title: Bimolecular fluorescence complementation demonstrates that the c-Fes protein-tyrosine kinase forms constitutive oligomers in living cells.
abstract: The c-fes proto-oncogene encodes a unique nonreceptor protein-tyrosine kinase (c-Fes) that contributes to the differentiation of myeloid hematopoietic, vascular endothelial, and some neuronal cell types. Although originally identified as the normal cellular homologue of the oncoproteins encoded by avian and feline transforming retroviruses, c-Fes has recently been implicated as a tumor suppressor in breast and colonic epithelial cells. Structurally, c-Fes consists of a unique N-terminal region harboring an FCH domain, two coiled-coil motifs, a central SH2 domain, and a C-terminal kinase domain. In living cells, c-Fes kinase activity is tightly regulated by a mechanism that remains unclear. Previous studies have established that c-Fes forms high molecular weight oligomers in vitro, suggesting that the dual coiled-coil motifs may regulate the interconversion of inactive monomeric and active oligomeric states. Here we show for the first time that c-Fes forms oligomers in live cells independently of its activation status using a YFP bimolecular fluorescence complementation assay. We also demonstrate that both N-terminal coiled-coil regions are essential for c-Fes oligomerization in transfected COS-7 cells as well as HCT 116 colorectal cancer and K-562 myeloid leukemia cell lines. Together, these data provide the first evidence that c-Fes, unlike c-Src, c-Abl, and other nonreceptor tyrosine kinases, is constitutively oligomeric in both its repressed and active states. This finding suggests that conformational changes, rather than oligomerization, may govern its kinase activity in vivo.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Shaffer
authors.forename: Sabine
authors.initials: S
authors.lastname: Hellwig
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000235:genetics; D020868:Gene Silencing; D005816:Genetic Complementation Test; D045325:HCT116 Cells; D006801:Humans; D015151:Immunoblotting; D020014:K562 Cells; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D000090063:Proto-Oncogene Mas; D051578:Proto-Oncogene Proteins c-fes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; C054562:yellow fluorescent protein, Bacteria; D051578:Proto-Oncogene Proteins c-fes