An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn
Navin Rao
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorAmiya K. Ghosh
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorPengcheng Zhou
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorPatrice Douillard
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorChristopher E. Andoniou
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorCorresponding Author
Hamid Band
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Brigham & Women's Hospital, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Harvard Medical School,Smith Building, Room 538C, One Jimmy Fund Way, Boston, MA 02115, USA. Phone: +1-617-525-1101, Fax: +1-617-525-1010Search for more papers by this authorNavin Rao
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorAmiya K. Ghosh
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorPengcheng Zhou
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorPatrice Douillard
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorChristopher E. Andoniou
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Search for more papers by this authorCorresponding Author
Hamid Band
Brigham and Women's Hospital, Harvard Medical School, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Boston, MA, USA
Brigham & Women's Hospital, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Harvard Medical School,Smith Building, Room 538C, One Jimmy Fund Way, Boston, MA 02115, USA. Phone: +1-617-525-1101, Fax: +1-617-525-1010Search for more papers by this authorAbstract
The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using mouse embryonic fibroblasts from Cbl+/+ and Cbl−/− mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases.
REFERENCES
- 1 Chow , L. M. , Veillette , A. (1995 ) The Src and Csk families of tyrosine protein kinases in hemopoietic cells. Semin Immunol 7 : 207 –207 .
- 2 Thomas , S. M. , Brugge , J.S. (1997 ) Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13 : 513 –513 .
- 3 Sicheri , F. , Moarefi , I. , Kuriyan , J. (1997 ) Crystal structure of the Src family tyrosine kinase Hck. Nature 385 : 602 –602 .
- 4 Xu , W. , Harrison , S. C. , Eck , M. J. (1997 ) Three-dimensional structure of the tyrosine kinase c-Src. Nature 385 : 595 –595 .
- 5 Seidel-Dugan , C. , Meyer , B. E. , Thomas , S. M. , Brugge , J. S. (1992 ) Effects of SH2 and SH3 deletions on the functional activities of wild- type and transforming variants of c-Src. Mol Cell Biol 12 : 1835 –1835 .
- 6 Briggs , S. D. , Sharkey , M. , Stevenson , M. , Smithgall , T. E. (1997 ) SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1. J Biol Chem 272 : 17899 –17899 .
- 7 Gonfloni , S. , Williams , J. C. , Hattula , K. , Weijland , A. , Wierenga , R. K. , Superti-Furga , G. (1997 ) The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src. Embo J 16 : 7261 –7261 .
- 8 Moarefi , I. , LaFevre-Bernt , M. , Sicheri , F. , Huse , M. , Lee , C.H. , Kuriyan , J. , Miller , W. T. (1997 ) Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature 385 : 650 –650 .
- 9 Imamoto , A. , Soriano , P. (1993 ) Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice. Cell 73 : 1117 –1117 .
- 10 Nada , S. , Yagi , T. , Takeda , H. , Tokunaga , T. , Nakagawa , H. , Ikawa , Y. , Okada , M. , Aizawa , S. (1993 ) Constitutive activation of Src family kinases in mouse embryos that lack Csk. Cell 73 : 1125 –1125 .
- 11 Porter , M. , Schindler , T. , Kuriyan , J. , Miller , W. T. (2000 ) Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand. J Biol Chem 275 : 2721 –2721 .
- 12 Bijlmakers , M. J. , Marsh , M. (2000 ) Hsp90 is essential for the synthesis and subsequent membrane association, but not the maintenance, of the Src-kinase p56(lck). Mol Biol Cell 11 : 1585 –1585 .
- 13 Andoniou , C. E. , Lill , N. L. , Thien , C. B. , Lupher , M. L., Jr. , Ota , S. , Bowtell , D. D. , Scaife , R. M. , Langdon , W. Y. , Band , H. (2000 ) The Cbl proto-oncogene product negatively regulates the Src-family tyrosine kinase Fyn by enhancing its degradation. Mol Cell Biol 20 : 851 –851 .
- 14 Broome , M. A. , Galisteo , M. L. , Schlessinger , J. , Courtneidge , S. A. (1999 ) The proto-oncogene c-Cbl is a negative regulator of DNA synthesis initiated by both receptor and cytoplasmic tyrosine kinases. Oncogene 18 : 2908 –2908 .
- 15 Lupher , M. L., Jr. , Rao , N. , Eck , M. J. , Band , H. (1999 ) The Cbl protooncoprotein: a negative regulator of immune receptor signal transduction. Immunol Today 20 : 375 –375 .
- 16 Thien , C. B. , Langdon , W. Y. (2001 ) Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol 2 : 294 –294 .
- 17 Murphy , M. A. , Schnall , R. G. , Venter , D. J. , Barnett , L. , Bertoncello , I. , Thien , C. B. , Langdon , W. Y. , Bowtell , D. D. (1998 ) Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl- deficient mice. Mol Cell Biol 18 : 4872 –4872 .
- 18 Naramura , M. , Kole , H. K. , Hu , R. J. , Gu , H. (1998 ) Altered thymic positive selection and intracellular signals in Cbl- deficient mice. Proc Natl Acad Sci U S A 95 : 15547 –15547 .
- 19 Chiang , Y. J. , Kole , H. K. , Brown , K. , Naramura , M. , Fukuhara , S. , Hu , R. J. , Jang , I. K. , Gutkind , J. S. , Shevach , E. , Gu , H. (2000 ) Cbl-b regulates the CD28 dependence of T-cell activation. Nature 403 : 216 –216 .
- 20 Krawczyk , C. , Bachmaier , K. , Sasaki , T. , Jones , R. G. , Snapper , S. B. , Bouchard , D. , Kozieradzki , I. , Ohashi , P. S. , Alt , F.W. , Penninger , J. M. (2000 ) Cbl-b Is a Negative Regulator of Receptor Clustering and Raft Aggregation in T Cells. Immunity 13 : 463 –463 .
- 21 Joazeiro , C. A. , Wing , S. S. , Huang , H. , Leverson , J. D. , Hunter , T. , Liu , Y. C. (1999 ) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2- dependent ubiquitin-protein ligase. Science 286 : 309 –309 .
- 22 Lee , P. S. , Wang , Y. , Dominguez , M. G. , Yeung , Y. G. , Murphy , M. A. , Bowtell , D. D. , Stanley , E. R. (1999 ) The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation. Embo J 18 : 3616 –3616 .
- 23 Levkowitz , G. , Waterman , H. , Zamir , E. , Kam , Z. , Oved , S. , Langdon , W. Y. , Beguinot , L. , Geiger , B. , Yarden , Y. (1998 ) c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev 12 : 3663 –3663 .
- 24 Hicke , L. , Zanolari , B. , Riezman , H. (1998 ) Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization. J Cell Biol 141 : 349 –349 .
- 25 Lupher , M. L., Jr. , Rao , N. , Lill , N. L. , Andoniou , C. E. , Miyake , S. , Clark , E. A. , Druker , B. , Band , H. (1998 ) Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323. J Biol Chem 273 : 35273 –35273 .
- 26 Rao , N. , Lupher , M. L., Jr. , Ota , S. , Reedquist , K. A. , Druker , B. J. , Band , H. (2000 ) The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. J Immunol 164 : 4616 –4616 .
- 27 Meng , W. , Sawasdikosol , S. , Burakoff , S. J. , Eck , M. J. (1999 ) Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase. Nature 398 : 84 –84 .
- 28 Lupher , M. L. Jr. , Songyang , Z. , Shoelson , S. E. , Cantley , L. C. , Band , H. (1997 ) The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70. J Biol Chem 272 : 33140 –33140 .
- 29 Lill , N. L. , Douillard , P. , Awwad , R. A. , Ota , S. , Lupher , M. L., Jr. , Miyake , S. , Meissner-Lula , N. , Hsu , V. W. , Band , H. (2000 ) The evolutionarily conserved N-terminal region of Cbl is sufficient to enhance down-regulation of the epidermal growth factor receptor. J Biol Chem 275 : 367 –367 .
- 30 Miyake , S. , Lupher , M. L., Jr. , Druker , B. , Band , H. (1998 ) The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor alpha. Proc Natl Acad Sci U S A 95 : 7927 –7927 .
- 31 Zheng , N. , Wang , P. , Jeffrey , P. D. , Pavletich , N. P. (2000 ) Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin- protein ligases. Cell 102 : 533 –533 .
- 32 Levkowitz , 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. (1999 ) Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell 4 : 1029 –1029 .
- 33 Ota , S. , Hazeki , K. , Rao , N. , Lupher , M. L., Jr. , Andoniou , C.E. , Druker , B. , Band , H. (2000 ) The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase. J Biol Chem 275 : 414 –414 .
- 34 Miyake , S. , Lupher , M. L., Jr. , Andoniou , C. E. , Lill , N. L. , Ota , S. , Douillard , P. , Rao , N. , Band , H. (1997 ) The Cbl protooncogene product: from an enigmatic oncogene to center stage of signal transduction. Crit Rev Oncog 8 : 189 –189 .
- 35 Tsygankov , A. Y. , Mahajan , S. , Fincke , J. E. , Bolen , J. B. (1996 ) Specific association of tyrosine-phosphorylated c-Cbl with Fyn tyrosine kinase in T cells. J Biol Chem 271 : 27130 –27130 .
- 36 Hakak , Y. , Martin , G.S. (1999 ) Ubiquitin-dependent degradation of active Src. Curr Biol 9 : 1039 –1039 .
- 37 Harris , K. F. , Shoji , I. , Cooper , E. M. , Kumar , S. , Oda , H. , Howley , P. M. (1999 ) Ubiquitin-mediated degradation of active Src tyrosine kinase. Proc Natl Acad Sci U S A 96 : 13738 –13738 .
- 38 Oda , H. , Kumar , S. , Howley , P. M. (1999 ) Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination. Proc Natl Acad Sci U S A 96 : 9557 –9557 .
- 39 Winberg , G. , Matskova , L. , Chen , F. , Plant , P. , Rotin , D. , Gish , G. , Ingham , R. , Ernberg , I. , Pawson , T. (2000 ) Latent membrane protein 2A of epstein-barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases. Mol Cell Biol 20 : 8526 –8526 .
- 40 Kulka , R. G. , Raboy , B. , Schuster , R. , Parag , H. A. , Diamond , G. , Ciechanover , A. , Marcus , M. (1988 ) A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1. J Biol Chem 263 : 15726 –15726 .
- 41 Wilson , I. A. , Niman , H. L. , Houghten , R. A. , Cherenson , A. R. , Connolly , M. L. , Lerner , R. A. (1984 ) The structure of an antigenic determinant in a protein. Cell 37 : 767 –767 .
- 42 Treier , M. , Staszewski , L. M. , Bohmann , D. (1994 ) Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78 : 787 –787 .
- 43 Feshchenko , E. A. , Shore , S. K. , Tsygankov , A.Y. (1999 ) Tyrosine phosphorylation of C-Cbl facilitates adhesion and spreading while suppressing anchorage-independent growth of V-Abl-transformed NIH3T3 fibroblasts. Oncogene 18 : 3703 –3703 .
- 44 Lupher , M. L., Jr. , Reedquist , K. A. , Miyake , S. , Langdon , W. Y. , Band , H. (1996 ) A novel phosphotyrosine-binding domain in the N-terminal transforming region of Cbl interacts directly and selectively with ZAP-70 in T cells. J Biol Chem 271 : 24063 –24063 .
- 45 Miyake , S. , Mullane-Robinson , K. P. , Lill , N. L. , Douillard , P. , Band , H. (1999 ) Cbl-mediated negative regulation of platelet-derived growth factor receptor-dependent cell proliferation. A critical role for Cbl tyrosine kinase-binding domain. J Biol Chem 274 : 16619 –16619 .
- 46
Mimnaugh , E. G.
, Bonvini , P.
, Neckers , L.
(1999
) The measurement of ubiquitin and ubiquitinated proteins.
Electrophoresis
20
: 418
–418
.
10.1002/(SICI)1522-2683(19990201)20:2<418::AID-ELPS418>3.0.CO;2-N CAS PubMed Web of Science® Google Scholar
- 47 Sanjay , A. , Houghton , A. , Neff , L. , DiDomenico , E. , Bardelay , C. , Antoine , E. , Levy , J. , Gailit , J. , Bowtell , D. , Horne , W. C. , Baron , R. (2001 ) Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility. J Cell Biol 152 : 181 –181 .
- 48 Alexandropoulos , K. , Baltimore , D. (1996 ) Coordinate activation of c-Src by SH3- and SH2-binding sites on a novel p130Cas-related protein, Sin. Genes Dev 10 : 1341 –1341 .
- 49 Yokouchi , M. , Kondo , T. , Sanjay , A. , Houghton , A. , Yoshimura , A. , Komiya , S. , Zhang , H. , Baron , R. (2001 ) Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins. J Biol Chem 276 : 35185 –35185 .
- 50 Scheffner , M. , Werness , B. A. , Huibregtse , J. M. , Levine , A. J. , Howley , P. M. (1990 ) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63 : 1129 –1136 .
- 51 Meng , F. , Lowell , C. A. (1998 ) A beta 1 integrin signaling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. Embo J 17 : 4391 –4391 .
- 52 Tanaka , S. , Amling , M. , Neff , L. , Peyman , A. , Uhlmann , E. , Levy , J. B. , Baron , R. (1996 ) c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption. Nature 383 : 528 –528 .
- 53 Ribon , V. , Printen , J. A. , Hoffman , N. G. , Kay , B. K. , Saltiel , A. R. (1998 ) A novel, multifuntional c-Cbl binding protein in insulin receptor signaling in 3T3-L1 adipocytes. Mol Cell Biol 18 : 872 –872 .
- 54 Scaife , R. M. , Langdon , W. Y. (2000 ) c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology. J Cell Sci 113 Pt 2 : 215 –226. .
