Inhibition of the motility and growth of B16F10 mouse melanoma cells by dominant negative mutants of Dok-1

Dok-1 (p62(Dok)) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed t o contribute to the control of cell growth and migration through associatio n with the Ras GTPase-activating protein and the adapter protein Nck, the r ole of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mu tants of this protein: one (DokPH+PTB) containing the pleckstrin homology a nd phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants i n highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyr osine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membrane s and the cytoskeleton. Overexpression of DokPH+PTB in these cells also mar kedly reduced both the rates of cell spreading, migration, and growth as we ll as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importan ce of the phosphotyrosine-binding domain. These results suggest that at lea st in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.