The FLT3 gene is one of the most frequently mutated genes in hematologic malignancies. Mutated FLT3 has been found to lead to strong constitutive activation of several signaling proteins such as STAT5, CBL, VAV, SHP2, NF-kB, and AKT. Recently, we have reported that FLT3 contributed to leukemogenesis through generating a ETV6/FLT3 fusion gene in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation, which is the first report to show that FLT3 fusion gene can be involved in leukemogenesis. The ETV6/FLT3 which contained the helix-loop-helix oligomerization domain of ETV6 fused to the juxtamembrane (JM) domain plus tyrosine kinase (TK) domains of FLT3, conferred interleukin-3-independent growth on Ba/F3 cells (
Vu HA, et al. LEUKEMIA 20:1414–21, 2006
). To assess a role of the JM domain of FLT3 in the ETV6/FLT3 protein, we generated a series of ETV6/FLT3 mutants (tyrosine to phenylalanine substitution and deletion of the JM domain or N-terminal region of the TK1 domain). Each mutant was introduced into Ba/F3 cells and downstream signaling as well as cell proliferation was investigated. We found that the wild-type ETV6/FLT3 in Ba/F3 cells was a constitutively activated tyrosine kinase that led to activation of STAT5, AKT, MAPK, and NF-kB pathways as well as up-regulation of PIM-1. Deletion of the JM domain retained compatible level of autophosphorylation of the fusion protein as well as activation of STAT5, AKT and MAPK, suggesting that the JM domain is dispensable for STAT5, MAPK and AKT activations. In contrast, deletion of the JM domain did abrogate interleukin-3-independent growth of Ba/F3 cells, PIM-1 up-regulation, and activation of NF-kB. Importantly, while substitutions of other 3 tyrosines (589, 597, and 599) had no affect on NF-kB activation, the substitution of tyrosine 591 to phenylalanine abrogated this activation. Our results suggest that the tyrosine 591 in JM domain of FLT3 in ETV6/FLT3 is critical for NF-kB activation to support cell survival and PIM-1 up-regulation.
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