The c-fes locus encodes a 93-kDa non-
receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced
leucine-to-
proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting
mutant proteins, together with wild-type Fes, were fused to
green fluorescent protein and expressed 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 mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and
kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the
mutant proteins in the
granulocyte-macrophage colony-stimulating factor (
GM-CSF)-dependent
myeloid 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 relative to that of wild-type Fes. In contrast, the double coiled-coil
mutant protein showed greatly reduced
kinase and
biological activities in TF-1 cells. 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 coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts 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 regulation of
c-Fes tyrosine kinase activity and signaling, a mechanism unique among
tyrosine kinases.