Identification of Novel Phosphorylation Sites and Kinetics in Flt3 Receptor Wt, ITD and D835Y

FMS-like tyrosine kinase-3 (Flt3) is a receptor tyrosine kinase, which is normally expressed in hematopoietic progenitor cells. It has been implicated as a major cause of transformation in acute myeloid leukemia (AML). There are two types of Flt3 gene mutations have been identified in AML: duplication of amino acids in the juxtamembrane region- Internal Tandem Duplication (ITD) and an activation loop point-mutation of D835 in kinase domain. These mutations cause constitutive activation or over expression of Flt3 receptor and therefore lead to alteration in signal transduction. These alterations occur in approximately 30% of AML patients. High occurrence of these mutations in the Flt3 receptor in AML patients makes it one of the most interesting therapeutic targets. In this study we have identified three novel in vivo phosphorylation sites of Flt3 receptor and further compared the activity of phosphorylation sites of Flt3 wild type, Flt3 ITD and D835Y mutations by using homemade phospho-specific antibodies directed against specific tyrosines. For this study murine hematopoietic Ba/F3 cells were stably transfected with wild-type Flt3, ITD and D835Y mutations. We have confirmed that the activation of the wild type Flt3 receptor is ligand dependent and response in a time dependent manner, but Flt3-ITD and D835Y are constitutive active and ligand independent. Phosphorylated tyrosines 589, 591, 599, 726, 768, 793, 842, and 955 of Flt3 receptor were investigated and shown to be differentially activated in wild-type versus the mutated receptor. Using this data we can further study the mechanisms of signaling pathways of the Flt3 receptor that are involved in many biological responses and understand the mechanism by which Flt3 ITD and D835Y functions in pathological conditions.