Bruton tyrosine kinase is tyrosine phosphorylated and activated in pre-B lymphocytes and receptor-ligated B cells.

Abstract

The gene encoding Bruton tyrosine kinase (Btk) is known to be mutated in human X chromosome-linked agammaglobulinemia and in the Xid mouse. This kinase was examined in B lymphocytes before and after antigen receptor ligation and also in pre-B cells. Btk was found to be catalytically activated and tyrosine phosphorylated in response to anti-IgM imulation in B cells. This kinase is also constitutively phosphorylated on tyrosine residues in pre-B cells. These findings point to a functional role for Btk in pre-antigen and antigen receptor signaling during B-cell development and provide a biochemical explanation for the X-linked genetic syndromes already linked to this kinase.Bruton tyrosine kinase (Btk) is a member of a small family of tyrosine kinases that share certain structural features, but whose biological roles are poorly understood. These kinases, which include Itk/Tsk in T lymphocytes (1, 2), TEC I and TEC II in liver cells as well as hematopoietic cells (3,4), and the DSrc28 kinase in Drosophila (5) (19).Anti-Btk Antibodies. Anti-Btk antibodies were raised in rabbits using a glutathione S-transferase (GST) Btk fusion as described by Tsukada et al. (7) followed by depletion over a GST-Sepharose immunoadsorbent and affinity purification using GST-Btk-Sepharose.Anti-Phosphotyrosine Immunoprecipitation and Anti-Btk Immunoblot Analysis. Anti-IgM crosslinking was performed and lysates from 5 x 107 cells (in 1% Nonidet P40) were prepared as described (19). Four-fifths of each lysate was used for the anti-phosphotyrosine immunoprecipitation step, using 50 ,ul of a 50% slurry of a monoclonal anti-phosphotyrosine antibody coupled to agarose (PY 20, Zymed).The remaining fifth of each lysate was immunoprecipitated with anti-Btk. Immunoprecipitates were washed four times with 0.2% Nonidet P40 in 10 mM Tris, pH 8.0/120 mM NaCl/0.4 mM EDTA, separated on a 10% polyacrylamide/ SDS gel, and transferred to an Immobilon-P membrane, and Btk was revealed by immunoblotting, using the ECL system (Amersham). In separate experiments, lysates were immunoprecipitated with anti-Btk as described above and an anti-phosphotyrosine immunoblot was performed as described (19).Assay for Catalytic Activation of Btk. WEHI 231 B cells (3 x 106) were used for each time point of the in vitro kinase assay. Cells (1.3 x 107) were stimulated with 2.5 pg of F(ab')2 anti-IgM (Zymed) in 100 ,l of serum-free RPMI medium at 37°C. At various time points lysates were made as described above and precleared using rabbit IgG and protein A-Sepharose. A portion of each lysate representing 3 x 106 cells was immunoprecipitated with anti-Btk, washed four times with the 0.2% Nonidet P-40 buffer described in the previous Abbreviations: Btk, Bruton tyrosine kinase; SH, Src homology. *To whom reprint requests should be addressed. 10606The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Constitutive membrane association potentiates activation of Bruton tyrosine kinase

“…Activation of Btk by receptor crosslinking, overexpression with Src kinases, or the Btk* mutation is correlated with increased tyrosine phosphorylation of Btk (Figure 4, Li et al, 1995;Rawlings et al, 1996;Mahajan et al, 1995;Hinshelwood et al, 1995;Aoki et al, 1994;de Weers et al, 1994;Saouf et al, 1994;Kawakami et al, 1994;Sato et al, 1994;Matsuda et al, 1995). This is a result of both transphosphorylation by Src family kinases at Y551 and autophosphorylation at Y223 .…”

“…In addition, activation signals result in an increase in the level of phosphotyrosine on Btk (Li et al, 1995;Rawlings et al, 1996;Mahajan et al, 1995;Hinshelwood et al, 1995;Aoki et al, 1994;de Weers et al, 1994;Saouf et al, 1994;Sato et al, 1994;Matsuda et al, 1995). These observations suggest that membrane targeting and tyrosine phosphorylation are tightly integrated steps in the activation of Btk.…”

“…These diseases are characterized by defects in B cell development and function. Genetic and biochemical analyses indicate that Btk is a component of the signal transduction pathways utilized by the B cell antigen receptor (BCR), IL-5R, IL-6R, IL-10R, CD38, and FceRI (Wicker and Scher, 1986;Hinshelwood et al, 1995;Saouf et al, 1994;Aoki et al, 1994;de Weers et al, 1994;Koike et al, 1995;Sato et al, 1994;Hitoshi et al, 1993;Matsuda et al, 1995;Santos-Argumedo et al, 1995;Go et al, 1990;Kawakami et al, 1994). Activation of Btk by either receptor crosslinking or a point mutation E41K in the PH domain (Btk*) is dependent on Src family kinases Afar et al, 1996;Saouf et al, 1994;Mahajan et al, 1995) and is correlated with increased tyrosine phosphorylation Li et al, 1995;Mahajan et al, 1995;Hinshelwood et al, 1995;Aoki et al, 1994;de Weers et al, 1994;Saouf et al, 1994;Sato et al, 1994;Kawakami et al, 1994;Matsuda et al, 1995) and translocation to the plasma membrane (Li et al, 1995;Kawakami et al, 1994).…”

Impaired Ca2+ mobilization by X-linked agammaglobulinaemia (XLA) B cells in response to ligation of the B cell receptor (BCR)

“…It is phosphorylated and its kinase activity increased on ligation of the BCR on murine and human cell lines [9,10,30,31]. BCR signalling is defective in the murine immunodeficiency xid which is caused by a point mutation in the pleckstrin homology domain of the Btk gene [41].…”

Vertebrate non‐receptor protein–tyrosine kinase families

“…The Glu241Lys mutation in the PH domain could increase its affinity for PIP 2 , and one might expect an interaction between the PH domain and the membrane to play a critical role in Btk signalling. In this regard, it has been shown that Btk is activated through the IL-5 receptor , the BCR (Aoki et al 1994;Saouaf et al 1994), and Fc RI . Although the signalling pathways linked to Btk are not known, B lymphocytes from Xid mice are unresponsive to CD38 signalling, suggesting a role in this pathway (SantosArgumedo et al 1995).…”