WW Domain HECT E3s Target Cbl RING Finger E3s for Proteasomal Degradation
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m308009200
ISSN1083-351X
AutoresAlessandra Magnifico, Seth A. Ettenberg, Cuihong Yang, Jennifer Mariano, Swati Tiwari, Shengyun Fang, Stan Lipkowitz, Allan M. Weissman,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoCbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases. Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases. Specificity in ubiquitylation is conferred by ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; HECT, homologous to E6-AP carboxyl terminus; ENaC, epithelial sodium channel; UIM, Ub-interacting motif; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, EGF receptor; UBA, ubiquitin-associated; GST, glutathione S-transferase; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin protein ligase; HA, hemagglutinin; PR, proline-rich; aa, amino acid(s); MAPK, mitogen-activated protein kinase.1The abbreviations used are: Ub, ubiquitin; HECT, homologous to E6-AP carboxyl terminus; ENaC, epithelial sodium channel; UIM, Ub-interacting motif; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; EGFR, EGF receptor; UBA, ubiquitin-associated; GST, glutathione S-transferase; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin protein ligase; HA, hemagglutinin; PR, proline-rich; aa, amino acid(s); MAPK, mitogen-activated protein kinase.-protein ligases (E3s). These mediate the transfer of Ub from Ub-conjugating enzymes (E2s) to substrates or to a Ub that is already linked to substrate. There are two major classes of E3s: those that are homologous to the E6-AP carboxyl terminus (HECT) and RING finger and RING finger-like E3s (1Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1256) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2896) Google Scholar, 3Patterson, C. (2002) Science's STKE http://stke.sciencemag.org/cgi/reprint/sigtrans;2002/116/pe4.pdf.Google Scholar, 4Coscoy L. Sanchez D.J. Ganem D. J. Cell Biol. 2001; 155: 1265-1273Crossref PubMed Scopus (248) Google Scholar). As part of the ubiquitylation process, HECT E3s form thiol-ester catalytic intermediates with Ub. For RING finger and related E3s, there is no evidence for such intermediates (1Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1256) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2896) Google Scholar). A large number of yeast and metazoan plasma membrane receptors and transporters are substrates for ubiquitylation, which generally leads to their degradation in the yeast vacuole or metazoan lysosome. In Saccharomyces cerevisiae, the HECT domain E3, Rsp5p, is implicated in the ubiquitylation of many membrane proteins (5Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar, 6Hicke L. Nat. Rev. Mol. Cell. Biol. 2001; 2: 195-201Crossref PubMed Scopus (985) Google Scholar). This E3 also contains multiple WW domains in its amino-terminal half that are responsible for substrate binding. WW domains interact with a variety of proline-based motifs, and certain WW domains also recognize proline-containing phosphoserine/phosphothreonine sequences (7Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 8Macias M.J. Wiesner S. Sudol M. FEBS Lett. 2002; 513: 30-37Crossref PubMed Scopus (391) Google Scholar). There are several metazoan Rsp5p homologs. The most closely related to Rsp5 and also the best characterized is Nedd4 (also known as Nedd4-1). A highly homologous protein, Nedd4-2, which is the product of a different gene, has more recently been identified (9Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar). Nedd4 and Nedd4-2 are implicated in the ubiquitylation and lysosomal targeting of the epithelial sodium channel (ENaC), as is the related family member WWP2 (9Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 10Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (597) Google Scholar, 11McDonald F.J. Western A.H. McNeil J.D. Thomas B.C. Olson D.R. Snyder P.M. Am. J. Physiol. 2002; 283: F431-F436Crossref PubMed Scopus (53) Google Scholar). Closely related to these are WWP1 and Itch, also known as Alp4, which has been implicated in the Notch signaling pathway (1Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1256) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2896) Google Scholar, 12Perry 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 (279) Google Scholar, 13Qiu 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 (265) Google Scholar). Other WW domain HECT E3s that share significant homology with these include SMURF-1 and -2, which are involved in degradation of SMAD proteins (1Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1256) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2896) Google Scholar). Most WW-HECT E3s also have a calcium-dependent lipid-binding (C2) domain at the extreme amino terminus that mediates plasma membrane association (14Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (687) Google Scholar). In addition to ENaC, Nedd4 ubiquitylates other substrates including some that reside at, or transit through, the plasma membrane such as the Ras guanine nucleotide exchange factor (15Pham N. Rotin D. J. Biol. Chem. 2001; 276: 46995-47003Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Nedd4-mediated ubiquitylation is also implicated in viral budding from the plasma membrane (16Patnaik A. Wills J.W. J. Virol. 2002; 76: 2789-2795Crossref PubMed Scopus (19) Google Scholar, 17Kikonyogo A. Bouamr F. Vana M.L. Xiang Y. Aiyar A. Carter C. Leis J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11199-11204Crossref PubMed Scopus (192) Google Scholar). EPS15 and other Ub-interacting motif (UIM) proteins involved in endocytosis and lysosomal trafficking of receptors are also Nedd4 substrates (18Polo S. Sigismund S. Faretta M. Guidi M. Capua M.R. Bossi G. Chen H. De Camilli P. Di Fiore P.P. Nature. 2002; 416: 451-455Crossref PubMed Scopus (549) Google Scholar, 19Katz M. Shtiegman K. Tal-Or P. Yakir L. Mosesson Y. Harari D. Machluf Y. Asao H. Jovin T. Sugamura K. Yarden Y. Traffic. 2002; 3: 740-751Crossref PubMed Scopus (120) Google Scholar). For another class of membrane proteins, the receptor tyrosine kinases (RTKs), the Cbl family of RING finger E3s are generally implicated in down-regulation with evidence that they mediate RTK ubiquitylation as part of this process (20Sanjay, A., Horne, W. C., and Baron, R. (2001) Science's STKE, http://stke.sciencemag.org/cgi/reprint/sigtrans;2001/110/pe40.pdf.Google Scholar, 21Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-307Crossref PubMed Scopus (515) Google Scholar, 22Rao N. Dodge I. Band H. J. Leukocyte Biol. 2002; 71: 753-763PubMed Google Scholar). RTKs for which Cbl proteins have been most clearly implicated as cognate E3s include the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (23Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (911) Google Scholar, 24Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (830) Google Scholar, 25Waterman H. Levkowitz G. Alroy I. Yarden Y. J. Biol. Chem. 1999; 274: 22151-22154Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 26Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Mammalian Cbl proteins include c-Cbl, Cbl-b, and Cbl-c (also known as Cbl-3). c-Cbl has also been shown to play a role in the degradation of nonreceptor tyrosine kinases including Src, Fyn, and Syk (20Sanjay, A., Horne, W. C., and Baron, R. (2001) Science's STKE, http://stke.sciencemag.org/cgi/reprint/sigtrans;2001/110/pe40.pdf.Google Scholar, 21Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-307Crossref PubMed Scopus (515) Google Scholar, 22Rao N. Dodge I. Band H. J. Leukocyte Biol. 2002; 71: 753-763PubMed Google Scholar). Mutant forms of c-Cbl that function as "dominant negatives" have been shown to have transforming activity, presumably as a consequence of decreased turnover and thus increased activity of tyrosine kinases (20Sanjay, A., Horne, W. C., and Baron, R. (2001) Science's STKE, http://stke.sciencemag.org/cgi/reprint/sigtrans;2001/110/pe40.pdf.Google Scholar, 21Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-307Crossref PubMed Scopus (515) Google Scholar). Recruitment of Cbl family members to activated RTKs occurs through interactions involving the highly conserved amino-terminal phosphotyrosine binding domains of the Cbl proteins. c-Cbl and Cbl-b are closely related proteins with extensive proline-rich regions that can bind a variety of Src homology 3-containing proteins implicated in signaling and receptor down-regulation (20Sanjay, A., Horne, W. C., and Baron, R. (2001) Science's STKE, http://stke.sciencemag.org/cgi/reprint/sigtrans;2001/110/pe40.pdf.Google Scholar, 21Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-307Crossref PubMed Scopus (515) Google Scholar, 27Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (486) Google Scholar, 28Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Both also have C-terminal Ub-associated (UBA) domains. Cbl-c is the most recently described member of the family (29Keane M.M. Ettenberg S.A. Nau M.M. Banerjee P. Cuello M. Penninger J. Lipkowitz S. Oncogene. 1999; 18: 3365-3375Crossref PubMed Scopus (98) Google Scholar, 30Kim M. Tezuka T. Suziki Y. Sugano S. Hirai M. Yamamoto T. Gene (Amst.). 1999; 239: 145-154Crossref PubMed Scopus (53) Google Scholar), and unlike c-Cbl and Cbl-b (22Rao N. Dodge I. Band H. J. Leukocyte Biol. 2002; 71: 753-763PubMed Google Scholar, 31Murphy M.A. Schnall R.G. Venter D.J. Barnett L. Bertoncello I. Thien C.B. Langdon W.Y. Bowtell D.D. Mol. Cell. Biol. 1998; 18: 4872-4882Crossref PubMed Scopus (332) Google Scholar, 32Naramura M. Kole H.K. Hu R.J. Gu H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15547-15552Crossref PubMed Scopus (289) Google Scholar, 33Bachmaier K. Krawczyk C. Kozieradzki I. Kong Y.Y. Sasaki T. Oliveira-dos-Santos A. Mariathasan S. Bouchard D. Wakeham A. Itie A. Le J. Ohashi P.S. Sarosi I. Nishina H. Lipkowitz S. Penninger J.M. Nature. 2000; 403: 211-216Crossref PubMed Scopus (558) Google Scholar, 34Liu Y.C. Gu H. Trends Immunol. 2002; 23: 140-143Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), no specific developmental function has been ascribed to it. Cbl-c has a more limited proline-rich region and contains neither a UBA domain nor the C-terminal tyrosine phosphorylation sites found on c-Cbl and Cbl-b (20Sanjay, A., Horne, W. C., and Baron, R. (2001) Science's STKE, http://stke.sciencemag.org/cgi/reprint/sigtrans;2001/110/pe40.pdf.Google Scholar, 21Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 294-307Crossref PubMed Scopus (515) Google Scholar, 22Rao N. Dodge I. Band H. J. Leukocyte Biol. 2002; 71: 753-763PubMed Google Scholar). Since Nedd4 and Cbl both ubiquitylate plasma membrane proteins and Cbl proteins all include potential sites of interaction with WW domains, we asked whether they might physically and functionally interact. We report here that Nedd4, as well as Itch, associates with all three mammalian Cbl proteins and regulates Cbl protein levels by targeting them for ubiquitindependent proteasomal degradation. Consequently, Nedd4 inhibits Cbl-b-mediated ubiquitylation and down-regulation of the EGFR and reverses effects on proximal events in signaling through this receptor. Similarly, Nedd4 reverses Cbl-mediated down-regulation of activated Src. These data demonstrate negative regulation of RING finger E3s by HECT family E3s and point to the potential for an additional level of complexity in the regulation of protein-tyrosine kinases. Plasmids—HA epitope-tagged Cbl-b, Cbl-bC373A, and Cbl-b N1/3 have been described (35Ettenberg S.A. Magnifico A. Cuello M. Nau M.M. Rubinstein Y.R. Yarden Y. Weissman A.M. Lipkowitz S. J. Biol. Chem. 2001; 276: 27677-27684Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Plasmid encoding the Cbl-b proline-rich (PR) domain (aa 482–721) was generated by digestion of wild type Cbl-b with BamHI and EcoRI and ligation into pCEV29N from BglII to EcoRI to generate pCEV29N-Cbl-b PR. Plasmid encoding the Cbl-b RING finger (aa 327–483) was generated by digestion of Cbl-b C-terminal 2/3 (35Ettenberg S.A. Magnifico A. Cuello M. Nau M.M. Rubinstein Y.R. Yarden Y. Weissman A.M. Lipkowitz S. J. Biol. Chem. 2001; 276: 27677-27684Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) with BamHI and XhoI and religation. Plasmid encoding Cbl-b without epitope tag was generated by site-specific mutagenesis by introduction of a stop codon before the HA tag. pcDNA-HA-Nedd4 was generated by subcloning of ps-HA-Nedd4 (36Hatakeyama S. Jensen J.P. Weissman A.M. J. Biol. Chem. 1997; 272: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) into pcDNA3.1 (–) using NotI and EcoRI. Plasmid encoding Nedd4 in which the active site cysteine was mutated to serine (pc-HA-Nedd4C744S) was generated by site-directed mutagenesis (QuikChange kit; Stratagene, La Jolla CA). N-terminal Myc-tagged mouse Itch was generated by subcloning of full-length Itch (12Perry 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 (279) Google Scholar) into pCI-neo using NheI (5′) and NotI (3′) sites; the Myc tag was generated by PCR. The Cys to Gly mutation of the active site Cys of Itch was carried out by site-specific mutagenesis. Glutathione S-transferase (GST)-Nedd4, GST-Nedd4-N, GST-Nedd4-C (36Hatakeyama S. Jensen J.P. Weissman A.M. J. Biol. Chem. 1997; 272: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar); GST-Itch (37Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14422-14427Crossref PubMed Scopus (355) Google Scholar); EGFR (38Di Fiore P.P. Pierce J.H. Fleming T.P. Hazan R. Ullrich A. King C.R. Schlessinger J. Aaronson S.A. Cell. 1987; 51: 1063-1070Abstract Full Text PDF PubMed Scopus (504) Google Scholar); SrcY527F (39Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar); Myc-E6-AP (36Hatakeyama S. Jensen J.P. Weissman A.M. J. Biol. Chem. 1997; 272: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar); HA-Cbl-c (29Keane M.M. Ettenberg S.A. Nau M.M. Banerjee P. Cuello M. Penninger J. Lipkowitz S. Oncogene. 1999; 18: 3365-3375Crossref PubMed Scopus (98) Google Scholar); and HA-Ub (40Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar) have been described. pEGFP-C2 encoding GFP was from Invitrogen. Antibodies and Reagents—Anti-Cbl-b (H121; H454), anti-GFP (FL), anti-c-Cbl (C-15), anti-Src (N16), anti-EGFR (1005), anti-Myc 9E10 (SC-40), and anti-MAPK (C-14) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFR (Ab-3) was from Oncogene Science (Boston, MA); anti-phospho MAPK was from New England Biolabs (Beverly, MA); and anti-HA epitope (12CA5) was from culture supernatant. Anti-Ub has been described (41Cenciarelli C. Wilhelm Jr., K.J. Guo A. Weissman A.M. J. Biol. Chem. 1996; 271: 8709-8713Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Anti-Nedd4 was raised in rabbit by immunization with a GST fusion of mouse Nedd4 (36Hatakeyama S. Jensen J.P. Weissman A.M. J. Biol. Chem. 1997; 272: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Unless otherwise noted, reagents were used at the following concentrations in cell experiments: lactacystin, 20 μm (Calbiochem); cycloheximide, 100 μm; ammonium chloride, 20 mm; recombinant human EGF, 100 ng/ml (Pepro Tech Inc., Rocky Hill, NJ). Cell Experiments—The Jurkat human T-cell leukemia cell line (41Cenciarelli C. Wilhelm Jr., K.J. Guo A. Weissman A.M. J. Biol. Chem. 1996; 271: 8709-8713Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and 293T human embryonic kidney cells expressing SV40 large T antigen (37Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14422-14427Crossref PubMed Scopus (355) Google Scholar) were maintained as described. Transfections in 293T cells were with calcium phosphate (5 Prime → 3 Prime, Inc., Boulder, CO), Superfect (Qiagen, Valencia, CA), or LipofectAMINE 2000 (Invitrogen). Transfections into HeLa were with LipofectAMINE Plus (Invitrogen). For experiments involving Cbl-c, cells were plated at 0.25 × 106 in 100-mm dishes. For all other experiments, 0.5 × 106 cells were plated in 100-mm dishes. Transfections were carried out the following day. Cbl constructs expressed at a considerably better level than Nedd4; therefore, in general, 0.5–3.0 μg of Cbl encoding plasmid and 5–12 μg of Nedd4 plasmids were used for transfections. In all transfection experiments, the total amount of plasmid was equalized among samples using the appropriate expression vector without insert (V) so that equivalent amounts of promoter were also expressed in each point. Cells were washed at 4 °C in PBS containing 0.5 mm sodium orthovanadate and, unless otherwise indicated, lysed at 4 °C in lysis buffer (50 mm Tris HCl, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 0.5% β-octyl glucoside (Sigma), 10% glycerol, 10 mm iodoacetamide, 2 mm orthovanadate, and protease inhibitors as described) (41Cenciarelli C. Wilhelm Jr., K.J. Guo A. Weissman A.M. J. Biol. Chem. 1996; 271: 8709-8713Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Lysates were cleared at 14,000 rpm for 15 min at 4 °C. Cell lysates were either first subjected to immunoprecipitation or directly resolved by SDS-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membrane. Immunoprecipitations were carried out using antibodies and protein A-agarose. The wash buffer used in immunoprecipitation differed from the lysis buffer in being 0.1% in both Triton X-100 and β-octyl glucoside. Proteins were detected by immunoblotting using standard methods, either ECL (Amersham Biosciences) or 125I-protein A after incubation with primary antibodies. 125I quantification was by Storm PhosphorImager and ImageQuant software (Amersham Biosciences). Proteasome and lysosomal inhibitors were added to cells for 8 h beginning 36 h after transfection. For experiments involving EGF, 293T cells (70% confluence) were incubated overnight in serum-free medium (reduced from 10%). To evaluate Cbl-b ubiquitylation, cells were lysed in buffer containing 1% SDS to disrupt protein-protein interactions. After clearing of lysates, SDS was diluted 10-fold in lysis buffer. For Cbl-b ubiquitylation, HA-Ub was co-expressed to increase signal, but immunoblotting was with polyclonal anti-Ub. In Vitro Binding and Ubiquitylation—GST fusion proteins expressed in Escherichia coli were prepared, stained using Coomassie Blue, and quantified by comparison with bovine serum albumin standards. Proteins were bound to glutathione-Sepharose as described (42Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (938) Google Scholar). [35S]Methionine-labeled proteins were generated in rabbit reticulocyte lysate (37Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14422-14427Crossref PubMed Scopus (355) Google Scholar). For binding experiments, in vitro translation products were incubated with bead-bound GST fusion proteins for 2 h at 4 °C prior to washing in lysis buffer. In vitro ubiquitylation was carried out using glutathione-Sepharose-bound fusion proteins and added translation products as described (43Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar) and was modified to include 100 μm MG132 (Biomol, Plymouth Meeting, PA) and 1 μm Ub aldehyde (Boston Biochemicals, Cambridge, MA). Nedd4 and Cbl-b Bind in Vitro—To ascertain whether Nedd4 and Cbl-b interact, GST fusion proteins of murine Nedd4 (36Hatakeyama S. Jensen J.P. Weissman A.M. J. Biol. Chem. 1997; 272: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) were evaluated for binding to in vitro translated [35S]methionine-labeled Cbl-b (Fig. 1B, upper panel; see Fig. 1A for schematics of Cbl-b and Nedd4). Significant binding of Cbl-b to Nedd4 (GST-Nedd4; aa 52–777) was observed. The N-terminal portion of Nedd4 (GST-Nedd4-N; aa 52–422), which contains the three WW domains but lacks the HECT domain, similarly bound Cbl-b, whereas there was no specific binding to the C-terminal HECT domain (GST-Nedd4-C; aa 423–777). The domains of Cbl-b required for Nedd4 binding were next mapped (Fig. 1C). Nedd4 and Nedd4-N but not Nedd4-C demonstrated substantial binding to the proline-rich region of Cbl-b (Cbl-b PR) and to the nonoverlapping N-terminal third of Cbl-b (Cbl-b N1/3), which includes the phosphotyrosine binding domain as well as several noncanonical WW domain binding sites and a potential site of serine phosphorylation-dependent WW domain binding. In contrast, the RING finger and surrounding regions of Cbl-b did not bind Nedd4. Thus, Cbl-b and Nedd4 associate in a manner that does not require either protein's Ub ligase domain. Nedd4 and Cbl-b Associate in Cells—To evaluate whether Nedd4 and Cbl-b associate in cells, lysates from the Jurkat T-cell leukemia cell line, which expresses easily detectable levels of both Nedd4 and Cbl-b, were immunoprecipitated with either a polyclonal antibody raised against Nedd4 or anti-Cbl-b, followed by immunoblotting with antibodies directed against one or the other (Fig. 2A). Substantial specific immunoprecipitation of Nedd4 using anti-Cbl-b was detected (lane 1), as was immunoprecipitation of Cbl-b with anti-Nedd4 (lane 3). Both Cbl-b and Nedd4 associate with lipid rafts (5Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar, 44Plant P.J. Lafont F. Lecat S. Verkade P. Simons K. Rotin D. J. Cell Biol. 2000; 149: 1473-1484Crossref PubMed Scopus (123) Google Scholar, 45Lafont F. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3180-3184Crossref PubMed Scopus (97) Google Scholar). Co-immunoprecipitation was not affected by the presence or absence of β-octylglucoside, which disrupts lipid rafts. The co-immunoprecipitation of Cbl-b and Nedd4 suggests that a significant percentage of these molecules associate with each other in cells. If this is the case, preclearing of cell lysates with anti-Nedd4 would deplete a substantial percentage of Cbl-b. To evaluate this, lysates were first subject to immunoprecipitation either with anti-Nedd4 or with a nonspecific antibody followed by reimmunoprecipitation with anti-Cbl-b or with nonspecific antibody. As can be seen by comparing Fig. 2B, lanes 3 and 5, substantially less Cbl-b is immunoprecipitated after first preclearing with anti-Nedd4. Association of Nedd4 and Cbl-b was also observed in 293T human embryonic kidney cells (293T cells) when Nedd4 and Cbl-b were co-transfected, since there is significant specific immunoprecipitation of Nedd4 with anti-Cbl-b (Fig. 2C, lane 4, middle panel). Thus, Cbl-b and Nedd4 demonstrate a strong physical association both in vitro and in cells. Nedd4 Down-regulates Cbl Protein Levels—In transfected 293T cells, we noted that Cbl-b levels decreased when Nedd4 was co-expressed (Fig. 2C, upper panel, compare lanes 2 and 4). In contrast, Nedd4 levels were unaffected by co-expression of Cbl-b (Fig. 2C, lower panel, compare lanes 3 and 4). This suggested that Nedd4 might mediate loss of Cbl-b. As shown in Fig. 3A, Cbl-b levels did not decrease when co-expressed with Nedd4 in which the catalytic cysteine was mutated (Nedd4C744S). In contrast, expression of wild type Nedd4 at a comparable level markedly decreased Cbl-b. Thus, the loss of Cbl-b that we observe requires the Ub ligase activity of Nedd4. The most likely explanation for our findings is that Cbl-b is being targeted for degradation by Nedd4. However, to exclude the possibility that the effect observed was occurring prior to protein synthesis, we measured the rate of loss of Cbl-b in the absence or presence of Nedd4, using cycloheximide to inhibit new protein synthesis (Fig. 3B). As is evident (Fig. 3B), in addition to decreasing the level of Cbl-b at the beginning of the cycloheximide treatment, expression of Nedd4 markedly accelerated the rate of Cbl loss (t > 20 h without Nedd4, ∼5 h with Nedd4). Levels of co-expressed c-Cbl as well as the smaller Cbl-c protein (data not shown and Fig. 3C) were similarly affected by Nedd4, and consistent with this, both of these proteins also bound Nedd4 (data not shown). Thus, Nedd4 functionally and physically interacts with all three mammalian Cbl proteins. To determine whether the Nedd4-mediated loss of transfected Cbl extends to endogenous Cbl proteins, we examined endogenous Cbl levels in HeLa cells transfected with Nedd4. Under conditions where >70% transfection efficiency was achieved, Nedd4, but not catalytically inactive Nedd4C744S, resulted in a striking loss of endogenous Cbl-b and c-Cbl (Fig. 3D, middle panels). Similar results were obtained in 293T cells for endogenous c-Cbl (data not shown), which is the only Cbl family member readily detectable in these cells. To evaluate whether the Nedd4-mediated loss of Cbl-b requires the E3 activity of Cbl-b, wild type Cbl-b and RING mutant Cbl-b (Cb1-bC373A), which is inactive as an E3, were compared. As is evident (Fig. 4A), Nedd4 enhanced the loss of RING finger mutant Cbl-b (Cb1-bC373A) as well as wild type Cbl-b. This establishes that the E3 activity of Cbl-b is not required for Nedd4 to target it for degradation. Nedd4 Ubiquitylates Cbl-b and Targets It for Proteasomal Degradation—To determine whether Nedd4-mediated loss of Cbl-b involves the Ub-proteasome pathway, we first assessed whether Nedd4 ubiquitylates Cbl-b. To preclude autoubiquitylation of Cbl-b, cells were transfected with RING mutant Cbl-b (Cb1-bC373A), with or without co-transfection of Nedd4. After treatment with lactacystin to inhibit proteasome function and lysis of cells in 1% SDS to disrupt the association between Cbl-b and Nedd4, Cbl-b and Nedd4 immunoprecipitates were evaluated for ubiquitylation by immunoblotting with anti-Ub (Fig. 4B). Expression of Nedd4 resulted in a marked increase in ubiquitylated species immunoprecipitated with anti-Cbl-b (Fig. 4B, left upper panel). Nedd4 was not detected on reprobing of blots (data not shown), indicating that the SDS did disrupt protein-protein interactions as expected and that the ubiquitylation observed does not represent co-associated Nedd4 (data not shown). As further confirmation that the ubiquitylation seen with Nedd4 co-expression was of Cbl-b and not Nedd4, anti-Nedd4 immunoprecipitates from an equal amount of the same lysates were evaluated for ubiquitylation. No increase in ubiquitylation was observed with Nedd4 expression (right upper panel). Similar results were obtained with co-expression of w
Referência(s)