The protein defective in X‐linked agammaglobulinemia, Bruton's tyrosine kinase, shows increased autophosphorylation activity <i>in vitro</i> when isolated from cells in which the B cell receptor has been cross‐linked

Hinshelwood, Steve; Lovering, Ruth C.; Genevier, Helen C.; Levinsky, Roland J.; Kinnon, Christine

doi:10.1002/eji.1830250439

Cited by 18 publications

(15 citation statements)

References 13 publications

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“…As shown in Fig. 1, Btk was inducibly tyrosine-phosphorylated following BCR stimulation in wild type DT40 cells, consistent with previous reports (5,(13)(14)(15). In contrast to wild type cells, Lyn/Syk double-deficient DT40 cells failed to exhibit any Btk tyrosine phosphorylation following BCR ligation, indicating requirement of Lyn and/or Syk for the BCR-induced tyrosine phosphorylation of Btk.…”

Section: Resultssupporting

confidence: 90%

Transphosphorylation of Bruton's Tyrosine Kinase on Tyrosine 551 Is Critical for B Cell Antigen Receptor Function

Kurosaki

1997

Journal of Biological Chemistry

126

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is essential for BCR function and that this phosphorylation is mediated through the concerted actions of Lyn and Syk.The B cell antigen receptor (BCR) 1 is composed of surface immunoglobulin noncovalently associated with a pair of Ig␣/ Ig␤ disulfide-linked heterodimers, which are essential for signal transduction. Stimulation of the BCR induces the enzymatic activation and tyrosine phosphorylation of three distinct families of nonreceptor cytoplasmic protein tyrosine kinases (PTKs), the Src family, Syk, and Btk. The Src family kinases are rapidly activated after BCR engagement, and their activation correlates with the initial tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif on the BCR Ig␣ and Ig␤ subunits (reviewed in Refs. 1-4).Temporally, activation of Src family kinases is followed by Btk and Syk (5). This sequential activation potentially places Btk and Syk downstream of Src family kinases. Utilizing cooverexpression system in fibroblasts and COS cells, it has been demonstrated that Lyn transphosphorylates Btk on Tyr 551 in the catalytic domain, a site homologous to the Src family kinase consensus autophosphorylation site (6, 7). This results in a 5-10-fold increase in Btk enzymatic activity (7). The increase in activity also leads to increased autophosphorylation at Tyr 223 in the SH3 domain of Btk (8). The identical phosphopeptides were generated after cross-linking of the BCR, indicating that these sites are also tyrosine phosphorylated in B cells (7 EXPERIMENTAL PROCEDURESCells, Antisera, and DNA Transfection-DT40 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine. Anti-chicken IgM mAb M4 and anti-phospholipase C (PLC)-␥2 Ab were described previously (10). The anti-phosphotyrosine mAb (4G10) and anti-T7 mAb were obtained from Upstate Biotechnology, Inc. and Novagen, respectively. T7-tagged and mutant Btk cDNAs were created by polymerase chain reaction, and the resulting constructs were confirmed by DNA sequencing. These cDNAs were cloned into pApuro expression vector (10). For DNA transfection into DT40 cells, DNA was linearized, electroporated, and selected in the presence of puromycin (0.5 g/ml). The expression of Btk was analyzed by Western blotting.Immunoprecipitation and Immunoblot Analysis-DT40 cells were stimulated by mAb M4 for indicated time. Cells were solubilized in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA) containing 50 mM NaF, 10 M molybdate, and 0.2 mM sodium vanadate supplemented with protease inhibitors described previously (10). Cell lysates were sequentially incubated (1 h at 4°C for each incubation with Ab and protein A-Sepharose). For immunoblotting, samples were separated on SDS-PAGE and transferred to nitrocellulose membrane (Amersham Corp.). Filters were incubated with mAb 4G10 or anti-T7 mAb. After washing, filters were developed using a sheep anti-mouse IgG Ab conjugated to horseradish peroxidase and enhanced chemiluminescence (ECL).For in v...

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Section: Resultssupporting

confidence: 90%

Transphosphorylation of Bruton's Tyrosine Kinase on Tyrosine 551 Is Critical for B Cell Antigen Receptor Function

Kurosaki

1997

Journal of Biological Chemistry

126

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“…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 .…”

Section: Alternative Strategies Target Btk To the Plasma Membranementioning

confidence: 99%

“…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.…”

Section: Tyrosine Phosphorylated Btk Is Found Predominantly In the Mementioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

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Constitutive membrane association potentiates activation of Bruton tyrosine kinase

Rawlings

Park

et al. 1997

Oncogene

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Mutations in the nonreceptor tyrosine kinase Btk result in the B cell immunode®ciencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunode®-ciency (xid) in mice. Genetic and biochemical evidence implicates Btk as a key component of several B cell signaling pathways. Activation of Btk by a point mutation (E41K) within the PH domain (Btk*) results in ®broblast transformation and is correlated with increased membrane localization of Btk. When wild type Btk is activated by coexpression with Lyn, the tyrosine phosphorylated pool of Btk is highly enriched in the membrane fraction. To determine whether membrane association is su cient to activate Btk, we targeted Btk to the plasma membrane using a series of fusion proteins including GagBtk, CD16Btk and CD4Btk. Constitutive membrane association greatly enhanced the ability of Btk to transform Rat2 ®broblasts in the presence of high levels of Src activity. All membrane targeted forms of Btk were highly tyrosine phosphorylated. Transformation required membrane localization, Btk kinase activity, transphosphorylation by Src family kinases, and an intact SH2 domain but not the PH or SH3 domains. These data suggest that membrane localization is a critical early step in Btk activation.

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“…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].…”

Section: Discussionmentioning

confidence: 99%

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

Genevier¹,

Callard²

1997

Clinical and Experimental Immunology

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SUMMARYXLA bone marrow samples were shown to contain B cells expressing IgM, and pre-B cells that express the m-surrogate light chain (mwLC) complex, albeit at a reduced frequency to that found in normal bone marrow. Antibody ligation of m heavy chain on these cells and an XLA B cell line did not induce a Ca 2þ flux, whereas ligation of m heavy chain on normal bone marrow cells, mwLC þ pre-B cell lines and an IgM þ B cell line did. The block in XLA B cells was not due to a defect in the basic mechanism of Ca 2þ flux generation, as the cells responded well to thapsigargin. In addition, the defect did not affect T cells, which were shown to respond to CD3 antibody with a Ca 2þ flux. Ligation of m heavy chain on XLA bone marrow cells did, however, activate tyrosine kinases, resulting in tyrosine phosphorylation of a cellular protein with a molecular weight of approximately 115 kD. These results indicate that Btk may be necessary for the generation of the Ca 2þ flux in response to ligation of m heavy chain on B cells and mwLC þ pre-B.

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The protein defective in X‐linked agammaglobulinemia, Bruton's tyrosine kinase, shows increased autophosphorylation activity in vitro when isolated from cells in which the B cell receptor has been cross‐linked

Cited by 18 publications

References 13 publications

Transphosphorylation of Bruton's Tyrosine Kinase on Tyrosine 551 Is Critical for B Cell Antigen Receptor Function

Transphosphorylation of Bruton's Tyrosine Kinase on Tyrosine 551 Is Critical for B Cell Antigen Receptor Function

Constitutive membrane association potentiates activation of Bruton tyrosine kinase

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

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