Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk

Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol-3,4,5-trisphos phate (PtdIns-3,4,5-P-3) through the Btk pleckstrin homology (PH) domain, a n interaction thought to be required for Btk membrane translocation during B cell receptor signaling. Here, we report that interaction of PtdIns-3,4,5 -P-3, with the PH domain of Btk directly induces Btk enzymatic activation i n an in vitro kinase assay. A point mutation that reduces interaction of Pt dIns-3,4,5-B, with the Btk PH domain blocks in vitro PtdIns-3,4,5-P-3-depen dent Btk activation, whereas the PH domain deletion enhances Btk basal acti vity but eliminates the PtdIns-3,4,5-P-3-dependent stimulation. Btk kinase activity and the Btk activation loop phosphorylation site are both required for the PtdIns-3,4,5-P-3-mediated stimulation of Btk kinase activity. Toge ther, these results suggest that the Btk PH domain is positioned such that it normally suppresses both Btk kinase activity and access to substrates; w hen interacting with PtdIns-3,4,5-P-3, this suppression is relieved, produc ing apparent Btk activation. In addition, using Src family kinase inhibitor s and Btk catalytically inactive mutants, we demonstrate that in vivo, the activation of Btk is due to both Lyn phosphorylation and PtdIns-3,4,5-P-3-m ediated direct activation. Thus, the Btk-PtdIns-3,4,5-P-3 interaction serve s to translocate Btk to the membrane and directly regulate its signaling fu nction.