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Division of Molecular Cytology, Institute for Enzyme Research, University of Tokushima, 3-18-15, Kuramoto, Tokushima, 770-8503, Japan
1 Harima Institute at Spring-8, RIKEN, Mikazuki, Sayo, Hyogo, 679-5148, Japan
2 Division of Pharmacy, Tokushima University Hospital, 2-50-1, Kuramoto, Tokushima, 770-8503, Japan
(Requests for offprints should be addressed to A Kurisaki; Email: akikuri{at}hotmail.com)
(T Yamaguchi is now at Division of Pharmacy, Ehime University Hospital, Shitsukawa, Toon, 791-0295, Ehime, Japan)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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FKBP12 is a cytoplasmic protein that binds to the immunosuppressant drugs, Tacrolimus (FK506) and rapamycin, with high affinity (Schreiber 1991, Snyder & Sabatini 1995). FKBP12 has been demonstrated to interact with the two major intracellular calcium channels, ryanodine receptor and inositol 1,4,5-triphosphate receptor (IP3R) as a physiological subunit of the channel protein complexes (Jayaraman et al. 1992, Cameron et al. 1995, Schiene-Fischer & Yu 2001). Dissociation of FKBP12 from these channels influences physiological calcium flux from the channels (Steinmann et al. 1991, Clipstone & Crabtree 1992). In addition, FKBP12 plays a role as an anchor for the calcium-activated phosphatase, calcineurin, to the tri-complex with IP3R and modulates the phosphorylation state of the receptor (Cameron et al. 1997).
FKBP12 also regulates TGF-ß superfamily signal transduction (Wang et al. 1994, 1996, Okadome et al. 1996, Chen et al. 1997, Wang & Donahoe 2004). FKBP12 interacts with the type I receptors of the TGF-ß family members, including BMP and activin (Wang et al. 1996, Kurozumi et al. 1998). FKBP12 binds to the GS domain in the cytoplasmic region of TGF-ß type I receptor and represses the signal. Overexpression of FKBP12 strongly inhibits TGF-ß signal in Mv1 Lu cells. On the other hand, the dissociation of FKBP12 from the type I receptor by FK506 derivatives enhances the TGF-ß signal. Indeed, upon ligand stimulation, phosphorylation of serine residues of the GS domain of type I receptors by TGF-ß type II receptor dissociates FKBP12 from the type I receptors to fully propagate the signals (Huse et al. 2001). These findings indicate that FKBP12 functions as a secure switch for the leaky signal in the absence of ligands. However, there are several contradictory reports. Physiological studies in FKBP12-deficient mice demonstrated that FKBP12 is dispensable for TGF-ß-mediated signaling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors (Shou et al. 1998). The biochemical analysis of mouse primary embryonic fibroblasts and thymocytes from the gene targeting mice failed to show any unique physiological role of FKBP12 in TGF-ß signaling (Bassing et al. 1998, Shou et al. 1998). The discrepancy between these reports may be explained by the existence of the related protein, FKBP12.6, and the other FKBP proteins (Gothel & Marahiel 1999).
In the present study, we investigated the roles of FKBP12 on activin signaling. We have shown that FKBP12 transiently dissociates from activin type I receptor upon ligand stimulation, but a few hours later FKBP12 associates again with the receptor. We have demonstrated that FKBP12 forms a complex with Smad7 on the activated type I receptor, and recruits the E3 ubiquitin ligase, Smurf1, to the receptor to promote its ubiquitination. Our data suggest a novel inhibitory function of FKBP12 as the adaptor molecule of the receptor in activin signaling.
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Materials and methods |
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Chinese hamster ovary (CHO) cells were cultured in 
-modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin at 37 °C, in a 5% CO2 atmosphere. Human embryonic kidney 293 (HEK293) cells and 293T cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS L-glutamine, and penicillin/streptomycin.
Plasmid constructions
The entire coding region of human FKBP12 was amplified by PCR, and subcloned into N-terminally tagged vector, pcDNA3-Flag or pcDNA3–6 myc. The mutant derivative of FKBP12 was generated by PCR with mismatch sense and antisense primers. The products were verified by DNA sequencing. Human constitutive active ALK4 (caALK4) was subcloned into pcDNA3 containing five repeats of the myc epitope sequence. N-terminally Flag-tagged human Smad6 and Smad7 in pcDNA3 were constructed by a similar PCR-based approach. For mammalian two-hybrid interaction assays, pBIND–FKBP12, pACT–ALK4, and pACT–Smad7 were constructed by subcloning of the full-length cDNAs into pBIND vector, which contains the yeast GAL4 DNA binding domain upsteam of a multiple cloning region or pACT vector, which contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region (Promega). The plasmids for the bacterial expression of glutathione-S-transferase (GST)–Smad7, GST–Smad7N (2–261 amino acids) and GST–Smad7C (204–426 amino acids) were generous gifts from Dr J Ericsson (Gronroos et al. 2002). The GST-fusion proteins were expressed in an Escherichia coli strain, BL21 (DE3) and purified on glutathione sepharose beads (Amersham).
Cell transfections and luciferase assays
Transient transfection was performed with Transfast (Promega) according to the manufacturer’s protocols. Transfection was also performed with calcium phosphate precipitation as previously described (Kurisaki et al. 2003). For luciferase assays, CHO cells were seeded in 24-well plates and transfected with the Transfast reagent (Promega). At 24 h after transfection, the cells were treated with 50 ng/ml activin A and/or 250 nM FK506 (Fujisawa Pharmaceutical Co., Tokyo, Japan) for 24 h, and then extracted in a lysis buffer (25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, and 1% Triton X-100). The luciferase activity was measured and normalized to the ß-galactosidase activity, as described previously (Shoji et al. 2000).
Mammalian two-hybrid assay
The mammalian two-hybrid assay was performed using the CheckMate Mammalian Two-Hybrid System (Promega) according to the manufacturer’s protocol. In brief, CHO cells were transfected with the plasmids of interest, a cytomegalovirus promoter-driven ß-gal (CMV-ß-gal) and a reporter plasmid, pG5 luc, which drives the luciferase gene under the control of the GAL4-responsive promoter. Luciferase activity was measured and normalized to the ß-galactosidase activity as described previously (Tsuchida et al. 1995, Shoji et al. 2000).
Immunoprecipitation and immunoblotting
HEK293 cells transfected with plasmids were harvested in lysis buffer A (20 mM Tris–HCl, pH7.4, 150 mM NaCl, 1% (w/v) Nonidet P-40, 1% (v/v) aprotinin, and 1 mM phenylmethylsulfonyl fluoride) at 24 h after transfection. After centrifugation, the lysates were incubated with first antibodies at 4 °C overnight, and then incubated with protein G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). For His-tagged ALK4, cell lysates were incubated with Ni-NTA agarose (Invitrogen) at 4 °C for 2 h. The precipitated beads were washed with the lysis buffer extensively, and subjected to SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes, and blotted with first antibodies followed by incubation with horseradish peroxidase-conjugated second antibodies. Labeled proteins were detected by chemiluminescence (ECL or ECL-Plus; Amersham). The antibodies used for immunoprecipitation and immunoblotting were as follows: mouse monoclonal antibodies including anti-myc (9E11; Neo Markers), anti-Flag (M2; Sigma), anti-HA (12CA5; Roche), and anti-His (Penta his; QIAGEN).
In vitro interaction assays
For in vitro interaction studies, HEK293T cells were transfected with Flag-FKBP12, wild-type (wt) ALK4-His or 5 myc-caALK4. Cells were lysed in lysis buffer A, and incubated with purified GST–Smad7 fusion proteins immobilized on glutathione beads at 4 °C for 2 h. The beads were washed with the lysis buffer and then subjected to SDS-PAGE followed by western blotting as described above.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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FKBP12 functions as a secure switch for TGF-ß signals, and has been shown to be released from type I receptors upon ligand stimulation (Chen et al. 1997, Huse et al. 2001). We investigated this interaction over a long-term period. To do this, 293T cells were transfected with Flag-FKBP12, activin type II receptor (ActRIIA), and His-tagged activin type I receptor (His-ALK4). The cells were stimulated with activin for various time-periods, and FKBP12 bound to ALK4 was analysed by pull-down assays with Ni-NT agarose beads (Fig. 1
). FKBP12 was gradually dissociated from ALK4 up to 1 h after stimulation with activin. However, the amount of FKBP12 bound to ALK4 was increased later. At 6 h after ligand treatment, a similar amount of FKBP12 bound to the receptor was observed as that prior to stimulation. This observation raised the possibility that FKBP12 works not only as a secure switch prior to ligand stimulation but also controls the signaling after long-term stimulation with activin.
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To confirm the inhibitory effect of FKBP12 on activin signaling, we performed reporter assays using an activin-responsive luciferase reporter, p3TP-Lux, in CHO cells. Activin induced a 3TP-Lux transcriptional response in CHO cells (Fig. 2A). Overexpression of FKBP12 suppressed activin-induced luciferase activity in a dose-dependent manner. Under these conditions, the endogenous mRNA levels of receptors were not affected (data not shown). Treatment of cells with FK506, a chemical ligand for FKBP12 that can release FKBP12 from the type I receptor in TGF-ß signaling, enhanced the luciferase activity 1.5-fold in the absence of activin (data not shown), as previously demonstrated in TGF-ß signaling (Wang et al. 1996). These results indicated that endogenous FKBP12 functions as a secure switch in activin signaling in the absence of ligand. On the other hand, in the presence of activin, the increase in the luciferase activities by FK506 was not dramatic, but still a substantial effect of this drug (1.1-fold) was observed (data not shown). These results further support the idea that FKBP12 also has an inhibitory effect on activin signaling in the presence of ligand.
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Since Smad7 also inhibits activin signaling, we next analysed the inhibitory effect of FKBP12 and Smad7 on activin signaling. CHO cells transfected with FKBP12 and Smad6 or Smad7 were subjected to the luciferase assay. FKBP12 alone repressed the luciferase activity induced by activin in a dose-dependent manner (Fig. 2B
). Overexpression of Smad7 inhibited the activin signal as reported previously (Liu et al. 2002). When FKBP12 was coexpressed with Smad7, FKBP12 further repressed the signal in a dose-dependent manner. Interestingly, this co-operative effect of FKBP12 with Smad7 was not observed when Smad6 was coexpressed with FKBP12.
FKBP12 physically interacts with Smad7 in the presence of activin
In order to explain this result, we hypothesized that FKBP12 may physically interact with Smad7 and repress activin signaling co-operatively. To test this possibility, we analysed the interaction with a mammalian two-hybrid assay. When CHO cells were transfected with Gal4-FKBP12, VP16-ALK4, and the Gal4-luciferase reporter, the luciferase activity was 9-fold higher than with Gal4-FKBP12 alone, indicating that interaction between FKBP12 and ALK4 was detectable with this mammalian two-hybrid system (Fig. 3A). When Smad7 was coexpressed with FKBP12, the luciferase activity was six times higher than with FKBP12 alone. However, coexpression of Smad6 with FKBP12 gave much weaker activity, suggesting that FKBP12 interacts more strongly with Smad7 than with Smad6. This interaction was further confirmed by a coimmunoprecipitation assay. HEK293 cells transfected with 6 myc-FKBP12 and Flag-Smad7 were lysed, and subjected to immunoprecipitation analysis as in Fig. 3B. Smad7 bound to FKBP12 was only detected after cells were stimulated with activin, suggesting that FKBP12 functions in a complex with Smad7 after activation of the signal.
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In order to define the responsible domain of this interaction, we performed an in vitro interaction assay with GST-Smad7 deletion mutants (Fig. 4A). HEK293 cells transfected with Flag-FKBP12 were lysed and subjected to the pull-down assay with GST-Smad7 deletion mutants. FKBP12 interacted with full-length Smad7 and weakly interacted with Smad7N but not with Smad7C (Fig. 4B, top panel). These interactions were remarkably enhanced when cells were activated by coexpression with caALK4 mutant but not transfected with wt ALK4 (Fig. 4B, middle and lower panels). This mutated (T206E) receptor, caALK4, shows constitutively high kinase activity and can activate the downstream signaling pathway in an activin-independent manner (Willis et al. 1996). Since Smad7 is quickly induced after ligand stimulation and associates with the type I receptors to inhibit the signal, our results raise the possibility that FKBP12 bound again to ALK4 after prolonged activin treatment may function to recruit Smad7 to the receptor. To verify this hypothesis, we investigated the effect of FKBP12 on the association of Smad7 to ALK4. We repeated the GST pull-down assay with the HEK293 cell lysate that overexpressed 5 myc-caALK4 with or without FKBP12. ALK4 associated with the full-length and the N-terminal part of Smad7 (Fig. 4C, top panel). When FKBP12 was overexpressed, ALK4 bound to the full-length Smad7 and Smad7N was remarkably increased (Fig. 4C, middle panel). ALK4 also bound to the C-terminal Smad7. However, treatment with FK506, which dissociates FKBP12 from ALK4, dramatically decreased the interaction of ALK4 with full-length Smad7 and Smad7N, although ALK4 still bound to Smad7C (Fig. 4C, bottom panel). These results indicated that FKBP12 is able to enhance the association between ALK4 and Smad7 by the additional interaction of FKBP12 to the N-terminal of Smad7.
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Smad7 is known to recruit Smurf1, an E3 ubiquitin ligase, to the type I receptor to promote ubiquitination of the receptor (Kavsak et al. 2000, Ebisawa et al. 2001). Since we showed that FKBP12 co-operates with Smad7 to inhibit the signaling, and enhances the association of Smad7 with ALK4, we next investigated whether FKBP12 affects the complex formation between Smad7, Smurf1, and ALK4. To do this, FKBP12 and Smurf1 bound to Smad7 were analysed by coimmunoprecipitation assay with 293T cells transfected with FKBP12, Smurf1, Smad7, and caALK4. Since overexpression of caALK4 activates the cellular activin-signaling pathway for a long period, this generates similar conditions as the later time-points described in Fig. 1. As shown in Fig. 5A, the interaction between Smad7 and Smurf1 was not clearly affected by the overexpression of FKBP12, although FKBP12 was clearly associated with Smad7 (Fig. 5A, panel a, lane 4). However, treatment of the cells with 250 nM FK506 for 4 h before harvest released FKBP12 from ALK4, and decreased the quantity of Smurf1 bound to Smad7 (Fig. 5A, panel a, lane 5). Under these conditions, Smad7 was dissociated from ALK4 (Fig. 5A, panel d, lane 5). These results indicated that FKBP12 is an important factor for the assembly of Smurf1, Smad7, and ALK4. We also immunoprecipitated ALK4 and analyzed the amount of Smurf1, Smad7, and FKBP12 bound to ALK4. The amount of Smad7 and Smurf1 bound to ALK4 was remarkably increased by the transfection of FKBP12 (Fig. 5B, lane 3). This interaction was weakened by the addition of FK506 (Fig. 5B, lane 4). These results indicated that FKBP12 is an important factor for the formation of these complexes on the type I receptor. A point mutated FKBP12 (F36Y) lacks peptidyl-prolyl isomerase activity (Wiederrecht et al. 1992). This mutant bound fairly weakly with Smad7. Overexpression of these mutants did not affect the interaction between Smurf1 and Smad7 (Fig. 5A, lane 6).
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Finally, we tested whether FKBP12 affects the ubiquitination of ALK4 induced by Smad7–Smurf1 complexes. HEK293T cells were transfected with FKBP12, Smad7, Smurf1, and ubiquitin together with caALK4, and the ubiquitinated proteins were immunoprecipitated. Ubiquitination of ALK4 was enhanced when the adaptor protein Smad7 and the E3 ligase Smurf1 were overexpressed (Fig. 6, lane 4), as shown in previous studies (Ebisawa et al. 2001). The level of ubiquitinated caALK4 was similar even though FKBP12 was further expressed (Fig. 6, lane 5). However, the release of FKBP12 from ALK4 by the addition of FK506 decreased the ubiquitination of ALK4 (Fig. 6, lane 6). These results support the importance of FKBP12 for the ubiquitination of ALK4 by the Smad7–Smurf1 complex.
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Discussion |
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). This finding is consistent with the phosphorylation status of R-Smads and their localization in the nucleus as previously observed in TGF-ß signaling (Inman et al. 2002). We confirmed that the overexpression of FKBP12 inhibits the activin-dependent transcriptional response (Fig. 2A), and that treatment with FK506, which releases endogenous FKBP12 from the type I receptor, activated the activin signaling.
Inhibitory Smads, Smad7 and Smad6, are the major feedback regulators in TGF-ß superfamily signaling (Hayashi et al. 1997, Nakao et al. 1997, Itoh et al. 1998). Accordingly, we hypothesized that these Smad proteins might participate in the inhibitory functions of FKBP12 on activin signaling after prolonged ligand stimulation. Consequently, we showed that FKBP12 synergistically inhibits activin-dependent transcriptional response with Smad7, but not with Smad6 (Fig. 2B). In addition, we found that FKBP12 associates with Smad7, especially when activin signaling is activated, and FKBP12 strengthens the interaction between Smad7 and ALK4 (Figs 3 and 4). These findings raised the possibility that FKBP12 may play an important role in the recruitment of Smad7 to the type I receptor. Previous studies have demonstrated that the MH2 domain of Smad7 is important for its association with the type I receptors (Hayashi et al. 1997, Souchelnytskyi et al. 1998). However, another study has demonstrated that the N-terminal part of Smad7 does not interact with the TGF-ß type I receptor, but it enhanced the inhibitory activity of the Mad homology domain 2 (MH2) by facilitating the interaction of the MH2 domain with the receptor (Hanyu et al. 2001). Very recently, Ogunjimi et al.(2005) have shown that the N-terminal domain of Smad7 stimulates Smurf activity by recruiting the E2 enzyme, UbcH7, to the HECT domain (homologous to E6-AP carboxyl terminus) of Smurf2. In our in vitro interaction assay, the N-terminal part of Smad7 associated with ALK4 weakly, and the interaction was remarkably enhanced when FKBP12 was overexpressed. We did not observe a clear interaction of Smad7 via its MH2 domain with activated type I receptor under normal conditions, but this interaction was distinctly observed when FKBP12 was released by the addition of FK506 (Fig. 4C). These results suggest that FKBP12 or related proteins might function as a rigid partner of ALK4 in 293T cells, and Smad7 could be easily recruited to the type I receptor through FKBP12. We noticed that FKBP12 also associated with Smad6 in the mammalian two-hybrid assay (Fig. 3A). However, this association was relatively weaker than that of Smad7, and Smad6 did not synergistically inhibit activin signaling with FKBP12 in the luciferase assay because Smad6 mainly inhibits BMP signaling (Hanyu et al. 2001). We therefore focused the further study on the effect of FKBP12 on Smad7.
To further clarify the roles of FKBP12 in the inhibitory mechanism of Smad7 in activin signaling, we also tested the effect of FKBP12 on the recruitment of Smurf1 to ALK4. Interestingly, when FKBP12 was dissociated from ALK4 by the addition of FK506, the protein level of Smurf1 bound to Smad7 was decreased (Fig. 5A), indicating that the FKBP12–ALK4 interaction is important for the recruitment of Smurf1 to Smad7. Another important inhibitory mechanism in activin signaling is the ubiquitination of the type I receptor induced by the Smad7–Smurf1 complex. As shown in Fig. 6, dissociation of FKBP12 from ALK4 by FK506 treatment dramatically decreased the ubiquitination of the receptor. This indicates that FKBP12 is an essential factor for the ubiquitination of type I receptor. However, we failed to observe a clear enhancement of the ubiquitination of ALK4 by the overexpression of FKBP12. This might be due to sufficient amounts of endogenous FKBP12 or related proteins expressed in this cell line.
FKBP12 is a peptidyl–prolyl cis-trans isomerase, and recognizes a proline preceded by a leucine or similarly branched hydrophobic residue (Albers 1990, Harrison & Stein 1990). We tested whether the isomerase activity is required for the recruitment of Smad7 and Smurf1 to the receptor by using an FKBP12 mutant, F36Y, which lacks the enzyme activity. However, we could not see any clear effects on the complex formation with the mutants. The isomerase activity of FKBP12 may not be required for the recruitment of Smurf1 to Smad7.
Recent work has revealed the importance of endocytosis in TGF-ß family signaling. Although type I receptor activation occurs at the plasma membrane, downstream signaling through the Smads requires receptor internalization (Hayes et al. 2002, Penheiter et al. 2002, Di Guglielmo et al. 2003). Ligand-induced internalization of TGF-ß receptor complexes occurs through two different internalization routes of the TGF-ß receptors (Di Guglielmo et al. 2003, ten Dijke & Hill 2004). Clathrin-dependent internalization into early endosomes promotes TGF-ß signaling, as Smad anchor for receptor activation (SARA) bound on phosphatidylinositol 3-phosphate-enriched early endosomes presents Smad2 and Smad3 to the activated receptor complex. On the other hand, caveolae-mediated internalization is involved in the degradation of TGF-ß receptors. In caveolae, Smad7–Smurf complexes target TGF-ß receptors for poly-ubiquitination and proteasomal degradation. In this paper, we have shown that FKBP12 forms a complex with Smad7, promotes recruitment of Smurf1, and enhances degradation of ALK4. FKBP12 therefore presumably functions as an adaptor of the Smad7–Smurf1 complex on the receptor in the caveolae-dependent pathway to negatively regulate activin signaling. Since the inhibitory effects of FKBP12 in the activin transcriptional response are similar to that of TGF-ß, FKBP12 may play similar roles in other TGF-ß superfamily signalings.
FKBP12 also functions as a negative regulator of TGF-ß receptor endocytosis. Yao et al.(2000) used a fibroblast cell line expressing the chimeric TGF-ß receptor system and demonstrated that the receptor internalization was enhanced when FKBP12 binding to the chimeric type I receptor was prevented by rapamycin. Interestingly, despite the faster rate of internalization, the rate of ligand degradation was unchanged by rapamycin. We assume that rapamycin enhances not only ligand-dependent endocytosis and subsequent degradation of TGF-ß ligand but also induces disassembly of ubiquitination machinery as we demonstrated with FK506 in Figs 5 and 6. These complex effects of rapamycin might compensate for the degradation of TGF-ß ligand induced by this drug.
In summary, we propose a novel role of FKBP12 in activin signaling as summarized in Fig. 7. FKBP12 functions as an adaptor protein for the efficient recruitment of the Smad7–Smurf1 complex to the activin type I receptor after prolonged activin treatment. FKBP12 seems to be a prerequisite molecule for proper regulation of activin signaling not only as a secure switch but also as an adaptor for Smad7 and Smurf1 to ALK4, which then promotes ubiquitination of the receptor to terminate the signal.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Bassing CH, Shou W, Muir S, Heitman J, Matzuk MM & Wang XF 1998 FKBP12 is not required for the modulation of transforming growth factor beta receptor I signaling activity in embryonic fibroblasts and thymocytes. Cell Growth and Differentiation 9 223–228.[Abstract]
Cameron AM, Steiner JP, Sabatini DM, Kaplin AI, Walensky LD & Snyder SH 1995 Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. PNAS 92 1784–1788.
Cameron AM, Nucifora FC Jr, Fung ET, Livingston DJ, Aldape RA, Ross CA & Snyder SH 1997 FKBP12 binds the inositol 1,4,5-trisphosphate receptor at leucine-proline (1400–1401) and anchors calcineurin to this FK506-like domain. Journal of Biological Chemistry 272 27582–27588.
Chen YG, Liu F & Massagué J 1997 Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO Journal 16 3866–3876.[CrossRef][ISI][Medline]
Clipstone NA & Crabtree GR 1992 Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357 695–697.[CrossRef][Medline]
Di Guglielmo GM, Le Roy C, Goodfellow AF & Wrana JL 2003 Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover. Nature Cell Biology 5 410–421.[CrossRef][ISI][Medline]
ten Dijke P & Hill CS 2004 New insights into TGF-beta-Smad signalling. Trends in Biochemical Science 29 265–273.
Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T & Miyazono K 2001 Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. Journal of Biological Chemistry 276 12477–12480.
Gothel SF & Marahiel MA 1999 Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. Cellular and Molecular Life Science 55 423–436.[CrossRef][ISI][Medline]
Gronroos E, Hellman U, Heldin CH & Ericsson J 2002 Control of Smad7 stability by competition between acetylation and ubiquitination. Molecular Cell 10 483–493.[CrossRef][ISI][Medline]
Hanyu A, Ishidou Y, Ebisawa T, Shimanuki T, Imamura T & Miyazono K 2001 The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling. Journal of Cell Biology 155 1017–1027.
Harrison RK & Stein RL 1990 Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion. Biochemistry 29 1684–1689.[CrossRef][Medline]
Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA Jr, Wrana JL et al. 1997 The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell 89 1165–1173.[CrossRef][ISI][Medline]
Hayes S, Chawla A & Corvera S 2002 TGF beta receptor internalization into EEA1-enriched early endosomes: role in signaling to Smad2. Journal of Cell Biology 158 1239–1249.
Heldin CH, Miyazono K & ten Dijke P 1997 TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390 465–471.[CrossRef][Medline]
Huse M, Muir TW, Xu L, Chen YG, Kuriyan J & Massagué J 2001 The TGF beta receptor activation process: an inhibitor- to substrate-binding switch. Molecular Cell 8 671–682.[CrossRef][ISI][Medline]
Inman GJ, Nicolas FJ & Hill CS 2002 Nucleocytoplasmic shuttling of Smads 2, 3, and 4 permits sensing of TGF-beta receptor activity. Molecular Cell 10 283–294.[CrossRef][ISI][Medline]
Itoh S, Landstrom M, Hermansson A, Itoh F, Heldin CH, Heldin NE & ten Dijke P 1998 Transforming growth factor beta1 induces nuclear export of inhibitory Smad7. Journal of Biological Chemistry 273 29195–29201.
Jayaraman T, Brillantes AM, Timerman AP, Fleischer S, Erdjument-Bromage H, Tempst P & Marks AR 1992 FK506 binding protein associated with the calcium release channel (ryanodine receptor). Journal of Biological Chemistry 267 9474–9477.
Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH & Wrana JL 2000 Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Molecular Cell 6 1365–1375.[CrossRef][ISI][Medline]
Kingsley DM 1994 The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes and Development 8 133–146.
Kurisaki K, Kurisaki A, Valcourt U, Terentiev AA, Pardali K, Ten Dijke P, Heldin CH, Ericsson J & Moustakas A 2003 Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation. Molecular and Cellular Biology 23 4494–4510.
Kurozumi K, Nishita M, Yamaguchi K, Fujita T, Ueno N & Shibuya H 1998 BRAM1, a BMP receptor-associated molecule involved in BMP signalling. Genes to Cells 3 257–264.[Abstract]
Liu X, Nagarajan RP, Vale W & Chen Y 2002 Phosphorylation regulation of the interaction between Smad7 and activin type I receptor. FEBS Letters 519 93–98.[CrossRef][ISI][Medline]
Massagué J 2000 How cells read TGF-beta signals. Nature Reviews in Molecular and Cellular Biology 1 169–178.
Miyazono K, Kusanagi K & Inoue H 2001 Divergence and convergence of TGF-beta/BMP signaling. Journal of Cell Physiology 187 265–276.[CrossRef][ISI][Medline]
Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH et al. 1997 Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature 389 631–635.[CrossRef][Medline]
Ogunjimi AA, Briant DJ, Pece-Barbara N, Le Roy C, Di Guglielmo GM, Kavsak P, Rasmussen RK, Seet BT, Sicheri F & Wrana JL 2005 Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain. Molecular Cell 19 297–308.[CrossRef][ISI][Medline]
Okadome T, Oeda E, Saitoh M, Ichijo H, Moses HL, Miyazono K & Kawabata M 1996 Characterization of the interaction of FKBP12 with the transforming growth factor-beta type I receptor in vivo. Journal of Biological Chemistry 271 21687–21690.
Penheiter SG, Mitchell H, Garamszegi N, Edens M, Dore JJ Jr & Leof EB 2002 Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway. Molecular and Cellular Biology 22 4750–4759.
Phillips DJ 2005 Activins, inhibins and follistatins in the large domestic species. Domestic Animal Endocrinology 28 1–16.[CrossRef][ISI][Medline]
Schiene-Fischer C & Yu C 2001 Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Letters 495 1–6.[CrossRef][ISI][Medline]
Schreiber SL 1991 Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251 283–287.
Shoji H, Tsuchida K, Kishi H, Yamakawa N, Matsuzaki T, Liu Z, Nakamura T & Sugino H 2000 Identification and characterization of a PDZ protein that interacts with activin type II receptors. Journal of Biological Chemistry 275 5485–5492.
Shou W, Aghdasi B, Armstrong DL, Guo Q, Bao S, Charng MJ, Mathews LM, Schneider MD, Hamilton SL & Matzuk MM 1998 Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12. Nature 391 489–492.[CrossRef][Medline]
Snyder SH & Sabatini DM 1995 Immunophilins and the nervous system. Nature Medicine 1 32–37.[CrossRef][ISI][Medline]
Souchelnytskyi S, Nakayama T, Nakao A, Moren A, Heldin CH, Christian JL & ten Dijke P 1998 Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-beta receptors. Journal of Biological Chemistry 273 25364–25370.
Steinmann B, Bruckner P & Superti-Furga A 1991 Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cis-trans-isomerase. Journal of Biological Chemistry 266 1299–1303.
Sugino H & Tsuchida K 2000 Activin and follistatin. In Skeletal Growth Factor, pp 251–263. Ed. E Canalis. Philadelphia: Lippincott Williams & Wilkins.
Tsuchida K 2004 Activins, myostatin and related TGF-beta family members as novel therapeutic targets for endocrine, metabolic and immune disorders. Current Drug Targets. Immune, Endocrine and Metabolic Disorders 4 157–166.[CrossRef]
Tsuchida K, Vaughan JM, Wiater E, Gaddy-Kurten D & Vale WW 1995 Inactivation of activin-dependent transcription by kinase-deficient activin receptors. Endocrinology 136 5493–5503.[Abstract]
Wang T & Donahoe PK 2004 The immunophilin FKBP12: a molecular guardian of the TGF-beta family type I receptors. Frontiers in Bioscience 9 619–631.[ISI][Medline]
Wang T, Donahoe PK & Zervos AS 1994 Specific interaction of type I receptors of the TGF-beta family with the immunophilin FKBP-12. Science 265 674–676.
Wang T, Li BY, Danielson PD, Shah PC, Rockwell S, Lechleider RJ, Martin J, Manganaro T & Donahoe PK 1996 The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors. Cell 86 435–444.[CrossRef][ISI][Medline]
Wiederrecht G, Hung S, Chan HK, Marcy A, Martin M, Calaycay J, Boulton D, Sigal N, Kincaid RL & Siekierka JJ 1992 Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex. Journal of Biological Chemistry 267 21753–21760.
Willis SA, Zimmerman CM, Li LI & Mathews LS 1996 Formation and activation by phosphorylation of activin receptor complexes. Molecular Endocrinology 10 367–379.[Abstract]
Yao D, Dore JJ Jr & Leof EB 2000 FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization. Journal of Biological Chemistry 275 13149–13154.
Received in final form 5 January 2006
Accepted 10 February 2006
Made available online as an Accepted Preprint 14 February 2006
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