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Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-gamma2 to Ras in B cell receptor signaling.

Oh-hora M, Johmura S, Hashimoto A, Hikida M, Kurosaki T - J. Exp. Med. (2003)

Bottom Line: The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2.Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells.This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan.

ABSTRACT
Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, we have generated Sos1/Sos2 double- or RasGRP3-deficient B cell lines and determined their ability to mediate Ras activation upon B cell receptor (BCR) stimulation. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation.

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Requirement for RasGRP3 in BCR-mediated ERK activation in primary B cells. (A) Expression of mGEFRasGRP3 in infected B cells. Splenic B cells from C57BL/6J mice were transduced with a GEF mutant of mouse RasGRP3 (mGEFRasGRP3) by lentivirus. Transduction efficiencies were monitored by EGFP expression. Fluorescence intensities of untransduced cells are shown in dashed lines (top). Expression levels of endogenous RasGRP3 and infected mGEFRasGRP3 were analyzed by RT-PCR as described in the Materials and Methods (bottom). (B) Attenuation of Erk activation by the expression of mGEFRasGRP3. Infected cells were stimulated with 12 μg/ml anti–mouse IgM and ERK activation was measured using anti-phospho–p44/p42 MAPK mAb (E10) (top) and anti-p44/p42 MAPK Ab (bottom).
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fig4: Requirement for RasGRP3 in BCR-mediated ERK activation in primary B cells. (A) Expression of mGEFRasGRP3 in infected B cells. Splenic B cells from C57BL/6J mice were transduced with a GEF mutant of mouse RasGRP3 (mGEFRasGRP3) by lentivirus. Transduction efficiencies were monitored by EGFP expression. Fluorescence intensities of untransduced cells are shown in dashed lines (top). Expression levels of endogenous RasGRP3 and infected mGEFRasGRP3 were analyzed by RT-PCR as described in the Materials and Methods (bottom). (B) Attenuation of Erk activation by the expression of mGEFRasGRP3. Infected cells were stimulated with 12 μg/ml anti–mouse IgM and ERK activation was measured using anti-phospho–p44/p42 MAPK mAb (E10) (top) and anti-p44/p42 MAPK Ab (bottom).

Mentions: To determine the importance of RasGRP family, particularly RasGRP3, in primary B cells as well, we took an approach of introducing a dominant-negative form of RasGRP3 into primary B cells by using a lentivirus system. Based on the data that overexpression of the GEF mutant of RasGRP3 in wild-type DT40 B cells inhibited BCR-mediated Ras activation (unpublished data), the similar mutant using the mouse RasGRP3 cDNA was subcloned into the HIV-based lentiviral vector containing a bicistronic mRNA encoding GFP. Because the Erk assay, rather than the Ras assay, needs a relatively small amount of infected B cells, we monitored BCR-mediated Erk activity in infected primary B cells. As shown in Fig. 4 B, primary B cells infected with a dominant negative form of RasGRP3 manifested a decrease in BCR-mediated Erk activation, compared with control viruses.


Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-gamma2 to Ras in B cell receptor signaling.

Oh-hora M, Johmura S, Hashimoto A, Hikida M, Kurosaki T - J. Exp. Med. (2003)

Requirement for RasGRP3 in BCR-mediated ERK activation in primary B cells. (A) Expression of mGEFRasGRP3 in infected B cells. Splenic B cells from C57BL/6J mice were transduced with a GEF mutant of mouse RasGRP3 (mGEFRasGRP3) by lentivirus. Transduction efficiencies were monitored by EGFP expression. Fluorescence intensities of untransduced cells are shown in dashed lines (top). Expression levels of endogenous RasGRP3 and infected mGEFRasGRP3 were analyzed by RT-PCR as described in the Materials and Methods (bottom). (B) Attenuation of Erk activation by the expression of mGEFRasGRP3. Infected cells were stimulated with 12 μg/ml anti–mouse IgM and ERK activation was measured using anti-phospho–p44/p42 MAPK mAb (E10) (top) and anti-p44/p42 MAPK Ab (bottom).
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Related In: Results  -  Collection

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fig4: Requirement for RasGRP3 in BCR-mediated ERK activation in primary B cells. (A) Expression of mGEFRasGRP3 in infected B cells. Splenic B cells from C57BL/6J mice were transduced with a GEF mutant of mouse RasGRP3 (mGEFRasGRP3) by lentivirus. Transduction efficiencies were monitored by EGFP expression. Fluorescence intensities of untransduced cells are shown in dashed lines (top). Expression levels of endogenous RasGRP3 and infected mGEFRasGRP3 were analyzed by RT-PCR as described in the Materials and Methods (bottom). (B) Attenuation of Erk activation by the expression of mGEFRasGRP3. Infected cells were stimulated with 12 μg/ml anti–mouse IgM and ERK activation was measured using anti-phospho–p44/p42 MAPK mAb (E10) (top) and anti-p44/p42 MAPK Ab (bottom).
Mentions: To determine the importance of RasGRP family, particularly RasGRP3, in primary B cells as well, we took an approach of introducing a dominant-negative form of RasGRP3 into primary B cells by using a lentivirus system. Based on the data that overexpression of the GEF mutant of RasGRP3 in wild-type DT40 B cells inhibited BCR-mediated Ras activation (unpublished data), the similar mutant using the mouse RasGRP3 cDNA was subcloned into the HIV-based lentiviral vector containing a bicistronic mRNA encoding GFP. Because the Erk assay, rather than the Ras assay, needs a relatively small amount of infected B cells, we monitored BCR-mediated Erk activity in infected primary B cells. As shown in Fig. 4 B, primary B cells infected with a dominant negative form of RasGRP3 manifested a decrease in BCR-mediated Erk activation, compared with control viruses.

Bottom Line: The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2.Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells.This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan.

ABSTRACT
Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, we have generated Sos1/Sos2 double- or RasGRP3-deficient B cell lines and determined their ability to mediate Ras activation upon B cell receptor (BCR) stimulation. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation.

Show MeSH