Atrophin-1-interacting Protein 4/Human Itch Is a Ubiquitin E3 Ligase for Human Enhancer of Filamentation 1 in Transforming Growth Factor-β Signaling Pathways
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m403221200
ISSN1083-351X
AutoresLibing Feng, Susana Guedes, Tongwen Wang,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoAtrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-β (TGF-β) signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-β type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-β stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. Here we report our studies that demonstrate the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-β-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-β stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-β signaling pathway and those involving HEF1 and AIP4. Atrophin-1-interacting protein 4 (AIP4) is the human homolog of the mouse Itch protein (hItch), an E3 ligase for Notch and JunB. Human enhancer of filamentation 1 (HEF1) has been implicated in signaling pathways such as those mediated by integrin, T cell receptor, and B cell receptor and functions as a multidomain docking protein. Recent studies suggest that HEF1 is also involved in the transforming growth factor-β (TGF-β) signaling pathways, by interacting with Smad3, a key signal transducer downstream of the TGF-β type I receptor. The interaction of Smad3 with HEF1 induces HEF1 proteasomal degradation, which was further enhanced by TGF-β stimulation. The detailed molecular mechanisms of HEF1 degradation regulated by Smad3 were poorly understood. Here we report our studies that demonstrate the function of AIP4 as an ubiquitin E3 ligase for HEF1. AIP4 forms a complex with both Smad3 and HEF1 through its WW domains in a TGF-β-independent manner and regulates HEF1 ubiquitination and degradation, which can be enhanced by TGF-β stimulation. These findings reveal a new mechanism for Smad3-regulated proteasomal degradation events and also broaden the network of cross-talk between the TGF-β signaling pathway and those involving HEF1 and AIP4. The transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; HEF1, human enhancer of filamentation 1; IOD, integrated optical density; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; AIP4, atrophin-1-interacting protein 4; HECT, homology to E6-AP carboxyl-terminal; Smurf1 and -2, Smad ubiquitination regulatory factor 1 and 2, respectively; HA, hemagglutinin; Ub, ubiquitin; GST, glutathione S-transferase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; LLnL, N-acetyl-l-leucinyl-l-leucinal-l-norleucinal; MG132, Nα-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal; SARA, Smad anchor for receptor activation; hItch, human Itch. signaling is involved in a broad range of cellular functions, including proliferation, adhesion, apoptosis, differentiation, and specification of developmental fate (1Massague J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar, 2Verrecchia F. Mauviel A. J. Invest Dermatol. 2002; 118: 211-215Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). The extracellular signals were transduced to the nucleus by the sequential association of type II and type I receptors and the Smad protein cascades (3Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar, 4Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar). The binding of ligands to the receptors leads to the phosphorylation of Smad2 and Smad3 at their SSXS motif within the COOH termini. The phosphorylated Smad2 or Smad3 forms complexes with Smad4 and translocates into the nucleus, where they function as DNA-binding transcription factors. Recently, Smad3 was discovered to have the novel ability of regulating the proteasomal degradation of the nuclear proto-onco-proteins SnoN and Ski (5Wan Y. Liu X. Kirschner M.W. Mol. Cell. 2001; 8: 1027-1039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Stroschein S.L. Bonni S. Wrana J.L. Luo K. Genes Dev. 2001; 15: 2822-2836Crossref PubMed Google Scholar) as well as the human enhancer of filmentation 1 (HEF1) (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). HEF1 is a member of a multiple domain docking protein Cas family including p130cas and Efs that have been implicated as signaling mediators of diverse processes including cellular attachment, motility, growth factor responses, apoptosis, and oncogenic transformation (8Law S.F. Zhang Y.Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (95) Google Scholar). HEF1 was first isolated in a screen for human proteins with the ability to alter Saccharomyces cerevisiae morphology from round to filamentous hyper-polarized cells (9Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Based upon its homology to p130cas, another group independently isolated HEF1, named Cas-L (10Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). Members of this family share similar domains, with an amino-terminal Src homology 3 domain that binds polyproline-containing protein, a large central domain encompassing multiple tyrosine motifs that are recognized by the Src homology 2 domain protein upon phosphorylation, a serine-rich domain, and a carboxyl-terminal domain containing a helix-loop-helix motif (9Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar, 11Law S.F. Zhang Y.Z. Fashena S.J. Toby G. Estojak J. Golemis E.A. Exp. Cell Res. 1999; 252: 224-235Crossref PubMed Scopus (41) Google Scholar). Earlier studies have showed that HEF1 is predominantly expressed in epithelial cells and lymphocytes, whereas p130cas is abundant in many cell types (8Law S.F. Zhang Y.Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (95) Google Scholar, 10Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). The expression of HEF1 is cell cycle-regulated, with p105HEF1 and p115HEF1 accumulating at the focal adhesion sites when cells go through S and G2 phase, whereas p55HEF1 is specifically produced and localized to the mitotic spindle during mitosis (8Law S.F. Zhang Y.Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (95) Google Scholar). So far, HEF1 has been implicated in integrin, T cell antigen receptor, B cell antigen receptor, and the G-protein coupled calcitonin receptor signaling pathways (10Minegishi M. Tachibana K. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar, 12Astier A. Manie S.N. Avraham H. Hirai H. Law S.F. Zhang Y. Golemis E.A. Fu Y. Druker B.J. Haghayeghi N. Freedman A.S. Avraham S. J. Biol. Chem. 1997; 272: 19719-19724Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Manie S.N. Beck A.R. Astier A. Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Kanda H. Mimura T. Morino N. Hamasaki K. Nakamoto T. Hirai H. Morimoto C. Yazaki Y. Nojima Y. Eur. J. Immunol. 1997; 27: 2113-2117Crossref PubMed Scopus (31) Google Scholar, 15Zhang Z. Hernandez-Lagunas L. Horne W.C. Baron R. J. Biol. Chem. 1999; 274: 25093-25098Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Molecular and genetic studies indicated that HEF1 overexpression leads to an increase in cell motility and apoptosis, consistent with a role of HEF1 in regulating integrin signaling (16Fashena S.J. Einarson M.B. O'Neill G.M. Patriotis C. Golemis E.A. J. Cell Sci. 2002; 115: 99-111PubMed Google Scholar, 17van Seventer G.A. Salmen H.J. Law S.F. O'Neill G.M. Mullen M.M. Franz A.M. Kanner S.B. Golemis E.A. van Seventer J.M. Eur. J. Immunol. 2001; 31: 1417-1427Crossref PubMed Scopus (76) Google Scholar, 18Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar, 19Kamiguchi K. Tachibana K. Iwata S. Ohashi Y. Morimoto C. J. Immunol. 1999; 163: 563-568PubMed Google Scholar). Protein ubiquitination is the type of post-translational modification in which a highly conserved 76-amino acid polypeptide, ubiquitin, is attached to proteins. A cascade of three enzymes mediates this process, the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzymes and the E3 ubiquitin ligase. E1 activates ubiquitin by generating a high energy E1-thiol ester-ubquitin intermediate. E2s transfer the activated ubiquitin to the cysteine residue on E3 before conjugating ubiquitin to the target proteins (20Hartmann-Petersen R. Seeger M. Gordon C. Trends Biochem. Sci. 2003; 28: 26-31Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Ubiquitin E3 ligase plays a vital role in substrate recognition specificity and subsequent protein degradation by the 26 S proteasome, which is a large multisubunit proteolytic complex. There are three major types of E3 ligase: homology to E6-AP carboxyl-terminal (HECT) domain E3s, RING finger motif-containing E3s, and E4/U box-containing proteins. HEF1 was found to be involved in the TGF-β signaling pathway. It was isolated as a Smad3-specific interactor in a yeast two-hybrid screen using Smad3 as bait (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). Further studies indicate that Smad3 interaction with HEF1 enhances HEF1 degradation in a proteasome-dependent fashion and that the activation of TGF-β signaling further enhances HEF1 degradation (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). In epithelial cell lines and a T lymphoid cell line (H9), HEF1 protein level was demonstrated to undergo rapid reduction in responding to TGF-β stimulation followed by a negative feedback-type increase of HEF1 mRNA (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). Zheng and McKeown-Longo (21Zheng M. McKeown-Longo P.J. J. Biol. Chem. 2002; 277: 39599-39608Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) also reported that TGF-β and cell adhesion regulate HEF1 expression and phosphorylation in dermal fibroblasts, adding more evidence to the functional link between TGF-β signaling pathways and HEF1 protein expression. Such a link provides a new molecular mechanistic explanation for the ability of TGF-β to regulate myriad biological functions through cross-talk to many of the HEF1-involved signaling pathways. However, the exact nature of the TGF-β signaling events associated with HEF1 degradation is still not fully understood, and the detailed molecular mechanisms of HEF1 degradation also need to be uncovered. Here we report the biochemical studies of the physical and functional interaction between HEF1 and atrophin-1-interacting protein 4 (AIP4), which is the human homolog of the mice Itch protein (hItch) (22Perry W.L. Hustad C.M. Swing D.A. O'Sullivan T.N. Jenkins N.A. Copeland N.G. Nat. Genet. 1998; 18: 143-146Crossref PubMed Scopus (284) Google Scholar), an HECT family E3 ligase for Notch and JunB (23Qiu L. Joazeiro C. Fang N. Wang H.Y. Elly C. Altman Y. Fang D. Hunter T. Liu Y.C. J. Biol. Chem. 2000; 275: 35734-35737Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 24Fang D. Elly C. Gao B. Fang N. Altman Y. Joazeiro C. Hunter T. Copeland N. Jenkins N. Liu Y.C. Nat. Immunol. 2002; 3: 281-287Crossref PubMed Scopus (295) Google Scholar). Lack of the Itch protein in non-agouti mice contributes to the autoimmune phenotypes of the Itch mice (22Perry W.L. Hustad C.M. Swing D.A. O'Sullivan T.N. Jenkins N.A. Copeland N.G. Nat. Genet. 1998; 18: 143-146Crossref PubMed Scopus (284) Google Scholar). AIP4 was originally cloned as an interactor of atrophin-1, the protein implicated in the neurodegenerative disease dentatorubral pallidoluysian atrophy (25Margolis R.L. Li S.H. Young W.S. Wagster M.V. Stine O.C. Kidwai A.S. Ashworth R.G. Ross C.A. Brain Res. Mol. Brain Res. 1996; 36: 219-226Crossref PubMed Scopus (36) Google Scholar, 26Wood J.D. Yuan J. Margolis R.L. Colomer V. Duan K. Kushi J. Kaminsky Z. Kleiderlein J.J. Sharp A.H. Ross C.A. Mol. Cell Neurosci. 1998; 11: 149-160Crossref PubMed Scopus (139) Google Scholar). Its protein sequence suggests that it belongs to the C2-WW subfamily within the HECT domain-containing E3 ligase family. The C2-WW subfamily is characterized with a calcium-dependent phospholipid-binding domain (or C2 domain) at the NH2 terminus followed by 2-4 WW domains and then the COOH-terminal HECT domain. The HECT domain is a ∼350-residue region that harbors a strictly conserved cysteine residue that forms an essential thiol ester intermediate during catalysis at the COOH terminus. Two known C2-WW subfamily members are Smad ubiquitination regulatory factor 1 (Smurf1) and Smad ubiquitination regulatory factor 2 (Smurf2), both of which function as constitutive E3 ligases for the bone morphogenetic protein pathway-restricted Smads (Smad1 and Smad5) and TGF-β pathway-restricted Smads (Smad2 and Smad3), respectively (27Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (693) Google Scholar, 28Bonni S. Wang H.R. Causing C.G. Kavsak P. Stroschein S.L. Luo K. Wrana J.L. Nat. Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (273) Google Scholar). In addition, Smurf2, when bound to Smad7, is recruited to the activated TGF-β receptors to form a complex in which Smurf2 ubiquitinates Smad7 (29Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). In response to TGF-β stimulation, Smurf2 can be recruited by Smad2/Smad3 to form a complex with and ubiquitinate SnoN (28Bonni S. Wang H.R. Causing C.G. Kavsak P. Stroschein S.L. Luo K. Wrana J.L. Nat. Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (273) Google Scholar). The later observation suggests that E3 ligases not only mediate the ubiquitination of Smads but also can be recruited by Smads to mediate the ubiquitination of Smad-interacting proteins. AIP4 was first isolated as an interactor of Smad3 but does not mediate the ubiquitination of Smad3. 2S. Guedes, J. Farley, X. Liu, and T. Wang, manuscript in preparation. Recently, it has been demonstrated that AIP4 does mediate the ubiquitination of atrophin-1 as well as the two scaffold proteins MAGI-1 and GAGI-2 that were previously shown to bind atrophin-1. 3J. D. Wood, Z. A. Keminsky, Y. Kim, S. Guedes, T. Wang, and C. A. Ross, submitted for publication. Since Smad3 interacts with HEF1 and regulates HEF1 degradation, we tested the possibility of AIP4 to be recruited by Smad3 to mediate the ubiquitination of HEF1. Our results show that AIP4 is an E3 ligase for HEF1 and, together with Smad3, regulates proteasomal degradation of HEF1. Antibodies and Reagents—Anti-Myc (9E10) and anti-HEF1 (N-17) were purchased from Santa Cruz Biotechnology. Anti-p130cas monoclonal antibody was purchased from Transduction Laboratories. Anti-T7 (69522-4) was purchased from Novagen. Anti-FLAG was obtained from Sigma, and anti-HA was purchased from Roche Applied Science. Nα-Benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (MG132) (c2211), N- acetyl-l-leucinyl-l-leucinal-l-norleucinal (LLnL) (A6185), phosphatase inhibitor (P5726), protease inhibitor mixture (P8340), and ubiquitin (U6253) were all purchased from Sigma. Cycloheximide (100183) was purchased from ICN Biomedicals Inc. MG132 and LLnL were dissolved in Me2SO and added directly into cell culture medium to a final concentration of 50 μm for 5 h before harvest. Constructs—The construction of full-length Myc-AIP4 has been described previously. 3J. D. Wood, Z. A. Keminsky, Y. Kim, S. Guedes, T. Wang, and C. A. Ross, submitted for publication. The pCMV-HEF1 expression vector has been described previously (8Law S.F. Zhang Y.Z. Klein-Szanto A.J.P. Golemis E.A. Mol. Cell. Biol. 1998; 18: 3540-3551Crossref PubMed Scopus (95) Google Scholar). All of the other mammalian expression constructs for HEF1 were constructed in our laboratory previously (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). Smad3 and HEF1 were subcloned into EcoRI/XhoI sites, and AIP4 was suncloned with BamHI and NotI sites in pGEX-5X-1 (Amersham Biosciences) using standard procedures (30.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, pp. 3.16, 9.1, and 10.8, New York, NYGoogle Scholar). Smad3 and HEF1 were subcloned into EcoRI/XhoI sites, and AIP4WT/CA were subcloned into SalI/NotI in pCS2+ vector containing a SP6 promoter for in vitro translation. Myc-Smurf1 and Myc-Smurf2 constructs were obtained from the laboratory of J. Wrana. The HA-Ub construct was a kind gift from Dr. M. Treier. Mammalian Cell Line—293 cells (human kidney cells transformed with adenovirus 5 DNA) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 25,000 units of penicillin, 25 mg of streptomycin, and 5 ml of 200 mm l-glutamine at 37 °C in the presence of 5% CO2. Mink lung epithelial cells (Mv1Lu or ML) were cultured in the same medium and under the same conditions as description above except using active fetal bovine serum. Transfection—293 cells were transfected using the standard CaPO4 procedure (30.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, pp. 3.16, 9.1, and 10.8, New York, NYGoogle Scholar), and cells were harvested 24 h after transfection. Immunoprecipitation and Western Blotting—Cells were incubated 30 min on ice with Hepes-buffered saline-lysis buffer (50 mm HEPES, 5 mm EDTA, 50 mm NaCl, 1% Triton X-100 supplemented with protease and phosphatase inhibitors just prior to use). Cell debris was pelleted by spinning in a microcentrifuge at 14,000 × g at 4 °C for 10 min, and supernatant was saved for immunoprecipitation and Western blot analysis. For immunoprecipitation, cell lysates were incubated with 2 μg of primary antibody for 2 h at 4 °C followed by an additional 2-h incubation with 40 μl of a 50% slurry of protein G-Sepharose 4 Fast Flow (Amersham Biosciences). Beads were then washed once using lysis buffer and three times with modified lysis buffer (lysis buffer containing 0.1% Triton X-100). The precipitated proteins were eluted in 2× SDS loading buffer (100 mm Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, and 20% glycerol) plus 10% β-mercaptoethanol, loaded on SDS-PAGE, and transferred onto polyvinylidene difluoride membrane (Millipore Corp.). Membranes were analyzed by Western blot (30.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, pp. 3.16, 9.1, and 10.8, New York, NYGoogle Scholar). Antibodies were diluted as follows: α-HEF1 (1:1000), α-p130cas (1:1000), α-FLAG (1:4000), α-T7 (1:10,000), α-Myc (1:1000), α-HA (1:2000). GST Pull-down Assay—GST-AIP4 and GST-HEF1 were expressed and purified from Escherichia coli strain BL-21. Briefly, the culture was induced at OD ∼0.6 with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 2-3 h. Cell were collected by spinning at 5,000 rpm at 4 °C for ∼15 min. The pellet was then resuspended in Prep buffer (100 mm NaCl, 100 mm Tris-HCl, pH 8.0, 50 mm EDTA, 2% Triton X-100) supplemented with 2 mm dithiothreitol and 1 mm phenylmethylsulfonyl fluoride. Lysis occurred using 10 mg/ml lysozyme (catalog no. BP 535-1; Fisher) in Prep buffer for 30 min on ice, and debris was spun down by spinning 30 min at 4,000 × g at 4 °C. Cell lysates were then incubated with a 50% slurry of glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences) for 1 h at 4 °C and washed three times with ice-cold PBS. About 4 μg of GST fusion proteins that were immobilized on beads were incubated with extracts in lysis buffer, washed three times with modified lysis buffer, and resuspended in SDS loading buffer. To test a direct protein-protein interaction, proteins were translated in vitro and 35S-labeled by using the TNT reticulocyte lysate system (Promega). The in vitro translated product (8 μl) was incubated with the GST beads in 200 μl of modified lysis buffer supplemented with protease inhibitors and washed as previously described. Yeast Two-hybrid Tests—Protein-protein interaction tests using the yeast two-hybrid system were carried out as described before (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). Briefly, full-length HEF1 was subcloned into the bait construct pEG202 and fused in frame with the DNA binding domain LexA. AIP4 and its deletions were subcloned into the prey construct pJG4-5 and fused in frame with the transcriptional activation domain B42. Yeast strain EGY48 was transformed with the bait construct first and then transformed with the prey construct. The interaction was monitored on glucose plate and galactose plate supplemented with X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Protein Degradation Assays—Mink lung epithelial cell (Mv1 Lu or ML) extracts were made as described before (5Wan Y. Liu X. Kirschner M.W. Mol. Cell. 2001; 8: 1027-1039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Briefly, mink lung epithelial cells were cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in 5% CO2. To stimulate cells with TGF-β, they were treated with 100 pm TGF-β (a gift from Anita Roberts). After 1 h, cells were washed with phosphate-buffered saline and harvested by scraping. Approximately 1 × 108 harvested cells were resuspended in 500 μl of hypotonic buffer (20 mm HEPES (pH 7.5), 5 mm KCl, 1.5 mm MgCl2, 1 mm dithiothreitol), 1× protease mixture (Roche Applied Science), and an energy regeneration mixture (31Murray A.W. Methods Cell Biol. 1991; 36: 581-605Crossref PubMed Scopus (805) Google Scholar) for 30 min to allow cells to swell. Cells were frozen by liquid nitrogen, thawed in a 37 °C water bath, and homogenized with 10 strokes using a Dounce homogenizer. Cell lysates were spun in an Eppendorf microcentrifuge at 14,000 rpm at 4 °C for 1 h. The clear supernatant was collected using a syringe needle and used directly for protein degradation assays. For protein degradation assays, ∼10 ng of 35S-labeled HEF1 synthesized in the TNT expression system and 8 ng of unlabeled in vitro translated Smad3 or AIP4CA or AIP4 were added to 20 μl of fresh Mv1Lu extracts supplemented with the degradation mixture (1.25 mg/ml ubiquitin, 1× energy regeneration, and 0.1 mg/ml cycloheximide). Aliquots were removed at different times and resolved by SDS-PAGE and autoradiography. AIP4 Interacts Directly with HEF1—Our previous studies have suggested that the Cas family multidomain docking protein HEF1 was subjected to rapid proteasomal degradation upon its interaction with the key signal transducer of the TGF-β pathway, Smad3. It was shown that the interaction between Smad3 and HEF1 via their amino-terminal domains is important for HEF1 degradation (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). However, it is not clear how Smad3 interaction with HEF1 can lead to HEF1 degradation by proteasome. The HECT family E3 ligase AIP4 was isolated together with HEF1 from the yeast two-hybrid system as a strong and specific interactor of Smad3 (7Liu X. Elia A.E. Law S.F. Golemis E.A. Farley J. Wang T. EMBO J. 2000; 19: 6759-6769Crossref PubMed Scopus (64) Google Scholar). Initially, we tested whether AIP4 is an E3 ligase for Smad3. However, unlike Smurf1 or Smurf2, AIP4 does not ubiquitinate Smad3. 2S. Guedes, J. Farley, X. Liu, and T. Wang, manuscript in preparation. Thus, we tested whether AIP4 mediates HEF1 ubiquitination by interacting with Smad3 and HEF1. The interaction between AIP4 and HEF1 was first tested in the yeast two-hybrid system. The full-length HEF1 was cloned into pEG202 to generate a LexA fusion protein. A truncated version of AIP4, AIP4Δ65, fused with the transcription activation domain B42, was obtained directly from the screen using Smad3 as bait. AIP4Δ65 contains four WW domains and part of HECT domain. After both constructs were made and transformed into the yeast, the transformants were selected on a glucose U-H-W- plate. The transformants were then spotted onto a galactose/raffinose-X-gal plate, which allows the tested clone to turn blue within 24 h if an interaction occurs between the two fusion proteins. The same transformants were also spotted onto a control plate of glucose/X-gal, which allows the tested clone to remain white due to the repression of the expression of the B42 fusion protein by glucose. As shown in Fig. 1A, the yeast clone containing LexA-HEF1 and B42-AIP4Δ65 turned strong blue on the galactose/raffinose X-gal plate but remained white on glucose X-gal plate, suggesting strong and specific interaction between these two proteins. To further confirm the interaction observed in the yeast two-hybrid system, we carried out the GST pull-down assays to test the interaction between HEF1 and AIP4 in vitro. FLAG-tagged Smad3 and Myc-tagged AIP4 were transiently expressed in the 293 cells respectively or together. Cell lysates were made and tested for the expression of both proteins (Fig. 1B, lanes 1-3) and incubated with GST bead-bound GST-HEF1 expressed and purified from E. coli BL21. After eluting the bound proteins from the beads, Western blot was performed to detect Myc-AIP4 and FLAG-Smad3 using anti-Myc antibody and anti-FLAG antibody, respectively (Fig. 1B, lanes 4-6). Both Myc-AIP4 and FLAG-Smad3, respectively, were found to bind HEF1 (Fig. 1B, lanes 4 and 5). When both Myc-AIP4 and FLAG-Smad3 were co-expressed, both proteins were detected to bind GST-HEF1 (Fig. 1B, lane 6). The FLAG-Smad3 band was even more intense than that detected when FLAG-Smad3 alone was incubated with GST-HEF1 (Fig. 1B, lane 6 compared with lane 5), suggesting that there is no competition between Smad3 and AIP4 to bind HEF1, but instead the interaction appears to be simultaneous or even cooperative. The specificity of such interactions was further tested by including HEF1 itself in the lysates (Fig. 1C). Excess of HEF1 expression together with FLAG-Smad3 and Myc-AIP4 totally blocked both of these proteins from binding to GST-HEF1 (Fig. 1C, compare lanes 5 and 6 with lanes 7 and 8). The above in vitro pull-down assay demonstrates that both AIP4 and Smad3 can form a complex, possibly a ternary complex with HEF1. We then tested via in vitro binding assay whether the interaction between AIP4 and HEF1 or between AIP4 and Smad3 is direct. 35S-labeled in vitro translated HEF1 or Smad3 proteins (Fig. 1D, lanes 1 and 2) were incubated with GST-AIP4 (Fig. 1D, lanes 3 and 4). GST alone was use as a negative control (lanes 5 and 6). 35S-Labeled Smad3 and HEF1 were found to bind GST-AIP4 (Fig. 1D, lanes 3 and 4). A separate study has shown that HEF1 interacts with Smad3 directly. 4Nourry, C., Maksumova, L., Liu, X., and Wang, T. (2004) BMC Cell Biol. 5, 20. These data pointed out that HEF1, AIP4, and Smad3 perform mutual direct interaction. Next we tested whether AIP4 and HEF1 interact in mammalian cells. Myc-tagged full-length AIP4 protein was transiently co-expressed with HEF1 protein in the 293 cells. HEF1 protein was immunoprecipitated with anti-p130cas antibody, and the co-precipitated Myc-tagged AIP4 was detected by Western blot using anti-Myc antibody. Myc-AIP4 was detected to co-precipitate with HEF1 (Fig. 1E, lane 4). A conserved cysteine residue in the HECT domain of AIP4 (C830) is considered to form an essential thiol ester intermediate during catalysis. To determine whether the ligase activity of AIP4 regulates the interaction, as shown to be the case for the Smurfs (27Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (693) Google Scholar), we mutated this conserved cysteine and created the ligase-dead mutant AIP4 (C830A) and tested its interaction with HEF1. No change was detected; thus, the ligase activity of AIP4 does not affect AIP4 binding to HEF1 (Fig. 1E, lane 5). The very weak band of AIP4 detected in lane 3 most likely represents the small amount of Myc-AIP4 co-precipitated with the endogenous HEF1. HEF1 Interacts with AIP4 through Its Carboxyl Terminus and Requires at Least Two WW Domain
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