Phosphorylation of the SSBP2 and ABL proteins by the ZNF198-FGFR1 fusion kinase seen in atypical myeloproliferative disorders as revealed by phosphopeptide-specific MS

Abstract

The ZNF198-fibroblast growth factor receptor-1 (FGFR1) fusion kinase is a constitutively activated tyrosine kinase associated with a specific atypical myeloproliferative disease. The chimeric protein localizes to the cytoplasm, unlike the wild type FGFR1 receptor kinase, and presumably inappropriately phosphorylates specific targets as part of the oncogenic signaling cascade. Other than known targets of the FGFR1 kinase itself, few specific targets of ZNF198-FGFR1 have been identified. Using a genetically engineered HEK 293 cell system, we have identified proteins that are specifically phosphorylated in the presence of the fusion kinase using anti-phosphotyrosine immunoprecipitation and MS. Compared with 293 cells expressing exongenous wild type FGFR1, ZNF198-FGFR1 is associated with phosphorylation of several proteins including SSBP2, ABL, FLJ14235, CALM and TRIM4 proteins. The specificity of the phosphorylation events in the SSBP2 and ABL proteins, which have previously been implicated in leukemogenesis, was further confirmed independently using immunoprecipitation with protein-specific antibodies and Western blotting. The MS analysis also identified the phosphorylation events in the ZNF198 moiety in the chimeric protein that might be related to its function. These studies identify the intersection of several different leukemia-related pathways in the development of this myeloproliferative disorder and provide new insights into the substrates of FGFR1 under defined conditions.

Figure 1

Analysis of SSBP2 protein phosphorylation in ZNF198-FGFR1 expressing cells. Western blot analysis of 293 cells shows a doublet at 40kDa in cells expressing the empty vector and the fusion kinase (ZF). High level expression is seen in K562 cells which is included as a positive control. When lysates from the same cell lines are used in IP studies using anti-SSBP2 antibodies and probed with anti-phosphotyrosine, the 40 kDa protein is seen only in the ZF cells and K562 control cells and not 293 cells expressing the empty vector.

Figure 2

Lysates from 293 cells transfected with exogenous ABL show high level of protein expression. 293 cells expressing the ZNF198-FGFR1 fusion kinase express ABL endogenously and increased expression is seen in these cells following transient expression of exogenous ABL (top). Non Specific bands are shown (*). Immunoprecipitation of ABL from transiently transfected 293 cells, when probed with antiphosphotyrosine antibodies, fails to show phospho-ABL despite high levels of ABL protein expression. In 293 cells expressing the ZNF198-FGFR1 kinase the 140 kDa Phospho-ABL is clearly seen in the host cells and after transient transfection with exogenous ABL. The 170 kDa fusion kinase is also seen demonstrating that ABL is in a complex with this protein.