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Cited by 199Protein Kinase Regulation and GTPase SignalingProtein Tyrosine PhosphatasesNF-κB Signaling Pathways

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Involvement of Guanosine Triphosphatases and Phospholipase C-γ2 in Extracellular Signal–regulated Kinase, c-Jun NH2-terminal Kinase, and p38 Mitogen-activated Protein Kinase Activation by the B Cell Antigen Receptor

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Hashimoto, Ari, Okada, Hidetaka, Jiang, Aimin, Kurosaki, Mari, Greenberg, Steven, Clark, Edward A., Kurosaki, Tomohiro·1998·The Journal of Experimental Medicine
DOI 10.1084/jem.188.7.1287PMC PMC2212492
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Abstract

Mitogen-activated protein (MAP) kinase family members, including extracellular signal–regulated kinase (ERK), c-Jun NH2-terminal kinase ( JNK), and p38 MAP kinase, have been implicated in coupling the B cell antigen receptor (BCR) to transcriptional responses. However, the mechanisms that lead to the activation of these MAP kinase family members have been poorly elucidated. Here we demonstrate that the BCR-induced ERK activation is reduced by loss of Grb2 or expression of a dominant-negative form of Ras, RasN17, whereas this response is not affected by loss of Shc. The inhibition of the ERK response was also observed in phospholipase C (PLC)-γ2–deficient DT40 B cells, and expression of RasN17 in the PLC-γ2–deficient cells completely abrogated the ERK activation. The PLC-γ2 dependency of ERK activation was most likely due to protein kinase C (PKC) activation rather than calcium mobilization, since loss of inositol 1,4,5-trisphosphate receptors did not affect ERK activation. Similar to cooperation of Ras with PKC activation in ERK response, both PLC-γ2–dependent signal and GTPase are required for BCR-induced JNK and p38 responses. JNK response is dependent on Rac1 and calcium mobilization, whereas p38 response requires Rac1 and PKC activation.

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Figures

Figure 1 Disruption of Grb2, Grap, and Shc genes in DT40 cells. (A) RNA expression of Grb2, Grap, and Shc was analyzed by Northern blot analysis using each chicken cDNA probe (top) or β-actin (bottom) (reference 64). Positions of 28S and 18S RNA are shown. (B) Grb2, Grap, and Shc protein expression in wild-type and targeted DT40 cells. Each protein was detected by Western blotting analysis using anti-Grb2, anti-Grap, and anti-Shc Abs. (C) Expression of dominant-negative Ras and dominant-negative Rac1.
Figure 1 Disruption of Grb2, Grap, and Shc genes in DT40 cells. (A) RNA expression of Grb2, Grap, and Shc was analyzed by Northern blot analysis using each chicken cDNA probe (top) or β-actin (bottom) (reference 64). Positions of 28S and 18S RNA are shown. (B) Grb2, Grap, and Shc protein expression in wild-type and targeted DT40 cells. Each protein was detected by Western blotting analysis using anti-Grb2, anti-Grap, and anti-Shc Abs. (C) Expression of dominant-negative Ras and dominant-negative Rac1.CC BY-NC-SA · PubMed Central
Figure 2 Cross-reactivity of various Abs. DT40 cell extracts were immunoprecipitated by anti-ERK2 Ab, anti-p38 Ab, and anti-JNK Ab. These immunoprecipitates were separated by 9% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated with indicated Abs (Blot Ab).
Figure 2 Cross-reactivity of various Abs. DT40 cell extracts were immunoprecipitated by anti-ERK2 Ab, anti-p38 Ab, and anti-JNK Ab. These immunoprecipitates were separated by 9% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated with indicated Abs (Blot Ab).CC BY-NC-SA · PubMed Central
Figure 3 BCR-induced ERK2 activation in various DT40 cells. Various DT40 cells were stimulated with M4 (4 μg/ ml) for indicated time. ERK2 was immunoprecipitated and the precipitates were assayed for kinase activity using GST–Elk1 fusion protein as a substrate. The kinase reaction products were resolved by 12.5% SDS-PAGE and their phosphorylation was quantified by autoradiography.
Figure 3 BCR-induced ERK2 activation in various DT40 cells. Various DT40 cells were stimulated with M4 (4 μg/ ml) for indicated time. ERK2 was immunoprecipitated and the precipitates were assayed for kinase activity using GST–Elk1 fusion protein as a substrate. The kinase reaction products were resolved by 12.5% SDS-PAGE and their phosphorylation was quantified by autoradiography.CC BY-NC-SA · PubMed Central
Figure 4 Time kinetics of ERK2, JNK, and p38 responses upon BCR engagement. After BCR stimulation, DT40 cell lysates were immunoprecipitated with anti-ERK2 Ab, anti-JNK1 Ab, or anti-p38 Ab. The kinase activities were assayed using GST–Elk1, GST–c-Jun, and GST–ATF2 as substrates for ERK2, JNK, and p38, respectively.
Figure 4 Time kinetics of ERK2, JNK, and p38 responses upon BCR engagement. After BCR stimulation, DT40 cell lysates were immunoprecipitated with anti-ERK2 Ab, anti-JNK1 Ab, or anti-p38 Ab. The kinase activities were assayed using GST–Elk1, GST–c-Jun, and GST–ATF2 as substrates for ERK2, JNK, and p38, respectively.CC BY-NC-SA · PubMed Central
Figure 5 Involvement of PLC-γ2 and Ras on BCR-induced ERK2 activation. BCR-stimulated DT40 cells were lysed and immunoprecipitated with anti-ERK2 Ab. Stimulation conditions and in vitro kinase assay were described as in Fig. 3.
Figure 5 Involvement of PLC-γ2 and Ras on BCR-induced ERK2 activation. BCR-stimulated DT40 cells were lysed and immunoprecipitated with anti-ERK2 Ab. Stimulation conditions and in vitro kinase assay were described as in Fig. 3.CC BY-NC-SA · PubMed Central
Figure 6 Effect of RasN17 and Rac1N17 on JNK and p38 responses. DT40 cells were stimulated with M4 (4 μg/ml) for indicated times. Cell lysates were immunoprecipitated with anti-JNK1 Ab and anti-p38 Ab, and kinase activities were assayed using GST–c-Jun and GST–ATF2 as substrates, respectively. After electrophoresis, the labeled GST–c-Jun and GST–ATF2 bands were visualized by autoradiography.
Figure 6 Effect of RasN17 and Rac1N17 on JNK and p38 responses. DT40 cells were stimulated with M4 (4 μg/ml) for indicated times. Cell lysates were immunoprecipitated with anti-JNK1 Ab and anti-p38 Ab, and kinase activities were assayed using GST–c-Jun and GST–ATF2 as substrates, respectively. After electrophoresis, the labeled GST–c-Jun and GST–ATF2 bands were visualized by autoradiography.CC BY-NC-SA · PubMed Central
Figure 7 BCR-induced JNK and p38 responses in PLC-γ2– and IP3R-deficient DT40 cells. Cells were stimulated with M4 (4 μg/ml) for indicated times and cell lysates were immunoprecipitated with anti-JNK1 and anti-p38 Ab. The kinase assays were done as in Fig. 6.
Figure 7 BCR-induced JNK and p38 responses in PLC-γ2– and IP3R-deficient DT40 cells. Cells were stimulated with M4 (4 μg/ml) for indicated times and cell lysates were immunoprecipitated with anti-JNK1 and anti-p38 Ab. The kinase assays were done as in Fig. 6.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Hashimoto, A., Okada, H., Jiang, A., Kurosaki, M., Greenberg, S., Clark, E. A., & Kurosaki, T.
Affiliations
Kansai Medical University (JP)
Corresponding author
Kurosaki, Tomohiro
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
1998-10-05
Volume
188
Issue
7
Pages
1287-1295
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.188.7.1287
PMC
PMC2212492
OpenAlex
W1965678722
Cited by
199
References
68
Citations by year
2026: 1 · 2025: 2 · 2024: 1 · 2021: 4 · 2020: 7 · 2019: 3 · 2018: 8 · 2017: 4 · 2016: 4 · 2015: 8 · 2014: 6 · 2013: 5 · 2012: 2
FWCI
5.59
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5p01gm042508-09)
  • National Institute of General Medical Sciences (GM42508, GM-37905, R01GM037905)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
Keywords
B cell receptor, extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, p38 MAPK, PLCγ2, Ras, Grb2, Shc, protein kinase C, Rac1, calcium mobilization, IP3 receptor, MAPK activation, signal transduction, B cell signaling
MeSH terms
Animals, Cell Line, Chickens, Enzyme Activation, Isoenzymes, Type C Phospholipases, Protein Kinase C, Proteins, Receptors, Antigen, B-Cell, Signal Transduction, Calcium-Calmodulin-Dependent Protein Kinases, GTP-Binding Proteins, Mitogen-Activated Protein Kinase 1, GTP Phosphohydrolases, rac GTP-Binding Proteins, Mitogen-Activated Protein Kinases, JNK Mitogen-Activated Protein Kinases, p38 Mitogen-Activated Protein Kinases, Adaptor Proteins, Signal Transducing, GRB2 Adaptor Protein, Phospholipase C gamma

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