The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase.
The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.
Ota
S
S
Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Hazeki
K
K
Rao
N
N
Lupher
M L
ML
Andoniou
C E
CE
Druker
B
B
Band
H
H
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D004792:Enzyme Precursors / Q000378:metabolism*; D019143:Evolution, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007075:Immunoglobulin M / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D016393:Lymphoma, B-Cell; D009154:Mutation; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020218:Response Elements; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D026362:Serum Response Factor; D015398:Signal Transduction; D000072377:Syk Kinase; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D004792:Enzyme Precursors; D007075:Immunoglobulin M; D047908:Intracellular Signaling Peptides and Proteins; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011947:Receptors, Antigen, B-Cell; D011994:Recombinant Proteins; D026362:Serum Response Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C067419:CBL protein, human