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-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 mass spectrometry. 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, were 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.

8p11 myeloproliferative syndrome: a review

“…A recent study using proteomics methods has identified a number of proteins that are specifically phosphorylated as a result of the t(8;13)(p11;q12) including SSBP2 and ABL [77]. SSBP2 is a DNAbinding protein that regulates gene expression, and ABL is involved in cytoskeletal organization.…”

Why and how to investigate the role of protein phosphorylation in ZIP and ZnT zinc transporter activity and regulation

“…Phosphorylation-specific antibodies have been used at both the protein and peptide levels. However, the poor specificity and sensitivity of anti-phosphoserine and antiphosphothreonine antibodies have limited their use leaving the more specific anti-phosphotyrosine antibodies as the preferred method for antibody-based enrichment [191][192][193][194][195][196][197][198]. In nature, however, the vast majority of protein phosphorylation is on serine and threonine residues [199].…”

Centrosomal targeting of tyrosine kinase activity does not enhance oncogenicity in chronic myeloproliferative disorders

“…6 At the molecular level, specific downstream phosphorylation targets have been identified for the ZNF-FGFR1 leukemic fusion as opposed to WT FGFR1. 48 In conclusion, the fusion of a tyrosine kinase to a centrosomal translocation partner is a recurring phenomenon in CMD. However, the translocation partner does not regularly target the chimeric fusion to the centrosome.…”