Abstract
Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.
Highlights
The Src tyrosine kinase is overexpressed in many tumor types, and its kinase activity may increase as the tumor stage advances [1,2]
Since Cub domain containing protein 1 (CDCP1) is a substrate of the Src kinase, we infected in a parallel experiment cells with two different viruses encoding the c-Src or wild type CDCP1 protein
In parental cells and those expressing only CDCP1, tyrosine phosphorylation at the exposure level shown was confined to one protein of 110 kDa whereas foci derived cells showed in addition phosphorylation of Src (60 kD) and CDCP1 (130 kD) proteins
Summary
The Src tyrosine kinase is overexpressed in many tumor types, and its kinase activity may increase as the tumor stage advances [1,2]. This is, not correlated with increased cell proliferation, and even cooperation of Src with the epidermal growth factor receptor rather enhances cell invasiveness [3]. While activation of c-Src is tightly regulated this is not possible for v-Src, because the protein generated by the Rous sarcoma virus lacks the carboxy-terminal sequence containing the negative regulatory tyrosine residue. Carboxy-terminal truncation of Src is found in rare cases of human tumors [6]
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