A Tumor-Suppressor Function for the c-Fes Tyrosine Kinase: Interaction with Bcr/Abl in CML
September 1, 2000
A Tumor-Suppressor Function for the c-Fes Tyrosine Kinase: Interaction with Bcr/Abl in CML
By Jack M. Lionberger, and Thomas E. Smithgall, PhD
Cells interpret environmental cues through signal transduction pathways that start at the plasma membrane and terminate in the nucleus, cytoskeleton, or other subcellular compartments. Instructions regulating cell behavior are transmitted by sequential interactions between proteins that are tightly regulated in normal cells. The signaling molecules discussed here are protein-tyrosine kinases, enzymes that transfer phosphate from ATP to tyrosine residues either on themselves (a process known as autophosphorylation) or onto substrate molecules. As a result, tyrosine-phosphorylated proteins may bind to other proteins or exhibit enhanced enzymatic activity, leading to secondary signaling events. In the hematopoietic system, cytokines and other soluble growth factors often control protein-tyrosine kinase activity.1
Introduction
Receptors on the plasma membrane of myeloid hematopoietic cells recruit and activate multiple protein-tyrosine kinases in response to ligands such as interleukin-3 (IL-3). Kinase activation initiates a cascade of signaling events that ultimately reaches the nucleus, influencing the transcription of genes that regulate proliferation, differentiation, and survival. Receptor-induced tyrosine kinase activation is critical to signal transduction from the plasma membrane, and tight regulation of this process is required to prevent aberrant growth-regulatory signals. Many oncogene products constitutively activate normal tyrosine kinase cascades and cause inappropriate survival and proliferative signals as part of the oncogenic process. The focus of this article is Bcr/Abl, the oncogenic protein-tyrosine kinase associated with chronic myelogenous leukemia (CML).2
Background
In greater than 95% of CML cases, the abnormal cytogenetic marker known as the Philadelphia chromosome (Ph1) is present.3 This reciprocal translocation juxtaposes the breakpoint cluster region (c-bcr) on chromosome 22 and the c-abl tyrosine kinase on chromosome 9, resulting in the expression of the Bcr/Abl fusion protein. Bcr/Abl exhibits constitutive tyrosine kinase activity and transmits mitogenic and anti-apoptotic signals that are the causative factors in the initiation of CML. Early in the disease process, the single somatic cell sustaining the translocation is believed to be a primitive hematopoietic stem cell. Bcr/Abl enables this cell to escape normal regulation and expand clonally, giving rise to increasing numbers of transformed progeny.
Unlike the acute leukemias, Ph1-positive blast cells initially retain the capacity to undergo terminal differentiation, which leads to the accumulation of functionally mature granulocytes. When these cells become sufficient in number (sometimes in excess of 200,000 per microliter), they cause the symptoms associated with CML, including lethargy, malaise, night sweats, and low-grade fever. At this point the leukemia may be diagnosed, even though the initiating molecular events occurred years before the symptoms arose.
The chronic phase continues after diagnosis for an average of 3-5 years. Ultimately, the cells lose the capacity for terminal differentiation and the disease progresses to the blast-crisis stage, in which immature blast cells predominate. This phase is fatal within 9-18 months without a bone marrow transplant. Two key questions concerning this disease are: 1) why does the chronic phase last so long despite unregulated proliferation; and 2) what causes the disease to progress to the aggressive blast crisis phase? Here, we discuss evidence that Bcr/Abl may actually initiate and maintain a differentiation signal during the chronic phase, allowing the tumor to remain indolent for many years. That an oncogene can simultaneously promote and attenuate the neoplastic process may seem contradictory at first glance. However, this phenomenon can be reconciled by considering that Bcr/Abl signal transduction has many features in common with cytokine receptor signaling.
Bcr/Abl as an Activator
Bcr/Abl activates many of the same signal transduction pathways as cytokine receptors. However, Bcr/Abl signaling is independent of cytokines because of the constitutively active tyrosine kinase domain. Thus, proliferative and survival pathways are continuously activated by Bcr/Abl, leading to the expansion of myeloid precursor cells as described above. During the chronic phase of the disease, Bcr/Abl also may mimic cytokine signals for differentiation, explaining the accumulation of mature granulocytes that are the hallmark of the disease. Part of the Bcr/Abl differentiation signal may be mediated by c-Fes, a cytoplasmic tyrosine kinase normally involved in leukocyte differentiation.4
c-Fes Expression
Early studies implicating c-Fes both in normal differentiation and as a suppressor for Bcr/Abl employed the erythroleukemia cell line K-562. These cells were derived from the blast crisis phase of CML and express Bcr/Abl, but show no detectable c-Fes expression. When Fes is introduced into K-562 cells with an expression vector, the cells undergo growth arrest and express markers of terminal myeloid differentiation, suggesting that Fes may attenuate Bcr/Abl-mediated tumorigenesis.5 More recently, our laboratory demonstrated that Fes physically interacts with the normal Bcr protein through the N-terminal portion retained in Bcr/Abl.6,7 These findings suggested that Fes might interact directly with Bcr/Abl as well. Such an interaction may suppress Bcr/Abl-induced tumorigenesis, activate a normal Fes-dependent differentiation pathway, or both, thereby suppressing the CML phenotype and explaining the K-562 result. Alternatively, Fes-induced growth suppression may not be the result of a specific Bcr/Abl-Fes interaction, but instead may result from the more general effects Fes might have in immature hematopoietic cells. To investigate these possibilities, we developed a system in which the effect of Fes expression on Bcr/Abl signaling could be assessed more directly than in the K-562 cell model. As described below, this system is based on the murine hematopoietic cell line, DAGM.
The DAGM cell line is dependent on IL-3 for growth and survival. Withdrawal of cytokine leads to rapid cell death though an apoptotic mechanism. Importantly, expression of the p210 form of Bcr/Abl induces cytokine independence in this cell line, providing a useful model of transformation by Bcr/Abl.8 To study the effect of Fes expression on Bcr/Abl signaling in DAGM cells, we first created three populations of cells that over-expressed either wild-type human c-Fes, a kinase-defective form of Fes, or b-galactosidase as a negative control. Importantly, neither form of Fes affected the growth responses of the cells in the presence or absence of IL-3, indicating that the negative regulation of Fes kinase activity was intact.9
Fes and Bcr/Abl Signaling
Introduction of Bcr/Abl into these cell lines produced a dramatic result. The presence of wild-type Fes strongly suppressed Bcr/Abl-induced transformation of the cells to cytokine independence. This suppressive effect required the kinase activity of Fes, as cells expressing the kinase-inactive Fes mutant grew at the same rate as the b-galactosidase control population following introduction of Bcr/Abl. All of the cells proliferated at the same rate in the presence of IL-3, indicating that the effect of Fes is linked to Bcr/Abl and is not a general effect on cell proliferation. These data indicate that Fes can suppress Bcr/Abl signals for cytokine independence in DAGM cells, and are reminiscent of previous work with K-562 CML cells summarized above.
We also investigated the phosphorylation state of Fes in the presence and absence of Bcr/Abl. Neither wild-type nor kinase-defective Fes exhibited evidence of tyrosine phosphorylation in the absence of Bcr/Abl. However, co-expression with Bcr/Abl induced strong tyrosine phosphorylation of both forms of Fes, strongly suggesting that Fes is a direct target of Bcr/Abl in vivo. Binding experiments demonstrated that the Bcr-derived sequences of Bcr/Abl directly interact with Fes, providing further support for this possibility. Fes isolated from cells in which it was co-expressed with Bcr/Abl exhibited higher tyrosine kinase activity in vitro, providing direct evidence that Bcr/Abl can stimulate Fes tyrosine kinase activity.
Summary
Taken together, data summarized here suggest that c-Fes may be an important modulator of Bcr/Abl signaling in CML cells, which may occur by several mechanisms. In one model, Bcr/Abl may phosphorylate and activate Fes, which in turn generates signals for differentiation downstream. Recently, we have observed that expression of mutationally activated forms of Fes in the human cytokine-dependent myeloid leukemia cell line TF-1 is sufficient for terminal differentiation, which is consistent with this model. Tyrosine phosphorylation of Fes within its kinase domain is essential for full catalytic activity toward substrates. Although this phosphorylation event normally results from autophosphorylation in response to cytokines, Bcr/Abl appears to directly activate Fes as well.
In a second but not mutually exclusive model, Fes-induced phosphorylation of Bcr/Abl may skew its signaling toward differentiation-related pathways, thus explaining the observed biological effects. Consistent with this idea is the finding that Fes strongly phosphorylates Bcr/Abl when the proteins are co-expressed in a model system.9 Furthermore, Fes-mediated tyrosine phosphorylation changes the pattern of signaling proteins that bind to the normal Bcr protein.7 Regardless of the mechanism, loss of Fes expression may uncouple Bcr/Abl from a differentiation-related signaling pathway, favoring disease progression. Future studies will focus on the precise mechanism by which Fes causes growth inhibition and terminal differentiation in the presence of Bcr/Abl. (Dr. Smithgall is Associate Professor, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; and Mr. Lionberger is an MD/PhD graduate student, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE. )
References
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6. Maru Y, Peters KL, Afar DEH, et al. Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS. Mol Cell Biol 1995;15:835-842.
7. Peters KL, Smithgall TE. Tyrosine phosphorylation enhances the SH2 domain-binding activity of Bcr and inhibits Bcr interaction with 14-3-3 proteins. Cell Signal 1999;11:507-514.
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9. Lionberger JM, Smithgall TE. The c-Fes protein-tyrosine kinase suppresses cytokine-independent outgrowth of myeloid leukemia cells induced by Bcr-Abl. Cancer Res 2000;60:1097-1103.
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