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 severe developmental blocks in human B-cell ontogeny leading to X-linked agammaglobulinemia. Here, we provide unique biochemical and genetic evidence that 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), and kinase domains of BTK are all individually important and apparently indispensable, but not sufficient, for its function as a negative regulator of Fas-mediated apoptosis. BTK associates with Fas via its kinase and PH domains and prevents the FAS-FADD interaction, which is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Fas-resistant DT-40 lymphoma B-cells rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout underwent apoptosis after Fas ligation, but wild-type DT-40 cells or BTK-deficient DT-40 cells reconstituted with wild-type human btk gene did not. Introduction of an Src homology 2 domain, a PH domain, or a kinase domain mutant humanbtk gene into BTK-deficient cells did not restore the resistance to Fas-mediated apoptosis. Introduction of wild-type BTK protein by electroporation rendered BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas ligation. 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 α-cyano-β-methyl-β-hydroxy-N-(2, 5-dibromophenyl)propenamide, a potent inhibitor of BTK, abrogated the BTK-Fas association without affecting the expression levels of BTK or Fas and rendered them sensitive to Fas-mediated apoptosis. The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell precursors during normal B-cell ontogeny may be reciprocally regulated by Fas and BTK.

[1]  S. Desiderio,et al.  BAP-135, a target for Bruton's tyrosine kinase in response to B cell receptor engagement. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  D J Rawlings,et al.  Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice. , 1993, Science.

[3]  K. Schulze-Osthoff,et al.  Divergent signalling via APO‐1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. , 1994, The EMBO journal.

[4]  S. Korsmeyer.  Regulators of cell death. , 1995, Trends in genetics : TIG.

[5]  R. Perlmutter,et al.  Impaired expansion of mouse B cell progenitors lacking Btk. , 1995, Immunity.

[6]  M. Pinkoski,et al.  A Physical Interaction between the Cell Death Protein Fas and the Tyrosine Kinase p59(*) , 1996, The Journal of Biological Chemistry.

[7]  V. Fadok,et al.  Apoptosis and programmed cell death in immunity. , 1992, Annual review of immunology.

[8]  F. Uckun,et al.  Cytotoxic activity of epidermal growth factor-genistein against breast cancer cells. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[9]  Ornella Parolini,et al.  Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia , 1993, Cell.

[10]  David Wallach,et al.  Involvement of MACH, a Novel MORT1/FADD-Interacting Protease, in Fas/APO-1- and TNF Receptor–Induced Cell Death , 1996, Cell.

[11]  D. Bentley,et al.  The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases , 1993, Nature.

[12]  R. Lefkowitz,et al.  Binding of G protein beta gamma-subunits to pleckstrin homology domains. , 1994, The Journal of biological chemistry.

[13]  G. Evan,et al.  A License to Kill , 1996, Cell.

[14]  Arul M. Chinnaiyan,et al.  FADD, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis , 1995, Cell.

[15]  H. Wortis,et al.  An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[16]  F. Rieux-Laucat,et al.  Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. , 1995, Science.

[17]  Stephen W. Fesik,et al.  NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain , 1996, Nature.

[18]  T. Kurosaki,et al.  Molecular mechanisms in B cell antigen receptor signaling. , 1997, Current opinion in immunology.

[19]  H. Sather,et al.  Cellular Expression of Antiapoptotic BCL-2 Oncoprotein in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: A Children's Cancer Group Study , 1997 .

[20]  O. Witte,et al.  Bruton's Tyrosine Kinase is a Key Regulator in B‐Cell Development , 1994, Immunological reviews.

[21]  L. Tuel-ahlgren,et al.  Engagement of the CD19 receptor on human B-lineage leukemia cells activates LCK tyrosine kinase and facilitates radiation-induced apoptosis. , 1993, Radiation research.

[22]  D. Berglund,et al.  Introduction of antibody into viable cells using electroporation. , 1991, Cytometry.

[23]  M. Saraste,et al.  Structure of the PH domain and Btk motif from Bruton's tyrosine kinase: molecular explanations for X‐linked agammaglobulinaemia , 1997, The EMBO journal.

[24]  A. Burkhardt,et al.  Signal transduction through the CD19 receptor during discrete developmental stages of human B-cell ontogeny. , 1993, The Journal of biological chemistry.

[25]  T. Kurosaki,et al.  BTK as a Mediator of Radiation-Induced Apoptosis in DT-40 Lymphoma B Cells , 1996, Science.

[26]  M. Vihinen.  BTKbase: a database of XLA-causing mutations , 1995 .

[27]  F. Alt,et al.  Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G. Linette,et al.  Differentiation and cell death: lessons from the immune system. , 1994, Current opinion in cell biology.

[29]  J. Tschopp,et al.  Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors , 1997, Nature.

[30]  Warren Strober,et al.  Dominant interfering fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome , 1995, Cell.

[31]  D. S. Hsu,et al.  Putative human blue-light photoreceptors hCRY1 and hCRY2 are flavoproteins. , 1996, Biochemistry.

[32]  S. Nagata,et al.  Apoptosis by Death Factor , 1997, Cell.

[33]  T. Pawson,et al.  SH2 and SH3 domains: From structure to function , 1992, Cell.

[34]  H. Steller.  Mechanisms and genes of cellular suicide , 1995, Science.

[35]  W. Fiers,et al.  Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis , 1995, Nature.

[36]  J. Fargnoli,et al.  Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase , 1995, Molecular and cellular biology.

[37]  T. Watanabe,et al.  Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in Lyn-deficient mice , 1996, The Journal of experimental medicine.

[38]  E. Gulbins,et al.  Activation of Src‐family tyrosine kinases during Fas‐induced apoptosis , 1996, Journal of leukocyte biology.

[39]  J. Camonis,et al.  A Novel Protein That Interacts with the Death Domain of Fas/APO1 Contains a Sequence Motif Related to the Death Domain (*) , 1995, The Journal of Biological Chemistry.

[40]  M. Peter,et al.  Cytotoxicity‐dependent APO‐1 (Fas/CD95)‐associated proteins form a death‐inducing signaling complex (DISC) with the receptor. , 1995, The EMBO journal.

[41]  A. Burkhardt,et al.  Physical and Functional Interactions between Lyn and p34 Kinases in Irradiated Human B-cell Precursors (*) , 1996, The Journal of Biological Chemistry.

[42]  A. Burkhardt,et al.  Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases , 1995, Science.

[43]  Guy S. Salvesen,et al.  X-linked IAP is a direct inhibitor of cell-death proteases , 1997, Nature.

[44]  T. Kurosaki,et al.  Stimulation of Src Family Protein-tyrosine Kinases as a Proximal and Mandatory Step for SYK Kinase-dependent Phospholipase Cγ2 Activation in Lymphoma B Cells Exposed to Low Energy Electromagnetic Fields* , 1998, The Journal of Biological Chemistry.

[45]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[46]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.