Bimolecular fluorescence complementation demonstrates that the c-Fes protein-tyrosine kinase forms constitutive oligomers in living cells.

The human c-fes proto-oncogene encodes a structurally unique, 93 kDa non-receptor protein-tyrosine kinase (c-Fes) expressed in myeloid hematopoietic, vascular endothelial, and some neuronal cells where it has been linked to signaling pathways controlling differentiation (1,2). Early work showed that restoring wild-type c-Fes expression in the chronic myelogenous leukemia cell line K-562 suppresses cell proliferation and primes the cells for differentiation to macrophages by phorbol esters (3,4). Similarly, active c-Fes mutants induced GM-CSF-independent proliferation in addition to cell attachment and spreading in the cytokine-dependent myeloid leukemia cell line TF-1, consistent with differentiation along the monocyte-macrophage pathway (5). In the monocytic precursor cell line U-937, an active c-Fes mutant also induced cell adherence, macrophage morphology, and differentiation marker expression (6). Over-expression of wild-type c-Fes accelerated NGF-induced neurite extension in PC12 cells, while active c-Fes mutants induced spontaneous neurite formation in this cell line, suggestive of a role in neuronal differentiation (7,8). Finally, over-expression of wild-type c-Fes induced FGF-2-independent tube formation by cultured brain capillary endothelial cells, implicating c-Fes in angiogenesis (9). More recently, c-Fes expression has been detected in epithelial cells, where it may serve a tumor suppressor function. Greer and colleagues determined that tumor onset in a breast cancer model occurred more rapidly in mice targeted with either null or kinase-inactivating c-fes mutations, and that a c-fes transgene restored the latency of tumor formation (10). Our group found that c-Fes protein is strongly expressed in normal human colonic epithelial cells, while expression was reduced or absent in 67% of colon tumor sections from the same individuals (11). In addition, we have established that the expression of functional c-fes transcripts is downregulated in colorectal cancer cell lines via promoter methylation (12). Furthermore, re-expression of wild-type or active c-Fes suppressed anchorage-independent growth of two colorectal cancer cell lines, HCT 116 and HT-29, both of which are negative for c-Fes protein expression (11). Kinase-inactivating mutations have also been reported for c-Fes in colorectal cancer cell lines (10,11,13), providing further support for a tumor suppressor function for c-Fes in some tissue types. Structurally, c-Fes consists of a long unique N-terminal region, with a tubulin-binding Fes/CIP4 homology (FCH) domain and two coiled-coil homology motifs, followed by a central Src-homology 2 (SH2) domain and a C-terminal kinase domain (1,2) (Figure 1). The c-Fes FCH and first coiled-coil domain together belong to the family of F-BAR homology domains, which have been linked to regulation of membrane curvature by other proteins (14). Unlike other non-receptor protein-tyrosine kinases, c-Fes lacks negative regulatory features such as an SH3 domain or the negative regulatory tail associated with Src-family kinases (15). Despite this, c-Fes kinase activity remains strictly regulated in mammalian cells. When wild-type c-Fes is ectopically expressed in rodent fibroblasts, little or no transforming activity is apparent (5,16,17). However, mutation or deletion of the first coiled-coil domain results in strong upregulation of kinase activity and release of transforming potential in fibroblasts (3,5). Similarly, en bloc substitution of the c-Fes SH2 domain with that of v-Src also causes loss of negative regulation in vitro (18), implicating the SH2 domain as well as the N-terminal coiled-coil motif in the regulation of kinase activity. Figure 1 c-Fes constructs and BiFC experimental design. A) The structure of the wild-type (WT) c-Fes protein is shown at the top, which includes a unique N-terminal region harboring a Fes/CIP4 homology (FCH) domain, two coiled-coil homology motifs (CC1 and CC2), ... Gel-filtration and cross-linking studies have established that c-Fes forms higher order oligomers in vitro (up to a pentamer), and that the two N-terminal coiled-coil domains are responsible for oligomerization (5,19). In particular, gel-filtration analyses involving a small fragment of the N-terminal region showed that point mutations disrupting both coiled-coil motifs prevent oligomerization (5). This result led to a hypothetical model for c-Fes activation in which the coiled-coil domains mediate interconversion between inactive monomeric and active oligomeric states (5). In this model, the more N-terminal coiled-coil was proposed to interact intramolecularly with the second to hold c-Fes in an inactive monomeric conformation. Supporting this model is the observation that negative regulation imparted by the first coiled-coil domain is dramatically perturbed by mutation (19,20). However, no evidence for an inactive monomer has been generated either in vitro or in vivo, and it is unclear how oligomerization modulates c-Fes activity in living cells. In this study, we investigated c-Fes oligomerization in live cells using a bimolecular fluorescence complementation (BiFC) assay based on yellow fluorescent protein (YFP) as originally developed by Kerppola and colleagues for the study of transcription factors (21,22). BiFC provides a useful technique to examine protein-protein interaction in a normal cellular environment, as it is sensitive enough to detect interactions between proteins expressed at physiological levels (21,22). In this approach, oligomerization partners (c-Fes in this case) are fused to non-fluorescent N- and C-terminal portions of the YFP coding sequence and co-expressed in the same cell. Oligomerization brings the two YFP fragments into close proximity, resulting in structural complementation and fluorescence. Using the BiFC technique, we made the surprising discovery that c-Fes exists as a constitutive oligomer in cells, regardless of its autophosphorylation state. We further demonstrated that both of the c-Fes coiled-coil regions are essential for oligomerization in transfected COS-7 cells as well as cell lines where c-Fes is known to exert biological effects (HCT 116 colorectal cancer cells and K-562 CML cells). Together, these data demonstrate that c-Fes oligomerization is independent of activation and suggest that conformational changes, rather than oligomerization, govern c-Fes kinase activation and downstream signaling in vivo. The discovery that c-Fes exists as a pre-formed oligomer in vivo makes it unique among non-receptor protein-tyrosine kinases, and suggests new strategies for the design of small molecules that may modulate its activity in vivo. Such compounds may be of utility for the differentiation therapy of certain types of tumors.