and CDH1 , as demonstrated here , will significantly advance our current understandings of the mechanism and regulation of HEF1 degradation in TGF-beta pathway . The physiological regulation of the complex formation between Smad3 , APC10 , HEF1 and CDH1 is likely very dynamic and complex . Since all these observations are made via in vitro systems , we do not know the detailed regulation of these interactions in specific cell types under different conditions . The physiological complex formation between Smad3 , HEF1 and APC10 is likely subjected to constant changes , depending upon the expression levels of these proteins . It is also possible that the formation of the complex involves sequential steps . For example , Smad3 could first binds to the APC10 before binding to HEF1 or vise versa and such binding could stabilizes Smad3 in a favorable conformation for subsequent interaction with other proteins , such as CDH1 . The regulation of the formation of this complex could also via PHOSphorylation of HEF1 and Smad3 and via competition involving other interacting proteins of Smad3 and HEF1 . Here we gain a glimpse of the complexity of the regulation of HEF1 ubiquitination and degradation in vivo . In 293 cells , we observed an enhancement effect of TGF-beta receptor activation on Smad3 interaction with APC10 , but not on HEF1 interaction with CDH1 ( Fig 5 ) . In Figure 7 , a cartoon is presented to summarize our data and present a model for the role of Smad3 interaction with APC10 in recruiting APC complex for ubiquitination and degradation of HEF1 , downstream of TGF-beta type I receptor activation . In this model , we propose three possible ways for HEF1 to be ubiquitinated by APC ligase via the formation of the multimeric complex of Smad3 , HEF1 , APC10 , CDH1 , the latter two of which bring in the entire APC ligase . The first scenario is that the PHOSphorylated Smad3 , released from SARA , forms a complex with APC10 , while CDH1 interacts with HEF1 constitutively . The two complexes come together via Smad3 interaction with HEF1 . APC10 assists CDH1 to bring Smad3-bound HEF1 to APC ligase for the subsequent ubiquitination . The second scenario is that HEF1 tethers Smad3 in a cytoplasmic complex and the type I receptor activation leads to phosphorylaTION of Smad3 and the subsequent conformational changes that can enhance its interaction with APC10 , which recruits the APC ligase and assists CDH1 to bind the SUBstrate HEF1 . The third scenario is that monomeric unPHOSphorylated Smad3 and HEF1 forms a constitutive complex to further recruit APC10 and CDH1 . Such a complex may mediate some basal level of constitutive ubiquitination of HEF1 . We recognize that the current observations need to be followed up in more physiological relevant conditions , such that the physiological functions of the observed interactions between these proteins can be validated .