Generation of Intracellular antibody directed against the non-Receptor Tyrosine Kinase Etk

564 Etk is a ∼70 KDa member of the Tec family of non-receptor Protein Tyrosine Kinases. It is expressed in a variety of hematopoietic, epithelial and endothelial cells and was shown to be involved in several cellular processes including proliferation, differentiation and motility. It was previously showed that dominant-negative inactivation of Etk results in a reduction in the transforming activity of v-Src in NIH3T3 cells. In this study, we constructed and expressed single domain antibodies directed against Etk kinase domain, isolated from phage display library. To assess binding and blocking activity of the antibodies, recombinant single domain antibodies were expressed in E.coli. Domain antibodies interacted with Etk kinase domain and blocked its enzymatic activity, as detected by Etk ability to phosphorylate the universal substrate, poly GluTyr, in an in-vitro kinase assay. Lipofectamin transfection and introduction of the single domain antibodies into NIH3T3 cells overexpressing v-Src results in an intracellular expression of the antibodies (Intrabodies). Intrabodies expression decreased Etk intrinsic kinase activity as demonstrated by an in vitro autophosphorylation of endogenous Etk, and led to a partial blockage (30-60%) of v-Src-induced colony formation on soft agar. Our data support the notion that Etk plays a role in Src-induced cellular transformation, and suggest the potential of intracellular antibodies in targeting cytoplasmic signaling molecules.