Induction of the plasminogen activator inhibitor-2 in cells expressing the ZNF198/FGFR1 fusion kinase that is involved in atypical myeloproliferative disease

Abstract

The ZNF198/FGFR1 fusion kinase associated with an atypical myeloproliferative disease is constitutively activated and regulates several STAT transcription factors. We used oligonucleotide microarrays to compare the gene-expression profiles between HEK-293 cells that stably express either the ZNF198/FGFR1 chimeric protein or the wild-type ZNF198 gene. Expression of the plasminogen activator inhibitor-2 (PAI-2/SERPINB2) was highly increased in cells expressing the fusion gene. Western blot analysis demonstrated that HEK-293 cells do not express PAI-2 endogenously, but in ZNF198/ FGFR1-expressing cells 2 molecular forms of PAI-2, which were 47 kDa and 32 kDa, were expressed intracellularly, and a 60-kDa form was secreted. Similarly, expression of ZNF198/FGFR1 in BaF/3 mouse hematopoietic cells also induced the expression of the PAI-2 protein. Immu IntroductionReciprocal chromosomal translocations in hematologic malignancies often result in the generation of novel fusion proteins. These chimeric proteins generally provide novel signaling functions that are considered to account for the oncogenic events in the leukemic cells. 1 One such chromosomal translocation was described in patients with an atypical form of myeloproliferative disease (MPD) that is associated with T-cell leukemia/lymphoma and peripheralblood eosinophilia 2 and involves a reciprocal t(8;13)(p11;q12) rearrangement. [3][4][5][6][7][8] In this translocation a zinc finger gene, ZNF198, is fused in-frame with the fibroblast growth factor receptor 1 (FGFR1). The resultant protein contains the zinc finger motif and proline-rich domain of ZNF198 and the intracellular tyrosine kinase domain of FGFR1. [9][10][11][12] The resulting ZNF198/FGFR1 fusion protein is a ligand-independent, constitutively active cytoplasmic tyrosine kinase, unlike FGFR1, which is a membrane-bound receptor tyrosine kinase. Although the signaling functions of the ZNF198/FGFR1 fusion kinase were proposed based on the functions of FGFR1, the fusion kinase has been suggested to have unique signaling functions that are different from FGFR1 because of its cytoplasmic localization and constitutive tyrosine kinase activity. [13][14] The oncogenic potential of the ZNF198/FGFR1 fusion kinase was demonstrated in IL-3-dependent Ba/F3 cells, where stable expression of the chimeric fusion protein produced IL-3-independent growth. [15][16][17] Although activation of FGFR1 is known to result from dimerization following ligand binding, the ZNF198/ FGFR1 fusion kinase is activated through homodimerization of the ZNF198 zinc finger domain. [14][15][16] ZNF198 is a widely expressed gene that encodes a 1377-amino acid nuclear protein with a molecular mass of 155 kDa. [9][10][11][12] Prominent features of ZNF198 are the 5 zinc finger motifs and a proline-rich domain (PRD) within the central part of the protein and an acidic domain at the C-terminal end of the protein. 9 On the basis of the structure and the type of zinc fingers present in ZNF198, it has been predicted to have a function in protein-prote...

Genetic fingerprinting of the development and progression of T-cell lymphoma in a murine model of atypical myeloproliferative disorder initiated by the ZNF198–fibroblast growth factor receptor-1 chimeric tyrosine kinase

“…The infection efficiency, as estimated by flow cytometry for GFP-positive cells, was approximately 16%, and the expression of the fusion protein in these transduced BM cells was confirmed either by Western blotting using the FGFR1 antibody described by Kasyapa et al 29 or PCR (data not shown). After 5-day culture, these infected BM cells were transplanted into recipient female mice ( Figure 1B).…”

8p11 myeloproliferative syndrome: a review

“…An additional pathway involving the plasminogen activator inhibitor 2 gene has also been implicated in cells with the ZNF198-FGFR1 fusion gene. By gene expression profiling, Kasyapa et al [68] demonstrated overexpression of plasminogen activator inhibitor 2, a proteinase inhibitor that prevents targeted proteolytic degradation, which function is not observed in activated native FGFR1 signaling. As such, plasminogen activator inhibitor 2 plays a role in protein stabilization but also confers resistance to tumor necrosis factor α-induced apoptosis; this latter feature is proposed to contribute to the aggressive nature of the cells carrying the ZNF198-FGFR1 fusion.…”

“…Xiao et al [9,24] demonstrated that the Nterminus proline-rich motifs and not the zinc finger motifs of ZNF198 are required for oligomerization of the fusion protein. Whereas native FGFR1 is tethered to the cell membrane, the ZNF198-FGFR1 fusion protein and other FGFR1 translocation products are found in the cytoplasm due to a loss of a putative nuclear localization signal located in the C-terminus end of ZNF198 [61,67,68]. The adaptor protein FRS2α binds to a sequence recognition motif of native FGFR1 near the juxtamembrane domain, between amino acids 407 and 433 [69].…”

Fibroblast Growth Factor Receptor and Platelet-Derived Growth Factor Receptor Abnormalities in Eosinophilic Myeloproliferative Disorders

“…Sequestration of these phosphorylated effectors by the 14-3-3 family of conserved, phosphoserine/threonine-binding proteins has been suggested to be an important mechanism by which ZNF198-FGFR1 abrogates apoptosis [11]. ZNF198-FGFR1 induces overexpression of the plasminogen activator inhibitor 2 (PAI-2/SERPINB2) in Ba/F3 and HEK-293 cells, and this has also been suggested to play a role in anti-apoptotic signalling [12]. …”