Properties of tripartite chimeras between Src and Lck

Abstract

To evaluate the possible regulatory effects of interactions between different domains of Src-family kinases, we have assembled five chimeric molecules containing parts of p60c-src (Src) and p56lck (Lck). Chimeras contained the N-terminal portion, kinase domain or C-terminal tail from either of the parent molecules. None of the four full-length hybrid proteins induced morphological transformation of NIH3T3 cells, nor stimulated phosphorylation of cell proteins on tyrosine residues, suggesting that their protein-tyrosine kinase activities were repressed appropriately in fibroblastic cells. However, two hybrid proteins, SLS and SLL, containing the Src N-terminal region, Lck kinase domain, and either Src or Lck tail, respectively, inefficiently induced anchorage-independent growth. Sites of phosphorylation in these molecules were determined by two-dimensional peptide mapping. SLS and SLL were both phosphorylated at their C-terminal tyrosine residues similarly to the parental molecules. Curiously, both proteins were also significantly phosphorylated at tyrosine 416, a feature of transforming variants of Src and Lck. A modified hybrid, SLO, containing the N-terminal region of Src, kinase domain of Lck and a truncated C-terminus, fully transformed cells and stimulated phosphorylation of cell proteins at tyrosine. Comparison of SLS, SLL and SLO suggests that the full-length hybrids are repressed by their C-terminal phosphorylated tyrosine residues in vivo. Consistent with this, SLO resembled activated Src in being cytoskeletal, and SLS and SLL resembled nontransforming Src and were not cytoskeletal. Hybrids with an Lck N-terminal region were cytoskeletal, like Lck itself, even though they were not transforming, suggesting that cytoskeletal localization of Lck is determined through its N terminus. Curiously, the hybrid molecules appeared not to be regulated in vitro. The specific activities of SLS, SLL and Src were approximately equal, but the specific activity of SLO was not increased, being much less than that of activated Src. Enzymatic dephosphorylation stimulated the in vitro kinase activity of parental Src, but not of SLS or SLL. These observations suggest that chimeric Src-Lck molecules are regulated in the cell, but not in vitro.