Molecular cloning of the chicken c-cbl gene

Cbl is the proto-oncogene from which v-Cbl is derived. v-Cbl induces myeloid and B cell lymphoid malignancies in rodents. Cbl is a signaling molecule with multiple functional domains: an SH2 domain which binds phosphotyrosine residues, a RING finger domain which acts as a ubiquitin ligase, a proline-rich region which serves as a docking site for SH3-containing proteins, phosphotyrosine residues which serve as docking sites for SH2-containing proteins such a CrkL and the p85 subunit of PI 3-kinase, and a nuclear localization signal. Cbl-related molecules include Cbl-b and Cbl-c. While all three members contain the SH2 and Ring finger domains, Cbl-b is missing the aforementioned phosphotyrosine residues and Cbl-c is missing both the phosphotyrosine residues and the proline rich region. To determine more precisely the role of c-Cbl, we wished to analyze Cbl in DT40 cells lacking Cbl. Using primers based on human cDNA sequence, we cloned partial 5′ and 3′ cDNAs corresponding to Cbl from DT40 cells. By Northern blot analysis, a single 2.8 kB transcript was identified. To complete the cloning, the 3′ PCR fragment was then used to screen a chicken liver cDNA library. To date, no clones corresponding to Cbl-b or Cbl-c nor pseudo-genes have been identified in DT40 cells or chicken liver cDNA. The full-length sequence of chicken c-Cbl encodes a protein of 902 amino acids, with conserved tyrosine residues and a proline-rich region. Therefore, the DT40 cell system should be an excellent system to study the role of Cbl in BcR or cytokine signaling. Mutants of Cbl are being prepared for reconstitution studies in cb1-deficient cells.