Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex

Bruton's tyrosine kinase (BTK) is a member of the Src-related Tec family of protein tyrosine kinases, Mutations in the btk gene have been linked to se vere developmental blocks in human B-cell ontogeny leading to X-linked agam maglobulinemia. Here, we provide unique biochemical and genetic evidence th at BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells. The Src homology 2, pleckstrin homology (PH), an d kinase domains of BTK are all individually important and apparently indis pensable, but not sufficient, for its function as a negative regulator of F as-mediated apoptosis, BTK associates with Fas via its kinase and PH domain s and prevents the FAS-FADD interaction, which is essential for the recruit ment and activation of FLICE by Fas during the apoptotic signal. Fas-resist ant DT-40 lymphoma B-cells rendered BTK-deficient through targeted disrupti on of the btk gene by homologous recombination knockout under went apoptosi s after Fas ligation, but wild-type DT-40 cells or BTK deficient DT-40 cell s reconstituted with wild-type human btk gene did not. Introduction of an S rc homology 2 domain, a PH domain, or a kinase domain mutant human btk gene into BTK-deficient cells did not restore the resistance to Fas-mediated ap optosis. introduction of wild type BTK protein by electroporation rendered BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas Ligatio n, BTK deficient RAMOS-1 human Burkitt's leukemia cells underwent apoptosis after Fas ligation, whereas BTK-positive NALM-6-UM1 human B-cell precursor leukemia cells expressing similar levels of Fas did not. Treatment of the anti-Fas-resistant NALM-6-UM1 cells with the leflunomide metabolite analog alpha-cyano-beta-methyl-beta-hydroxy-N-(2, 5-dibromophenyl)propenamide, a p otent inhibitor of BTK, abrogated the BTK-Fas association without affecting the expression levels of BTK or Fas and rendered them sensitive to Fas-med iated apoptosis, The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell pr ecursors during normal E-cell ontogeny may be reciprocally regulated by Fas and BTK.