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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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Both catalytic activity and C1 domain of RasGRP3 are required in BCR-mediated Ras activation. (A) Schematic diagram of various mutant RasGRP3 constructs. (B) Protein expression analyses of various mutant DT40 cell lines. Wild-type, RasGRP3-, or PLC-γ2–deficient DT40 B cells expressing various RasGRP3 mutants are shown. Endogenous RasGRP3 is also shown in wild-type or PLC-γ2–deficient DT40 cells. Whole cell lysates prepared from 2 × 106 cells were analyzed by Western blotting using anti-RasGRP3 Ab or anti-BLNK Ab. (C) Analysis of BCR-mediated Ras activation in RasGRP3-deficient cells expressing wild-type RasGRP3 and its mutants was performed as described in Fig. 1. Each experiment was repeated at least three times.
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fig6: Both catalytic activity and C1 domain of RasGRP3 are required in BCR-mediated Ras activation. (A) Schematic diagram of various mutant RasGRP3 constructs. (B) Protein expression analyses of various mutant DT40 cell lines. Wild-type, RasGRP3-, or PLC-γ2–deficient DT40 B cells expressing various RasGRP3 mutants are shown. Endogenous RasGRP3 is also shown in wild-type or PLC-γ2–deficient DT40 cells. Whole cell lysates prepared from 2 × 106 cells were analyzed by Western blotting using anti-RasGRP3 Ab or anti-BLNK Ab. (C) Analysis of BCR-mediated Ras activation in RasGRP3-deficient cells expressing wild-type RasGRP3 and its mutants was performed as described in Fig. 1. Each experiment was repeated at least three times.

Mentions: Generation of Sos1/Sos2 double-, RasGRP1/RasGRP3 double-, and RasGRP3-deficient DT40 B cells. (A) RNA expression of Sos1, Sos2, RasGRP1, and RasGRP3 was analyzed by Northern blot analysis using each chicken cDNA probe (top) or β-actin (bottom). The specific radio activity of each probe was roughly the same. Positions of 28S rRNA are shown. (B) Sos1, Sos2, RasGRP1, and RasGRP3 protein expression in wild-type and various mutant DT40 cells. Each protein was immunoprecipitated and detected by Western blotting with each specific Abs. Sos1−Sos2−, Sos1/Sos2 double-deficient DT40 cells. GRP3−, RasGRP3-deficient DT40 cells. GRP1−GRP3−, RasGRP1/RasGRP3 double-deficient DT40 cells. (C) Cell surface expression of BCR on wild-type and various DT40 mutants (see Fig. 6 A for designation). (D) Cell surface expression of transfected EGFR on wild-type and mutant DT40 cells.


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)

Both catalytic activity and C1 domain of RasGRP3 are required in BCR-mediated Ras activation. (A) Schematic diagram of various mutant RasGRP3 constructs. (B) Protein expression analyses of various mutant DT40 cell lines. Wild-type, RasGRP3-, or PLC-γ2–deficient DT40 B cells expressing various RasGRP3 mutants are shown. Endogenous RasGRP3 is also shown in wild-type or PLC-γ2–deficient DT40 cells. Whole cell lysates prepared from 2 × 106 cells were analyzed by Western blotting using anti-RasGRP3 Ab or anti-BLNK Ab. (C) Analysis of BCR-mediated Ras activation in RasGRP3-deficient cells expressing wild-type RasGRP3 and its mutants was performed as described in Fig. 1. Each experiment was repeated at least three times.
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Related In: Results  -  Collection

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fig6: Both catalytic activity and C1 domain of RasGRP3 are required in BCR-mediated Ras activation. (A) Schematic diagram of various mutant RasGRP3 constructs. (B) Protein expression analyses of various mutant DT40 cell lines. Wild-type, RasGRP3-, or PLC-γ2–deficient DT40 B cells expressing various RasGRP3 mutants are shown. Endogenous RasGRP3 is also shown in wild-type or PLC-γ2–deficient DT40 cells. Whole cell lysates prepared from 2 × 106 cells were analyzed by Western blotting using anti-RasGRP3 Ab or anti-BLNK Ab. (C) Analysis of BCR-mediated Ras activation in RasGRP3-deficient cells expressing wild-type RasGRP3 and its mutants was performed as described in Fig. 1. Each experiment was repeated at least three times.
Mentions: Generation of Sos1/Sos2 double-, RasGRP1/RasGRP3 double-, and RasGRP3-deficient DT40 B cells. (A) RNA expression of Sos1, Sos2, RasGRP1, and RasGRP3 was analyzed by Northern blot analysis using each chicken cDNA probe (top) or β-actin (bottom). The specific radio activity of each probe was roughly the same. Positions of 28S rRNA are shown. (B) Sos1, Sos2, RasGRP1, and RasGRP3 protein expression in wild-type and various mutant DT40 cells. Each protein was immunoprecipitated and detected by Western blotting with each specific Abs. Sos1−Sos2−, Sos1/Sos2 double-deficient DT40 cells. GRP3−, RasGRP3-deficient DT40 cells. GRP1−GRP3−, RasGRP1/RasGRP3 double-deficient DT40 cells. (C) Cell surface expression of BCR on wild-type and various DT40 mutants (see Fig. 6 A for designation). (D) Cell surface expression of transfected EGFR on wild-type and mutant DT40 cells.

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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