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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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Related in: MedlinePlus

Wortmannin inhibits the antigen-induced increase  in PI3-kinase activity, [Ca2+]i  mobilization and IP3 generation  in the J558Lμm3 CD45+CD19+  plasmacytoma. (A) Cells were  preincubated with DMSO (vehicle) or wortmannin at various  concentrations (in 0.2% final  DMSO concentration) for 25  min at 25°C, prewarmed for 4  min at 37°C, and then stimulated  for 2 min with 1 μg NP9BSA/4  × 106 cells/ml before analysis of  PI3-kinase activation as described in Fig, 4. Shown is the  mean fold increase in activity  over basal from four independent  experiments ± standard error of  the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical  software. (B) To study the effects  of wortmannin on the calcium response, J558Lμm3CD45+CD19+  cells were Indo-1 AM loaded  and preincubated with DMSO  (vehicle) or wortmannin at various concentrations (resulting in  0.2% final DMSO) for 25 min at  25°C, prewarmed for 4 min at  37°C, and stimulated at arrow  with 250 ng NP9BSA/106 cells/ml. Shown is the mean [Ca2+]i over time  with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM. (C)  J558Lμm3CD45+CD19+ cells were preincubated with DMSO or increasing concentrations of wortmannin, prewarmed, and stimulated with  2.5 μg NP9BSA/107/ml for 1 min before IP3 production was measured  by the IP3 receptor binding inhibition assay. Shown is the mean fold increase over basal in three independent experiments ± standard error of  the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical software.
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Figure 6: Wortmannin inhibits the antigen-induced increase in PI3-kinase activity, [Ca2+]i mobilization and IP3 generation in the J558Lμm3 CD45+CD19+ plasmacytoma. (A) Cells were preincubated with DMSO (vehicle) or wortmannin at various concentrations (in 0.2% final DMSO concentration) for 25 min at 25°C, prewarmed for 4 min at 37°C, and then stimulated for 2 min with 1 μg NP9BSA/4 × 106 cells/ml before analysis of PI3-kinase activation as described in Fig, 4. Shown is the mean fold increase in activity over basal from four independent experiments ± standard error of the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical software. (B) To study the effects of wortmannin on the calcium response, J558Lμm3CD45+CD19+ cells were Indo-1 AM loaded and preincubated with DMSO (vehicle) or wortmannin at various concentrations (resulting in 0.2% final DMSO) for 25 min at 25°C, prewarmed for 4 min at 37°C, and stimulated at arrow with 250 ng NP9BSA/106 cells/ml. Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM. (C) J558Lμm3CD45+CD19+ cells were preincubated with DMSO or increasing concentrations of wortmannin, prewarmed, and stimulated with 2.5 μg NP9BSA/107/ml for 1 min before IP3 production was measured by the IP3 receptor binding inhibition assay. Shown is the mean fold increase over basal in three independent experiments ± standard error of the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical software.

Mentions: To begin to dissect the relationship among the observed CD19 effects, we assessed the effects of the PI3-kinase inhibitor wortmannin on signaling events. Wortmannin has been shown to inhibit PI3-kinase activity by covalent modification of a lysine residue in the PI3-kinase catalytic subunit, p110 (43). We also used the competitive PI3-kinase inhibitor Ly294002 (44) and obtained similar results as in our studies with wortmannin (results not shown). The effect of wortmannin on antigen-induced activation of PI3-kinase was determined. The results shown in Fig. 6 A demonstrate the inhibitory effect of wortmannin on antigen-mediated PI3-kinase activation in the J558Lμm3CD45+ CD19+ plasmacytoma. Cells were pretreated for 30 min with DMSO (vehicle) or increasing concentrations of wortmannin, stimulated with antigen for 2 min, washed, and then lysed before PI3-kinase was immunoprecipitated and activity measured in an in vitro kinase assay with phosphatidylinositol (PI) containing vesicles as substrate. Consistent with its previously established IC50, 10 nM wortmannin inhibited the antigen-mediated PI3-kinase activation by 54%, and 25 nM wortmannin inhibited 75% of antigen-induced PI3-kinase activity. Wortmannin added to the cells immediately before washing and lysis did not effect the PI3-kinase activity (data not shown) indicating that the inhibitor was acting intracellularly.


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)

Wortmannin inhibits the antigen-induced increase  in PI3-kinase activity, [Ca2+]i  mobilization and IP3 generation  in the J558Lμm3 CD45+CD19+  plasmacytoma. (A) Cells were  preincubated with DMSO (vehicle) or wortmannin at various  concentrations (in 0.2% final  DMSO concentration) for 25  min at 25°C, prewarmed for 4  min at 37°C, and then stimulated  for 2 min with 1 μg NP9BSA/4  × 106 cells/ml before analysis of  PI3-kinase activation as described in Fig, 4. Shown is the  mean fold increase in activity  over basal from four independent  experiments ± standard error of  the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical  software. (B) To study the effects  of wortmannin on the calcium response, J558Lμm3CD45+CD19+  cells were Indo-1 AM loaded  and preincubated with DMSO  (vehicle) or wortmannin at various concentrations (resulting in  0.2% final DMSO) for 25 min at  25°C, prewarmed for 4 min at  37°C, and stimulated at arrow  with 250 ng NP9BSA/106 cells/ml. Shown is the mean [Ca2+]i over time  with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM. (C)  J558Lμm3CD45+CD19+ cells were preincubated with DMSO or increasing concentrations of wortmannin, prewarmed, and stimulated with  2.5 μg NP9BSA/107/ml for 1 min before IP3 production was measured  by the IP3 receptor binding inhibition assay. Shown is the mean fold increase over basal in three independent experiments ± standard error of  the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical software.
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Figure 6: Wortmannin inhibits the antigen-induced increase in PI3-kinase activity, [Ca2+]i mobilization and IP3 generation in the J558Lμm3 CD45+CD19+ plasmacytoma. (A) Cells were preincubated with DMSO (vehicle) or wortmannin at various concentrations (in 0.2% final DMSO concentration) for 25 min at 25°C, prewarmed for 4 min at 37°C, and then stimulated for 2 min with 1 μg NP9BSA/4 × 106 cells/ml before analysis of PI3-kinase activation as described in Fig, 4. Shown is the mean fold increase in activity over basal from four independent experiments ± standard error of the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical software. (B) To study the effects of wortmannin on the calcium response, J558Lμm3CD45+CD19+ cells were Indo-1 AM loaded and preincubated with DMSO (vehicle) or wortmannin at various concentrations (resulting in 0.2% final DMSO) for 25 min at 25°C, prewarmed for 4 min at 37°C, and stimulated at arrow with 250 ng NP9BSA/106 cells/ml. Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM. (C) J558Lμm3CD45+CD19+ cells were preincubated with DMSO or increasing concentrations of wortmannin, prewarmed, and stimulated with 2.5 μg NP9BSA/107/ml for 1 min before IP3 production was measured by the IP3 receptor binding inhibition assay. Shown is the mean fold increase over basal in three independent experiments ± standard error of the mean. Asterisk indicates a statistical significance of P <0.05 determined by JMP 3.16 statistical software.
Mentions: To begin to dissect the relationship among the observed CD19 effects, we assessed the effects of the PI3-kinase inhibitor wortmannin on signaling events. Wortmannin has been shown to inhibit PI3-kinase activity by covalent modification of a lysine residue in the PI3-kinase catalytic subunit, p110 (43). We also used the competitive PI3-kinase inhibitor Ly294002 (44) and obtained similar results as in our studies with wortmannin (results not shown). The effect of wortmannin on antigen-induced activation of PI3-kinase was determined. The results shown in Fig. 6 A demonstrate the inhibitory effect of wortmannin on antigen-mediated PI3-kinase activation in the J558Lμm3CD45+ CD19+ plasmacytoma. Cells were pretreated for 30 min with DMSO (vehicle) or increasing concentrations of wortmannin, stimulated with antigen for 2 min, washed, and then lysed before PI3-kinase was immunoprecipitated and activity measured in an in vitro kinase assay with phosphatidylinositol (PI) containing vesicles as substrate. Consistent with its previously established IC50, 10 nM wortmannin inhibited the antigen-mediated PI3-kinase activation by 54%, and 25 nM wortmannin inhibited 75% of antigen-induced PI3-kinase activity. Wortmannin added to the cells immediately before washing and lysis did not effect the PI3-kinase activity (data not shown) indicating that the inhibitor was acting intracellularly.

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
Related in: MedlinePlus