Saito K, Scharenberg A M, Kinet J P
Division of Allergy and Immunology, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 2001 May 11;276(19):16201-6. doi: 10.1074/jbc.M100873200. Epub 2001 Jan 30.
Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) through the Btk pleckstrin homology (PH) domain, an 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 in an in vitro kinase assay. A point mutation that reduces interaction of PtdIns-3,4,5-P(3) with the Btk PH domain blocks in vitro PtdIns-3,4,5-P(3)-dependent Btk activation, whereas the PH domain deletion enhances Btk basal activity 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. Together, these results suggest that the Btk PH domain is positioned such that it normally suppresses both Btk kinase activity and access to substrates; when interacting with PtdIns-3,4,5-P(3), this suppression is relieved, producing apparent Btk activation. In addition, using Src family kinase inhibitors 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)-mediated direct activation. Thus, the Btk-PtdIns-3,4,5-P(3) interaction serves to translocate Btk to the membrane and directly regulate its signaling function.
布鲁顿酪氨酸激酶（Btk）通过Btk普列克底物蛋白同源（PH）结构域与磷脂酰肌醇-3,4,5-三磷酸（PtdIns-3,4,5-P(3)）结合，这种相互作用被认为是B细胞受体信号传导过程中Btk膜转位所必需的。在此，我们报道在体外激酶测定中，PtdIns-3,4,5-P(3)与Btk的PH结构域相互作用直接诱导Btk酶激活。一个降低PtdIns-3,4,5-P(3)与Btk PH结构域相互作用的点突变会阻断体外PtdIns-3,4,5-P(3)依赖性Btk激活，而PH结构域缺失会增强Btk基础活性，但消除PtdIns-3,4,5-P(3)依赖性刺激。Btk激酶活性和Btk激活环磷酸化位点都是PtdIns-3,4,5-P(3)介导的Btk激酶活性刺激所必需的。这些结果共同表明，Btk的PH结构域的定位使其通常抑制Btk激酶活性和对底物的接近；当与PtdIns-3,4,5-P(3)相互作用时，这种抑制被解除，产生明显的Btk激活。此外，使用Src家族激酶抑制剂和Btk催化失活突变体，我们证明在体内，Btk的激活是由于Lyn磷酸化和PtdIns-3,4,5-P(3)介导的直接激活。因此，Btk与PtdIns-3,4,5-P(3)的相互作用有助于将Btk转运到膜上并直接调节其信号功能。
