Src and CDCP1 dependent focus formation require tyrosine phosphorylation of CDCP1.

<p>(A) NIH3T3 cells were infected as above with Src alone or with Src and the indicated CDCP1 forms. After three weeks, the cells were stained with crystal violett. Y734 - all codons for tyrosine residues but Y734 were mutated to encode Phe; Y734/743/762F - the codons for these tyrosines were mutated to Phe. (B) CDCP1 and the indicated tyrosine mutants were transiently overexpressed in 293 cells. After treatment with peroxovanadate the cells were lysed and CDCP1 immunoprecipitated with the Cub1 antibody. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with a phosphotyrosine-antibody (αPY; upper panel). In parallel, an aliquot from the cell lysate was analyzed for CDCP1 expression with an antibody against CDCP1 (lower panel). (C) Similar to (B), 293 cells were transfected with plasmids encoding CDCP1 and its mutants, and in addition c-Src. Cells were analyzed as above and the Cub1 immunoprecipitates detected with a phosphotyrosine specific antibody, whereas in an aliquot of the cell lysate CDCP1 and c-Src expression were tested (lower panel). αPY, anti-phosphotyrosine.