A point mutation in the N-terminal coiled-coil domain releases c-Fes tyrosine kinase activity and survival signaling in myeloid leukemia cells

The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascula r endothelial cells. Unique to Fes is a long N-terminal sequence with two r egions of strong homology to coited-coil oligomerization domains. We introd uced leucine-to-proline substitutions into the coiled coils that were predi cted to disrupt the coiled-coil structure. The resulting mutant proteins, t ogether with wild-type Fes, were fused to green fluorescent protein and exp ressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mut ation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase act ivities by approximately 50% relative to the levels of activity produced wi th the L145P mutation alone. To study the effects of the coiled-coil mutati ons in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent my eloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation rel ative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cel ls. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coi led coil in either oligomerization or recruitment of signaling partners. Ge l filtration experiments showed that the unique N-terminal region interconv erts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated re gulation of c-Fes tyrosine kinase activity and signaling, a mechanism uniqu e among tyrosine kinases.