Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

Fms-like tyrosine kinase-3 (Flt3), a growth factor receptor normally expressed in hematopoietic progenitor cells, has been shown to have an important role in development of acute myeloid leukemia (AML) due to activating mutations. Flt3 mutations are found in approximately one-third of AML patients and correlate with a poor prognosis, thus making the Flt3 receptor a potential therapeutic target. The aim of the investigation was to analyze the kinetics and specificity of Flt3 autophosphorylation in wild-type Flt3 as well as in oncogenic Flt3 mutants.

We have used Ba/F3 cells stably expressing either wild-type, internal tandem duplication, or D835Y mutants of Flt3 in order to compare the site selectivity of tyrosine phosphorylation sites. By the use of a panel of phosphospecific antibodies directed against potential tyrosine phosphorylation sites in Flt3, we identified several novel phosphorylation sites in Flt3 and studied the kinetics and specificity of ligand-induced phosphorylation in living cells.

Eight phosphorylated tyrosines (pY589, pY591, pY599, pY726, pY768, pY793, pY842, and pY955) were investigated and shown to be differentially phosphorylated in the wild-type versus the mutated receptors. Furthermore, we show that tyrosines 726, 793, and 842 are novel phosphorylation sites of Flt3 in intact cells.

In this study, we have looked at the site-specific phosphorylation in the wild-type Flt3 in comparison to the mutants found in AML. We observed not only quantitative changes but, more importantly, qualitative differences in the phosphorylation patterns of the wild-type and the mutated Flt3 receptors, which might enhance the understanding of the mechanisms by which Flt3 contributes to AML in patients with mutations in Flt3.