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Differential effects of B cell receptor and B cell receptor-FcgammaRIIB1 engagement on docking of Csk to GTPase-activating protein (GAP)-associated p62.

Vuica M, Desiderio S, Schneck JP - J. Exp. Med. (1997)

Bottom Line: Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins.The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2.The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. mvuica@wlchlink.welch.jhu.edu

ABSTRACT
The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcgamma receptors of the IIB1 type (FcgammaRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein-associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab')2 resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcgammaRIIB1-deficient cell line IIA1.6 and recovered when FcgammaRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.

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Stimulation-dependent association of p62 and Csk  in vivo. A20 cells were unstimulated (lanes 1–3), stimulated with  intact anti–mouse IgG (lanes 4–6),  or stimulated with F(ab′)2 (lanes  7–9). Precleared cell lysates were  incubated with normal rabbit  immunoglobulin (lanes 2, 5, and  8) or affinity-purified rabbit antibodies against Csk (lanes 3, 6,  and 9). One-tenth of the total  amount of lysate used in each  experiment was fractionated in  parallel (lanes 1, 4, and 7). Immunoprecipitated proteins were  fractionated on a 10% SDS– polyacrylamide gel, were transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine  antibody (PY20). The membrane was stripped and Csk was detected by  immunoblotting.
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Figure 1: Stimulation-dependent association of p62 and Csk in vivo. A20 cells were unstimulated (lanes 1–3), stimulated with intact anti–mouse IgG (lanes 4–6), or stimulated with F(ab′)2 (lanes 7–9). Precleared cell lysates were incubated with normal rabbit immunoglobulin (lanes 2, 5, and 8) or affinity-purified rabbit antibodies against Csk (lanes 3, 6, and 9). One-tenth of the total amount of lysate used in each experiment was fractionated in parallel (lanes 1, 4, and 7). Immunoprecipitated proteins were fractionated on a 10% SDS– polyacrylamide gel, were transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody (PY20). The membrane was stripped and Csk was detected by immunoblotting.

Mentions: As a first step toward identifying proteins associated with Csk under conditions of BCR and FcγRIIB stimulation, A20 cells were incubated with intact anti-IgG antibody or with F(ab′)2 fragments of similar specificity, and phosphotyrosine-containing proteins were detected in anti-Csk immunoprecipitates by immunoblotting. Several phosphotyrosine-containing species, including proteins of 62, 97, and 125 kD (p62, p97, and p125, respectively), were observed to immunoprecipitate with Csk (Fig. 1). The amount of p62 detectable in Csk immunoprecipitates by antiphosphotyrosine immunoblotting increased significantly after stimulation, indicating an elevation in phosphotyrosine content, association with Csk, or both. However, the increase in antiphosphotyrosine reactivity of p62 was substantially greater after stimulation with intact antibodies than with F(ab′)2. The antiphosphotyrosine signals associated with p97 and p125, in contrast, showed relatively little change after stimulation with anti-IgG antibodies.


Differential effects of B cell receptor and B cell receptor-FcgammaRIIB1 engagement on docking of Csk to GTPase-activating protein (GAP)-associated p62.

Vuica M, Desiderio S, Schneck JP - J. Exp. Med. (1997)

Stimulation-dependent association of p62 and Csk  in vivo. A20 cells were unstimulated (lanes 1–3), stimulated with  intact anti–mouse IgG (lanes 4–6),  or stimulated with F(ab′)2 (lanes  7–9). Precleared cell lysates were  incubated with normal rabbit  immunoglobulin (lanes 2, 5, and  8) or affinity-purified rabbit antibodies against Csk (lanes 3, 6,  and 9). One-tenth of the total  amount of lysate used in each  experiment was fractionated in  parallel (lanes 1, 4, and 7). Immunoprecipitated proteins were  fractionated on a 10% SDS– polyacrylamide gel, were transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine  antibody (PY20). The membrane was stripped and Csk was detected by  immunoblotting.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198989&req=5

Figure 1: Stimulation-dependent association of p62 and Csk in vivo. A20 cells were unstimulated (lanes 1–3), stimulated with intact anti–mouse IgG (lanes 4–6), or stimulated with F(ab′)2 (lanes 7–9). Precleared cell lysates were incubated with normal rabbit immunoglobulin (lanes 2, 5, and 8) or affinity-purified rabbit antibodies against Csk (lanes 3, 6, and 9). One-tenth of the total amount of lysate used in each experiment was fractionated in parallel (lanes 1, 4, and 7). Immunoprecipitated proteins were fractionated on a 10% SDS– polyacrylamide gel, were transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody (PY20). The membrane was stripped and Csk was detected by immunoblotting.
Mentions: As a first step toward identifying proteins associated with Csk under conditions of BCR and FcγRIIB stimulation, A20 cells were incubated with intact anti-IgG antibody or with F(ab′)2 fragments of similar specificity, and phosphotyrosine-containing proteins were detected in anti-Csk immunoprecipitates by immunoblotting. Several phosphotyrosine-containing species, including proteins of 62, 97, and 125 kD (p62, p97, and p125, respectively), were observed to immunoprecipitate with Csk (Fig. 1). The amount of p62 detectable in Csk immunoprecipitates by antiphosphotyrosine immunoblotting increased significantly after stimulation, indicating an elevation in phosphotyrosine content, association with Csk, or both. However, the increase in antiphosphotyrosine reactivity of p62 was substantially greater after stimulation with intact antibodies than with F(ab′)2. The antiphosphotyrosine signals associated with p97 and p125, in contrast, showed relatively little change after stimulation with anti-IgG antibodies.

Bottom Line: Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins.The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2.The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. mvuica@wlchlink.welch.jhu.edu

ABSTRACT
The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcgamma receptors of the IIB1 type (FcgammaRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein-associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab')2 resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcgammaRIIB1-deficient cell line IIA1.6 and recovered when FcgammaRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.

Show MeSH
Related in: MedlinePlus