Possible Role of the Activation Loop Tyrosine Residue (Y842) in FLT3/ITD Signaling May Be Mediated by SHP-2.

Internal tandem duplication (ITD) mutations in the FLT3 juxtamembrane domain lead to constitutive activation of FLT3 tyrosine kinase activity and cooperate in the oncogenic transformation of hematopoietic cells. Although numerous signaling molecules are known to be involved in FLT3/ITD signaling, the sites of interaction and mechanisms are not fully understood. In this study, FLT3/ITD protein was immunoprecipitated from TF-1/ITD cells and subjected to nanoLC-MS/MS analysis for determination of tyrosine phosphorylation sites. We found that tyrosine residue 842 (Y842) in the FLT3/ITD activation loop is constitutively phosphorylated. Mutation of this tyrosine residue to phenylalanine (FLT3/ITDY842F) reduces FLT3/ITD phosphorylation and activation of FLT3 downstream targets, including STAT5, AKT and ERK/MAPK. CEP-701, a selective FLT3 inhibitor, inhibits the proliferation of BaF3 cells expressing FLT3/ITD or FLT3/ITDY842F mutant with a similar potency. This implies CEP-701 targets FLT3/ITDY842F as effectively as FLT3/ITD. To identify the signaling pathway(s) initiated by FLT3/ITD Y842, SHP-2 and other molecules downstream of FLT3 were investigated. SHP-2 is known to be activated by FLT3 signaling and its interaction with Gab2 regulates the ERK/MAPK pathway. Previous studies also suggested that SHP-2 is possibly involved in STAT5 activation. Co-immunoprecipitation experiments revealed that SHP-2 association with the FLT3/ITDY842F mutant was considerably diminished as compared to the FLT3/ITD receptor. In addition, the interaction of SHP-2 with Gab2 adaptor protein was greatly compromised in FLT3/ITDY842F expressing BaF3 cells. Taken together, our data suggests that SHP-2 binds FLT3/ITD, either directly or indirectly, and contributes to the signaling pathways activated by mutant FLT3.