THE ROLE OF THE SRC-LIKE ADAPTOR PROTEINS, SLAP AND SLAP2,IN FLT3 RECEPTOR TYROSINE KINASE SIGNALING

Abstract

Introduction: The Src-like Adaptor Proteins, SLAP and SLAP2, are primarily expressed in hematopoietic cells and have an established role in the negative regulation of T-cell signaling. SLAP and SLAP2 act by recruiting the E3 ubiquitin ligase, c-Cbl, to components of the T-cell receptor leading to receptor ubiquitination and down-regulation. In addition to having a role in the negative regulation of T cell signaling, our lab has previously shown that SLAP and SLAP2 interact with the CSF-1 receptor, Fms, and interfere with CSF-1R function. The CSF-1 receptor is part of the type III family of receptor tyrosine kinases (RTKs) which includes the PDGF, c-Kit and Flt3 receptors. The Flt3 receptor plays an important role in both B-cell and Dendritic cell development and is commonly deregulated in acute myeloid leukemia (AML). The objective of this study is to determine whether SLAP and SLAP2 are involved in the negative regulation of Flt3 signaling. Methods: To study Flt3 signaling in vivo, murine Dendritic cells(DCs) were utilized. We first analyzed the splenic DC populations in SLAP1/2-/- double knock-out mice and determinedthat there is a defect in the myeloid DC population (CD11b/CD11c+). To study receptor signaling, we generated DCs in vitro from the bone marrow of these mice using Flt3 ligand(Flt3L). SLAP1/2-/- mice producedless BM-DCs than WT mice and the functionality of these DCs is currently being investigated. Results: SLAP and SLAP2 interact with the Flt3 receptor in vitro and bind to the receptor within the juxtamembrane region. BaF3/Flt3 cells that stably express SLAP, SLAP2 and SH2 mutant forms of these proteins were generated. The expression of SLAP2 in these lines resulted in a change in the phosphorylation kinetics of the receptor. In addition, growth of SLAP2-expressingcells in response to Flt3 ligand was enhanced in comparison to cells expressinga GFP vector alone. Conclusions: SLAP and SLAP2 may be involved in Flt3 regulation and appear to play arole in Dendritic cell differenation, a process that is dependent on Flt3 signalling.