Structure, transforming properties, and expression of TEL/PDGFβR mutants.
(A) Schematic diagram of wild-type (WT) PDGFβR and TEL/PDGFβR mutants. At left is depicted the wild-type PDGFβR protein with the major tyrosine phosphorylation sites denoted. Wild-type and mutant TEL/PDGFβR variants are shown that contain Tyr→Phe substitutions at sites corresponding to phosphorylation sites in the native PDGFβR protein. Boxed areas indicate the TEL domain; oval hatched areas denote the split tyrosine kinase domain. Numbering of tyrosine residues corresponds to positions in the wild-type PDGFβR protein. (B) Growth properties of Ba/F3 hematopoietic cells stably transformed with wild-type and mutant TEL/PDGFβR fusion genes. Ba/F3 cells transformed by pMSCV vector with or without wild-type TEL/PDGFβR, F2, F5, F7, or F8 were grown in the absence of IL-3. Cells were maintained at a density of 1 × 105 to 1 × 106/mL. (C) Expression of wild-type and mutant TEL/PDGFβR proteins in stably transformed Ba/F3 cells. Cells were lysed and were immunoprecipitated with antibody recognizing the cytoplasmic domain of the PDGFβR. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with antibody against the PDGFβR cytoplasmic domain. Multiple bands in each lane are the consequence of 2 translational start sites within theTEL gene and of autophosphorylation of TEL/PDGFβR. (D) Tyrosine phosphorylation of wild-type and mutant TEL/PDGFβR proteins. TEL/PDGFβR variants were immunoprecipitated as in panel C and immunoblotted with a monoclonal antibody against protein phosphotyrosine.
