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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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The 62-kD protein  bound to Csk is associated with  RasGAP. (A) Lysates of A20  cells stimulated with intact anti-IgG were incubated with agarose-conjugated normal mouse  antibodies (lane 1), mAbs against  RasGAP (lane 2), normal rabbit  antibodies (lane 3), or affinity-purified antibodies against Csk  (lane 4). Alternatively, lysates  were incubated with GST (lane  5) or GST-CskSH2 (lane 6). (B)  Lysates were immunodepleted  with normal rabbit serum (lane  1), antibodies against Csk (lane  2), RasGAP (lane 3), Sam68  (lane 4), normal mouse immunoglobulin (lane 5) or p62  (Transduction Laboratories; lane  6, 2C4; lane 7). Depleted lysates  were then precipitated with  GST-CskSH2 (lanes 1–6). (C)  RasGAP was precipitated as described above (lanes 2–4). The  sample was boiled in lysis buffer  containing 1% SDS. One-third  was reserved for analysis of the RasGAP immunoprecipitate. The remaining two-thirds of the sample was diluted 10-fold in lysis buffer, split into two aliquots, and incubated either with GST (lane 3) or GST-CskSH2 (lane 4). A control immunoprecipitation was performed with normal mouse immunoglobulin (lane 1). All precipitates (A–C) were separated by 9% SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibodies. Membranes were stripped and reprobed either with mAbs against Csk or rabbit serum against RasGAP. (D) RasGAP and Csk immunoprecipitates were  subjected to an in vitro kinase assay; 32P-labeled p62 was isolated from each precipitate by gel electrophoresis, excised, and analyzed by partial V-8 proteolysis as described in Materials and Methods. Phosphopeptides were visualized by autoradiography.
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Figure 3: The 62-kD protein bound to Csk is associated with RasGAP. (A) Lysates of A20 cells stimulated with intact anti-IgG were incubated with agarose-conjugated normal mouse antibodies (lane 1), mAbs against RasGAP (lane 2), normal rabbit antibodies (lane 3), or affinity-purified antibodies against Csk (lane 4). Alternatively, lysates were incubated with GST (lane 5) or GST-CskSH2 (lane 6). (B) Lysates were immunodepleted with normal rabbit serum (lane 1), antibodies against Csk (lane 2), RasGAP (lane 3), Sam68 (lane 4), normal mouse immunoglobulin (lane 5) or p62 (Transduction Laboratories; lane 6, 2C4; lane 7). Depleted lysates were then precipitated with GST-CskSH2 (lanes 1–6). (C) RasGAP was precipitated as described above (lanes 2–4). The sample was boiled in lysis buffer containing 1% SDS. One-third was reserved for analysis of the RasGAP immunoprecipitate. The remaining two-thirds of the sample was diluted 10-fold in lysis buffer, split into two aliquots, and incubated either with GST (lane 3) or GST-CskSH2 (lane 4). A control immunoprecipitation was performed with normal mouse immunoglobulin (lane 1). All precipitates (A–C) were separated by 9% SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibodies. Membranes were stripped and reprobed either with mAbs against Csk or rabbit serum against RasGAP. (D) RasGAP and Csk immunoprecipitates were subjected to an in vitro kinase assay; 32P-labeled p62 was isolated from each precipitate by gel electrophoresis, excised, and analyzed by partial V-8 proteolysis as described in Materials and Methods. Phosphopeptides were visualized by autoradiography.

Mentions: The ability of the R107K mutant to bind p62 was unexpected and reminiscent of the binding of GAP-A.p62 to the Csk SH2 domain, which is known to persist after mutation of R107 (25). To test whether the 62-kD species might represent GAP-A.p62, A20 cells were stimulated with intact anti-IgG; tyrosine phosphorylated co-migrating 62-kD proteins were detected in RasGAP, Csk, and Csk SH2 precipitates (Fig. 3 A). Next, A20 cells were stimulated with intact anti-IgG and lysates were immunodepleted using antibodies specific for Csk, RasGAP, the 62-kD phosphoprotein recognized by mAb 2C4 (30), or Sam68, a protein initially identified as related to RasGAP-associated p62 (31). Immunodepleted or undepleted lysates were then assayed for protein binding to GST (data not shown) or GST-CskSH2. The amount of tyrosine phosphorylated p62 recovered from GST-CskSH2 after immunodepletion of RasGAP was reduced significantly in comparison to a control immunodepletion with nonimmune antibody (Fig. 3 B). The recovery of p62 was also reduced after immunodepletion of Csk, as expected. In contrast, immunodepletion of Sam68 had no effect on the recovery of p62 from the GST-CskSH2 eluate. Because the 2C4 antibody does not detect its ligand on immunoblot, we were not able to estimate how much of the 2C4-binding p62 species was depleted in this experiment. These results indicate that p62 interacts with RasGAP in A20 cell lysates, and are consistent with the interpretation that the Csk-associated p62 is related to or identical to GAP-A.p62.


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)

The 62-kD protein  bound to Csk is associated with  RasGAP. (A) Lysates of A20  cells stimulated with intact anti-IgG were incubated with agarose-conjugated normal mouse  antibodies (lane 1), mAbs against  RasGAP (lane 2), normal rabbit  antibodies (lane 3), or affinity-purified antibodies against Csk  (lane 4). Alternatively, lysates  were incubated with GST (lane  5) or GST-CskSH2 (lane 6). (B)  Lysates were immunodepleted  with normal rabbit serum (lane  1), antibodies against Csk (lane  2), RasGAP (lane 3), Sam68  (lane 4), normal mouse immunoglobulin (lane 5) or p62  (Transduction Laboratories; lane  6, 2C4; lane 7). Depleted lysates  were then precipitated with  GST-CskSH2 (lanes 1–6). (C)  RasGAP was precipitated as described above (lanes 2–4). The  sample was boiled in lysis buffer  containing 1% SDS. One-third  was reserved for analysis of the RasGAP immunoprecipitate. The remaining two-thirds of the sample was diluted 10-fold in lysis buffer, split into two aliquots, and incubated either with GST (lane 3) or GST-CskSH2 (lane 4). A control immunoprecipitation was performed with normal mouse immunoglobulin (lane 1). All precipitates (A–C) were separated by 9% SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibodies. Membranes were stripped and reprobed either with mAbs against Csk or rabbit serum against RasGAP. (D) RasGAP and Csk immunoprecipitates were  subjected to an in vitro kinase assay; 32P-labeled p62 was isolated from each precipitate by gel electrophoresis, excised, and analyzed by partial V-8 proteolysis as described in Materials and Methods. Phosphopeptides were visualized by autoradiography.
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Figure 3: The 62-kD protein bound to Csk is associated with RasGAP. (A) Lysates of A20 cells stimulated with intact anti-IgG were incubated with agarose-conjugated normal mouse antibodies (lane 1), mAbs against RasGAP (lane 2), normal rabbit antibodies (lane 3), or affinity-purified antibodies against Csk (lane 4). Alternatively, lysates were incubated with GST (lane 5) or GST-CskSH2 (lane 6). (B) Lysates were immunodepleted with normal rabbit serum (lane 1), antibodies against Csk (lane 2), RasGAP (lane 3), Sam68 (lane 4), normal mouse immunoglobulin (lane 5) or p62 (Transduction Laboratories; lane 6, 2C4; lane 7). Depleted lysates were then precipitated with GST-CskSH2 (lanes 1–6). (C) RasGAP was precipitated as described above (lanes 2–4). The sample was boiled in lysis buffer containing 1% SDS. One-third was reserved for analysis of the RasGAP immunoprecipitate. The remaining two-thirds of the sample was diluted 10-fold in lysis buffer, split into two aliquots, and incubated either with GST (lane 3) or GST-CskSH2 (lane 4). A control immunoprecipitation was performed with normal mouse immunoglobulin (lane 1). All precipitates (A–C) were separated by 9% SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine antibodies. Membranes were stripped and reprobed either with mAbs against Csk or rabbit serum against RasGAP. (D) RasGAP and Csk immunoprecipitates were subjected to an in vitro kinase assay; 32P-labeled p62 was isolated from each precipitate by gel electrophoresis, excised, and analyzed by partial V-8 proteolysis as described in Materials and Methods. Phosphopeptides were visualized by autoradiography.
Mentions: The ability of the R107K mutant to bind p62 was unexpected and reminiscent of the binding of GAP-A.p62 to the Csk SH2 domain, which is known to persist after mutation of R107 (25). To test whether the 62-kD species might represent GAP-A.p62, A20 cells were stimulated with intact anti-IgG; tyrosine phosphorylated co-migrating 62-kD proteins were detected in RasGAP, Csk, and Csk SH2 precipitates (Fig. 3 A). Next, A20 cells were stimulated with intact anti-IgG and lysates were immunodepleted using antibodies specific for Csk, RasGAP, the 62-kD phosphoprotein recognized by mAb 2C4 (30), or Sam68, a protein initially identified as related to RasGAP-associated p62 (31). Immunodepleted or undepleted lysates were then assayed for protein binding to GST (data not shown) or GST-CskSH2. The amount of tyrosine phosphorylated p62 recovered from GST-CskSH2 after immunodepletion of RasGAP was reduced significantly in comparison to a control immunodepletion with nonimmune antibody (Fig. 3 B). The recovery of p62 was also reduced after immunodepletion of Csk, as expected. In contrast, immunodepletion of Sam68 had no effect on the recovery of p62 from the GST-CskSH2 eluate. Because the 2C4 antibody does not detect its ligand on immunoblot, we were not able to estimate how much of the 2C4-binding p62 species was depleted in this experiment. These results indicate that p62 interacts with RasGAP in A20 cell lysates, and are consistent with the interpretation that the Csk-associated p62 is related to or identical to GAP-A.p62.

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