One of the challenges related to a better understanding of the function of novel fusion kinase genes has been to identify the substrate proteins that are inappropriately activated by them, since it is likely that these events are important in the transforming ability of the fusion kinase. Usually these studies have been largely guided by a prior knowledge of the proteins that are known to be activated by the wild type kinase domain involved in the chromosome translocation. In this case, for example, the role of PLCG in downstream signaling of FGFR1 has been extensively investigated and was clearly identified in this study. The recent development of an unbiased way of screening for tyrosine phosphorylation events in cells expressing these chimeric kinases, however, has provided new opportunities to define the role of these fusion genes in cancer development (31
We have identified a change in the profile of tyrosine phosphorylation events that appear to be specific to various circumstances of FGFR1 expression, as well as specifically related to the presence of the ZNF198-FGFR1 oncogene. It is important to understand at the outset, however, that the analysis of tyrosine phosphorylation events in these experiments is not comprehensive and was not designed to be quantitative. By virtue of the technology, not all phosphorylated peptides will be identified in a given experiment, and it is likely that the more abundant proteins will be identified preferentially, although this does not necessarily mean that low abundance proteins will be overlooked. This concept was clearly seen in the comparison between phosphorylation events resulting from over expression of wild type FGFR1 and ligand-induced FGFR1 activity. In this comparison, although common targets were identified, a spectrum of differentially phosphorylated proteins were also identified in each case. Thus, within any given analysis, the ability to identify any particular phosphorylated protein is a balance between the presence/absence of the event and the frequency of that event in the cells being studied. Thus, the analysis does not represent the sum total of events in each cell line but provides clues about the dominant events that are associated with the presence of a specific type of kinase activity. In our experimental design, we selected proteins that were apparently exclusively phosphorylated in cells expressing the fusion kinase, although valuable information about FGFR1 targets was also obtained.
The function of the FGFR1-fusion proteins that are associated with atypical myeloproliferative disorder have been largely studied in experimental cell systems, because of the lack of cell lines that specifically carry the common variants of this chromosome rearrangement. In a recent survey of leukemia cell lines, however, Gu et al (33
) identified an AML derived line, KG-1, that apparently carried a rare variant, FGFR1OP2, as the FGFR1 fusion gene partner. We have used the model 293 cell system which, although not specifically from a hematopoietic lineage, shows activation of the STAT and PLCG proteins by the fusion kinase as well as induction of SERPINB2, which was also seen in BaF3 hematopoietic cells expressing the ZNF198-FGFR1 gene (23
). In this case, therefore, many of the known events demonstrated a normally functioning kinase. The advantage of the 293 system, is that we can specifically investigate novel events that are the consequences of the expression of ZNF198-FGFR1, since these cells can be compared directly to parental cells which differ only by the fact that they do not express the fusion kinase. In keeping with this concept, the ZNF198 component of the fusion kinase gene was only shown to be autophosphorylated in this cell system, demonstrating the specificity of this event. Thus, these cells provide an easily manipulated substrate in which to investigate downstream targets of the fusion kinase with the caveat that this survey is not comprehensive, since some lineage specific proteins may not be represented. Despite this potential limitation, we have successfully identified several proteins which appear to be specific targets of ZNF198-FGFR1, as well as obtaining some insights into the targets of FGFR1. The MS/PY approach has the added advantage that the specific phosphorylation sites within the substrate proteins are also identified, which allowed us to determine which of the tyrosines in the ZNF198 moiety in the chimeric protein are phosphorylated. It will now be possible, through site directed mutagenesis, to alter these sites to determine whether they have any effect on the function of the fusion kinase, specifically the dimerization event that is facilitated by the zinc finger motifs retained in the chimeric protein which are essential for the activation of the kinase domain (14
Within this overall cell system we also demonstrated that some of the signaling events associated with FGFR1 could be readily detected, such as phosphorylation of PLC-gamma as well as autophosphorylation events at Y653/654 in FGFR1, which occur within the kinase domain and which are essential for its activation (34
). It was interesting that the Y766 phosphorylation event was not observed in any of these cell clones, since this is an essential event for binding to PLC-gamma and efficient endocytosis of FGFR1, although not critical for mitogenic responses (34
). Endocytosis, however, is not relevant for the chimeric kinase which, unlike wild type FGFR1, is located constitutively within the cytoplasm and is no longer tethered to the membrane (17
Two genes identified by MS analysis as apparently specifically activated by the ZNF198-FGFR1 kinase, have previously been implicated in leukemia biology. The specificity of the phosphorylation events in both of these cases was subsequently independently confirmed. Thus, the SSBP2 gene, encodes a protein presumed to function as a single stranded DNA binding protein that can regulate gene expression and, by modulating LIM domain proteins, affects growth and differentiation of hematopoietic cells by interacting with LIM binding protein 1 (30
). SSBP2 was found in a multiprotein complex with the SCL/Tal1 transcription factor (38
) and was also shown to be downregulated in AML (39
). Recently it has also been suggested that SSBP2 is involved in prostate cancer development where it is preferentially inactivated in advanced stage tumors and reexpression of the gene in prostate cells induces cell cycle arrest (40
). More importantly in HEK 293 cells endogenous SSBP2 localizes to aggresomes, due its direct interaction with the adenoviral oncoprotein E1b55K (41
). Differential phosphorylation of SSBP2 by the fusion kinase despite the altered subcellular localization of SSBP2 suggests a potentially important role for this interaction. The implications from our analysis is that, in cells expressing ZNF198-FGFR1, the phosphorylation of SSBP2 is important in a signaling cascade that is initiated by the chimeric kinase. One of the potential predicted functions of SSBP2 is to interact with the cytoskeleton, mediated through the FISH domain in this protein, which is characteristic of proteins that interact with the cytoskeleton.
Interestingly, the other protein that was identified and independently confirmed as a target of the ZNF198-FGFR1 kinase, ABL, has also been implicated in cytoskeleton reorganization, although its primary role has been defined as a constitutively active kinase that also affects downstream targets. In the ZNF198-FGFR1 expressing cells, ABL is phosphorylated at Y412, which is well known to be required for full activation of the kinase function.
The suggestion from our MS data is that, through their involvement as downstream targets of ZNF198-FGFR1, SSBP2 and ABL demonstrates the intersection of different pathways known to be implicated in leukemogenesis in the development of this stem cell leukemia. Further evidence of this intersection comes from observations implicating ZNF198-FGFR1 in the regulation of PML body formation (17
). The formation of PML bodies requires sumoylation of the PML protein and we have previously demonstrated that the wild type ZNF198 protein is essential for PML body formation (23
). The presence of the ZNF198-FGFR1 protein in the cytoplasm in some way inhibits SUMO1 localization to the nucleus and is associated with absence of PML bodies. It is possible, therefore, that in addition to activating key signal transduction pathways, ZNF198-FGFR1 also acts in a manner similar to the PML-RARA fusion protein in promyelocytic leukemia by preventing PML body formation which contributes to the leukemogenesis process.