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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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Differential requirement for RasGRP and Sos family in BCR- or EGFR-mediated Ras activation. (A) BCR-mediated Ras and ERK activation in wild-type, Sos1/Sos2 double-, RasGRP3-, and RasGRP1/RasGRP3 double-deficient DT40 cells. Ras and ERK activation was measured as described in Fig. 1. (B) EGFR-mediated Ras and ERK activation in each deficient DT40 cell. Cells were stimulated by 50 ng/ml EGF and analyzed as in Fig. 1. These experiments were performed at least three times and the representative results are shown.
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fig3: Differential requirement for RasGRP and Sos family in BCR- or EGFR-mediated Ras activation. (A) BCR-mediated Ras and ERK activation in wild-type, Sos1/Sos2 double-, RasGRP3-, and RasGRP1/RasGRP3 double-deficient DT40 cells. Ras and ERK activation was measured as described in Fig. 1. (B) EGFR-mediated Ras and ERK activation in each deficient DT40 cell. Cells were stimulated by 50 ng/ml EGF and analyzed as in Fig. 1. These experiments were performed at least three times and the representative results are shown.

Mentions: As shown in Fig. 3 A, BCR-mediated Ras activation was unaffected in Sos1/Sos2 double-deficient DT40 cells, but was decreased by ∼60% in RasGRP3-deficient cells. This defective Ras activation was exacerbated to a small degree by double knockouts of RasGRP3 and RasGRP1, although RasGRP1 single-deficient DT40 cells exhibited apparently normal BCR-mediated Ras activation (unpublished data). Hence, we conclude that BCR-mediated Ras activation requires RasGRP3, but not the Sos family, in DT40 B cells. In addition, RasGRP1 appears to function as a redundant molecule of RasGRP3 and the relative importance of RasGRP3 in DT40 B cells is presumably attributable to dominant expression of RasGRP3 rather than RasGRP1 particularly in this cell line (Fig. 2 A). Consistent with the defective Ras activation in RasGRP3- or RasGRP1/RasGRP3 double-deficient DT40 cells, Erk activation was decreased in these mutants. However, the kinetics of Erk activation in these mutants was distinct from that of Ras, being marked at 1 min. These data imply that a Ras-independent pathway may compensate for the defective Erk activation in an attempt to cope with the deficit, particularly at the later time point.


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)

Differential requirement for RasGRP and Sos family in BCR- or EGFR-mediated Ras activation. (A) BCR-mediated Ras and ERK activation in wild-type, Sos1/Sos2 double-, RasGRP3-, and RasGRP1/RasGRP3 double-deficient DT40 cells. Ras and ERK activation was measured as described in Fig. 1. (B) EGFR-mediated Ras and ERK activation in each deficient DT40 cell. Cells were stimulated by 50 ng/ml EGF and analyzed as in Fig. 1. These experiments were performed at least three times and the representative results are shown.
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Related In: Results  -  Collection

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fig3: Differential requirement for RasGRP and Sos family in BCR- or EGFR-mediated Ras activation. (A) BCR-mediated Ras and ERK activation in wild-type, Sos1/Sos2 double-, RasGRP3-, and RasGRP1/RasGRP3 double-deficient DT40 cells. Ras and ERK activation was measured as described in Fig. 1. (B) EGFR-mediated Ras and ERK activation in each deficient DT40 cell. Cells were stimulated by 50 ng/ml EGF and analyzed as in Fig. 1. These experiments were performed at least three times and the representative results are shown.
Mentions: As shown in Fig. 3 A, BCR-mediated Ras activation was unaffected in Sos1/Sos2 double-deficient DT40 cells, but was decreased by ∼60% in RasGRP3-deficient cells. This defective Ras activation was exacerbated to a small degree by double knockouts of RasGRP3 and RasGRP1, although RasGRP1 single-deficient DT40 cells exhibited apparently normal BCR-mediated Ras activation (unpublished data). Hence, we conclude that BCR-mediated Ras activation requires RasGRP3, but not the Sos family, in DT40 B cells. In addition, RasGRP1 appears to function as a redundant molecule of RasGRP3 and the relative importance of RasGRP3 in DT40 B cells is presumably attributable to dominant expression of RasGRP3 rather than RasGRP1 particularly in this cell line (Fig. 2 A). Consistent with the defective Ras activation in RasGRP3- or RasGRP1/RasGRP3 double-deficient DT40 cells, Erk activation was decreased in these mutants. However, the kinetics of Erk activation in these mutants was distinct from that of Ras, being marked at 1 min. These data imply that a Ras-independent pathway may compensate for the defective Erk activation in an attempt to cope with the deficit, particularly at the later time point.

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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