Limits...
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.

Show MeSH
Tyrosine phosphorylation of signaling molecules upon  antigen stimulation of the CD19-negative and CD19-positive cell  lines. J558Lμm3 CD45+ variants  were stimulated for 1 min with  2.5 μg NP9BSA/107cells/ml,  lysed, and intermediary signaling  molecules immunoprecipitated  with various antibodies and fractionated on 10% SDS-PAGE.  After electrophoretic transfer to  PVDF membrane, both anti-phosphotyrosine and anti-effector immunoblotting were performed. Data are representative  of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199152&req=5

Figure 4: Tyrosine phosphorylation of signaling molecules upon antigen stimulation of the CD19-negative and CD19-positive cell lines. J558Lμm3 CD45+ variants were stimulated for 1 min with 2.5 μg NP9BSA/107cells/ml, lysed, and intermediary signaling molecules immunoprecipitated with various antibodies and fractionated on 10% SDS-PAGE. After electrophoretic transfer to PVDF membrane, both anti-phosphotyrosine and anti-effector immunoblotting were performed. Data are representative of at least three independent experiments.

Mentions: Antigen stimulation is known to induce recruitment of Src- and Syk-family tyrosine kinases to Ig-α and Ig-β components of the BCR (37–39). The tyrosine kinases bind via their SH2-domains to immunoreceptor tyrosine based activation motif (ITAM) phosphotyrosines and this leads to secondary recruitment, phosphorylation, and activation of downstream signaling molecules such as PLCγ1, PLCγ2, and PI3-kinase (reviewed in reference 40). To define the effects of CD19 expression on these phosphorylation events we stimulated CD19-negative and -positive cell lines, immunoprecipitated intracellular signaling molecules and conducted anti-phosphotyrosine immunoblotting analysis. The spectra of phosphoproteins in whole cell lysates from the J558Lμm3CD45+CD19- and the J558Lμm3CD45+ CD19+ cell lines looked similar with the exception of the presence of a tyrosine phosphorylated CD19 in the CD19-positive cell lysate (results not shown). Induced phosphorylation of several signaling molecules was analyzed after stimulation with normalization of immunoprecipitation and lane loading by sequential antieffector immunoblotting. We studied tyrosine phosphorylation after 1 min of antigen stimulation since we have shown previously that effector enzymes under study are maximally phosphorylated at this time in the J558Lμm3CD45+ plasmacytoma (41). As shown in Fig. 4, CD19 expression led to modest increases in antigen-induced tyrosine phosphorylation of Ig-α, Lyn, Syk, PLCγ1, and PLCγ2. Quantitation of tyrosine phosphorylation by scanning densitometry (at least three independent experiments for each effector enzyme) and normalization to effector levels revealed increased phosphorylation of Ig-α, Lyn, and Syk by 1.5–2-fold in the CD19-positive line. Induced PLCγ1 and PLCγ2 tyrosine phosphorylation was 2.5–3-fold higher in the CD19-positive cell line than in the CD19-negative cell line. These results suggest that CD19 functions in part by increasing the magnitude of certain proximal BCR coupled tyrosine phosphorylation events.


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)

Tyrosine phosphorylation of signaling molecules upon  antigen stimulation of the CD19-negative and CD19-positive cell  lines. J558Lμm3 CD45+ variants  were stimulated for 1 min with  2.5 μg NP9BSA/107cells/ml,  lysed, and intermediary signaling  molecules immunoprecipitated  with various antibodies and fractionated on 10% SDS-PAGE.  After electrophoretic transfer to  PVDF membrane, both anti-phosphotyrosine and anti-effector immunoblotting were performed. Data are representative  of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199152&req=5

Figure 4: Tyrosine phosphorylation of signaling molecules upon antigen stimulation of the CD19-negative and CD19-positive cell lines. J558Lμm3 CD45+ variants were stimulated for 1 min with 2.5 μg NP9BSA/107cells/ml, lysed, and intermediary signaling molecules immunoprecipitated with various antibodies and fractionated on 10% SDS-PAGE. After electrophoretic transfer to PVDF membrane, both anti-phosphotyrosine and anti-effector immunoblotting were performed. Data are representative of at least three independent experiments.
Mentions: Antigen stimulation is known to induce recruitment of Src- and Syk-family tyrosine kinases to Ig-α and Ig-β components of the BCR (37–39). The tyrosine kinases bind via their SH2-domains to immunoreceptor tyrosine based activation motif (ITAM) phosphotyrosines and this leads to secondary recruitment, phosphorylation, and activation of downstream signaling molecules such as PLCγ1, PLCγ2, and PI3-kinase (reviewed in reference 40). To define the effects of CD19 expression on these phosphorylation events we stimulated CD19-negative and -positive cell lines, immunoprecipitated intracellular signaling molecules and conducted anti-phosphotyrosine immunoblotting analysis. The spectra of phosphoproteins in whole cell lysates from the J558Lμm3CD45+CD19- and the J558Lμm3CD45+ CD19+ cell lines looked similar with the exception of the presence of a tyrosine phosphorylated CD19 in the CD19-positive cell lysate (results not shown). Induced phosphorylation of several signaling molecules was analyzed after stimulation with normalization of immunoprecipitation and lane loading by sequential antieffector immunoblotting. We studied tyrosine phosphorylation after 1 min of antigen stimulation since we have shown previously that effector enzymes under study are maximally phosphorylated at this time in the J558Lμm3CD45+ plasmacytoma (41). As shown in Fig. 4, CD19 expression led to modest increases in antigen-induced tyrosine phosphorylation of Ig-α, Lyn, Syk, PLCγ1, and PLCγ2. Quantitation of tyrosine phosphorylation by scanning densitometry (at least three independent experiments for each effector enzyme) and normalization to effector levels revealed increased phosphorylation of Ig-α, Lyn, and Syk by 1.5–2-fold in the CD19-positive line. Induced PLCγ1 and PLCγ2 tyrosine phosphorylation was 2.5–3-fold higher in the CD19-positive cell line than in the CD19-negative cell line. These results suggest that CD19 functions in part by increasing the magnitude of certain proximal BCR coupled tyrosine phosphorylation events.

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