Another group of proteins, however, although showing phosphorylation in both the parental cells, and cells expressing FGFR1, were not detected in cells expressing the fusion kinase (table 2). this myeloproliferative disorder and provide new insights into the substrates of FGFR1 under defined conditions. strong class=”kwd-title” Keywords: Mass spectrometry, Phosphopeptide fingerprinting, FGFR1 fusion kinase, Abl kinase, Myeloproliferative disease INTRODUCTION The fusion of the zinc finger domain name of the ZNF198 gene with the kinase domain name of the FGFR1 (fibroblast growth factor receptor-1) gene was originally defined (1C4) in an AMPD (atypical myeloproliferative disease) with poor prognosis (1C3;5;6). Since then, variant translocations have been described (7C13) where, although the partner gene fused with FGFR1 is different, in all cases analyzed so far, the specific partner gene is usually anticipated to facilitate dimerization of the FGFR1 kinase domain name resulting in its constitutive activation (14C16). Aescin IIA The chimeric kinase protein, unlike FGFR1, is usually no longer tethered to the cytoplasmic membrane but resides predominantly in the cytoplasm (14;17) and acts as a constitutive tyrosine kinase. Members of the STAT family of transcription factors represent one of the well characterized protein families phosphorylated in the presence of the ZNF198/FGFR1 fusion kinase and are constitutively activated in the presence of the various fusion kinases (14;18;19). In the BaF3 growth factor-dependent hematopoietic cells, the fusion kinases can confer growth factor independence, although when expressed in primary mouse hematopoietic stem cells, is usually apparently not sufficient in itself to cause cell transformation (20). Various approaches have been taken to investigate the function of the FGFR1 fusion kinases more extensively. The constitutive dimerization of the wild type ZNF198 gene suggests that this event is usually important in the function of the protein. We have shown Aescin IIA that this fusion kinase can dimerize with the endogenous wild type gene (14;17) and thereby potentially affect the wild type protein in a dominant negative way. Thus, one of the early observations was that ZNF198 was found in a protein complex with the UBE2 (HHR6) and RAD18 proteins (21), which are essential for the correct repair of UVB- induced chromosome breaks and cell survival. In the presence of the fusion kinase this function is usually compromised and cells were more sensitive to this DNA damaging agent. Mass spectrometry studies also Aescin IIA exhibited that ZNF198 existed in a complex with a number of proteins predicted to be involved in RNA processing and transcription (22), and that some of these interactions were lost in the presence of the fusion kinase. In normal cells, ZNF198 is usually localized to punctate structures in the nucleus of most cells (15;17). In the presence of the fusion kinase, these structures are lost, presumably because the Aescin IIA cytoplasmic location of the kinase sequesters the wild type gene in the cytoplasm (17). We recently exhibited that ZNF198 interacts with SUMO1 and is localized in PML bodies. Mutation of the sumoylation site in ZNF198 results in loss of PML bodies and a predominantly cytoplasmic membrane localization of ZNF198. In the presence of the fusion kinase, Itgal the PML bodies are also lost. Thus, from all of these observations suggest that the expression of the fusion kinase may interfere with important protein-protein interactions in the cell, which may contribute to the oncogenicity of this protein. Gene expression studies have also shown that this SERPINB2 and HSPA1A genes are upregulated by ZNF198/FGFR1 and their presence is required to maintain stability of the chimeric protein (23;24). The observations that this STAT proteins are constitutively activated in the presence of the fusion kinase points to another of its potential transforming activities – the inappropriate activation of target genes in the cytoplasm through phosphorylation of crucial tyrosine residues. Identifying these target proteins, which can be clearly identified in western blots from whole cell lysates expressing the kinase gene (H Baumann, Roswell Park Malignancy Institute, pers comm.), has been difficult and largely restricted to candidate gene approaches. Several proteins activated by the FGFR1 kinase domain name are also activated in cells expressing the fusion kinase, although it is usually presumably the proteins that are specifically activated by the chimeric protein which are likely involved in transformation of the leukemic stem cell. Recently, Rush et al (25) described an approach to specifically identify proteins undergoing tyrosine phosphorylation using a combination of IP (immunoprecipitation) and MS (mass spectrometry). Proteins from whole cell lysates expressing a chimeric tyrosine kinase were Aescin IIA compared to cells not expressing the fusion.