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Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Buhl AM, Pleiman CM, Rickert RC, Cambier JC - J. Exp. Med. (1997)

Bottom Line: PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515.Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses.Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.

ABSTRACT
Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

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In the CD19-positive J558Lμm3CD45+ cells, antigen-induced tyrosine phosphorylation of PLCγ1 and PLCγ2 is  slightly inhibited by wortmannin, while Lyn, Ig-α and Syk  inductive tyrosine phosphorylation is insensitive to wortmannin.  J558Lμm3CD45+CD19+ cells  were preincubated with DMSO  or increasing concentrations of  wortmannin, prewarmed and  stimulated for 1 min with  NP9BSA (2.5 μg/107 cells/ml)  and lysed. Immunoprecipitation,  SDS-PAGE fractionation and electrophoretic transfer were performed. For each immunoprecipitation, both anti-phosphotyrosine  and anti-effector immunoblots  are shown. Results are representative of at least three independent experiments.
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Figure 7: In the CD19-positive J558Lμm3CD45+ cells, antigen-induced tyrosine phosphorylation of PLCγ1 and PLCγ2 is slightly inhibited by wortmannin, while Lyn, Ig-α and Syk inductive tyrosine phosphorylation is insensitive to wortmannin. J558Lμm3CD45+CD19+ cells were preincubated with DMSO or increasing concentrations of wortmannin, prewarmed and stimulated for 1 min with NP9BSA (2.5 μg/107 cells/ml) and lysed. Immunoprecipitation, SDS-PAGE fractionation and electrophoretic transfer were performed. For each immunoprecipitation, both anti-phosphotyrosine and anti-effector immunoblots are shown. Results are representative of at least three independent experiments.

Mentions: To investigate the potential role of CD19-activated PI3-kinase on early BCR-mediated protein tyrosine phosphorylation events, CD19-positive cells were preincubated for 30 min with DMSO (vehicle) or increasing concentrations of wortmannin and stimulated for 1 min with antigen before effectors were immunoprecipitated and analyzed. As shown in Fig. 7, antigen-induced tyrosine phosphorylation of Ig-α, Lyn, and Syk was insensitive to wortmannin. PLCγ1 and PLCγ2 tyrosine phosphorylation was somewhat sensitive to wortmannin preincubation although the decrease in induced tyrosine phosphorylation was modest (35% inhibition at 25 nM wortmannin, Table 2) compared with the degree of wortmannin inhibition of IP3 generation and calcium mobilization (91.5% inhibition, Table 2). It seems very unlikely that the >90% inhibition of PLCγ activity could be caused by a 35% inhibition of PLCγ phosphorylation Thus, as summarized in Table 1, CD19 expression by the J558Lμm3 CD45+ plasmacytoma is required for optimal antigen stimulation of multiple intracellular signaling events, but only some of these are secondary to the nearly absolute dependence of PI3-kinase activation on CD19.


Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Buhl AM, Pleiman CM, Rickert RC, Cambier JC - J. Exp. Med. (1997)

In the CD19-positive J558Lμm3CD45+ cells, antigen-induced tyrosine phosphorylation of PLCγ1 and PLCγ2 is  slightly inhibited by wortmannin, while Lyn, Ig-α and Syk  inductive tyrosine phosphorylation is insensitive to wortmannin.  J558Lμm3CD45+CD19+ cells  were preincubated with DMSO  or increasing concentrations of  wortmannin, prewarmed and  stimulated for 1 min with  NP9BSA (2.5 μg/107 cells/ml)  and lysed. Immunoprecipitation,  SDS-PAGE fractionation and electrophoretic transfer were performed. For each immunoprecipitation, both anti-phosphotyrosine  and anti-effector immunoblots  are shown. Results are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: In the CD19-positive J558Lμm3CD45+ cells, antigen-induced tyrosine phosphorylation of PLCγ1 and PLCγ2 is slightly inhibited by wortmannin, while Lyn, Ig-α and Syk inductive tyrosine phosphorylation is insensitive to wortmannin. J558Lμm3CD45+CD19+ cells were preincubated with DMSO or increasing concentrations of wortmannin, prewarmed and stimulated for 1 min with NP9BSA (2.5 μg/107 cells/ml) and lysed. Immunoprecipitation, SDS-PAGE fractionation and electrophoretic transfer were performed. For each immunoprecipitation, both anti-phosphotyrosine and anti-effector immunoblots are shown. Results are representative of at least three independent experiments.
Mentions: To investigate the potential role of CD19-activated PI3-kinase on early BCR-mediated protein tyrosine phosphorylation events, CD19-positive cells were preincubated for 30 min with DMSO (vehicle) or increasing concentrations of wortmannin and stimulated for 1 min with antigen before effectors were immunoprecipitated and analyzed. As shown in Fig. 7, antigen-induced tyrosine phosphorylation of Ig-α, Lyn, and Syk was insensitive to wortmannin. PLCγ1 and PLCγ2 tyrosine phosphorylation was somewhat sensitive to wortmannin preincubation although the decrease in induced tyrosine phosphorylation was modest (35% inhibition at 25 nM wortmannin, Table 2) compared with the degree of wortmannin inhibition of IP3 generation and calcium mobilization (91.5% inhibition, Table 2). It seems very unlikely that the >90% inhibition of PLCγ activity could be caused by a 35% inhibition of PLCγ phosphorylation Thus, as summarized in Table 1, CD19 expression by the J558Lμm3 CD45+ plasmacytoma is required for optimal antigen stimulation of multiple intracellular signaling events, but only some of these are secondary to the nearly absolute dependence of PI3-kinase activation on CD19.

Bottom Line: PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515.Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses.Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.

ABSTRACT
Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Show MeSH