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Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT.

Asada H, Ishii N, Sasaki Y, Endo K, Kasai H, Tanaka N, Takeshita T, Tsuchiya S, Konno T, Sugamura K - J. Exp. Med. (1999)

Bottom Line: Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity.Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant.Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Tohoku University School of Medicine, Sendai 980-8575, Japan.

ABSTRACT
We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

Show MeSH
Coimmunoprecipitation of  SLP-76 and LAT with Grf40 in Jurkat cells.  Jurkat cells were stimulated with (+) or  without (−) OKT3 for 3 min, and their lysates were immunoprecipitated (IP) with  anti-Grf40 or preimmune (control) serum.  The immunoprecipitates were separated by  SDS-PAGE and then immunoblotted (IB)  with antiphosphotyrosine mAbs (A), and  with anti–SLP-76 antiserum or anti-LAT  Ab (B). COS7 cells were transiently transfected with 10 μg expression plasmids for  Myc-tagged wild-type Grf40 (wild) or four  Myc-tagged Grf40 mutants (dSH3C, dSH2,  dSH3N, and dSH3NC), together with 10  μg expression plasmids for SLP-76 by electroporation, and then incubated for 48 h.  Their lysates were immunoprecipitated with  anti–SLP-76 or preimmune (control) serum, and then immunoblotted with anti-Myc mAb or anti–SLP-76 antiserum (C).  COS7 cells were transiently transfected with  10 μg expression plasmids for Flag-tagged  wild-type SLP-76 (wild), four Flag-tagged  SLP-76 mutants (157-533, 217-533, 241-533, and 281-533), or an empty vector  (control), together with 10 μg expression  plasmids for Myc-tagged Grf40 by electroporation, and then incubated for 48 h.  Their lysates were immunoprecipitated with  anti-Flag mAb, and then immunoblotted  with anti-Myc or anti-Flag mAb (D).
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Figure 2: Coimmunoprecipitation of SLP-76 and LAT with Grf40 in Jurkat cells. Jurkat cells were stimulated with (+) or without (−) OKT3 for 3 min, and their lysates were immunoprecipitated (IP) with anti-Grf40 or preimmune (control) serum. The immunoprecipitates were separated by SDS-PAGE and then immunoblotted (IB) with antiphosphotyrosine mAbs (A), and with anti–SLP-76 antiserum or anti-LAT Ab (B). COS7 cells were transiently transfected with 10 μg expression plasmids for Myc-tagged wild-type Grf40 (wild) or four Myc-tagged Grf40 mutants (dSH3C, dSH2, dSH3N, and dSH3NC), together with 10 μg expression plasmids for SLP-76 by electroporation, and then incubated for 48 h. Their lysates were immunoprecipitated with anti–SLP-76 or preimmune (control) serum, and then immunoblotted with anti-Myc mAb or anti–SLP-76 antiserum (C). COS7 cells were transiently transfected with 10 μg expression plasmids for Flag-tagged wild-type SLP-76 (wild), four Flag-tagged SLP-76 mutants (157-533, 217-533, 241-533, and 281-533), or an empty vector (control), together with 10 μg expression plasmids for Myc-tagged Grf40 by electroporation, and then incubated for 48 h. Their lysates were immunoprecipitated with anti-Flag mAb, and then immunoblotted with anti-Myc or anti-Flag mAb (D).

Mentions: Since Grb2 has been shown to bind to SLP-76 and LAT, which are 76- and 36/38-kD tyrosine-phosphorylated proteins essential for TCR-mediated signaling, respectively (10, 11, 18), we asked ourselves whether or not Grf40 is also associated with SLP-76 and LAT. We detected 76- and 36/38-kD tyrosine-phosphorylated proteins that coimmunoprecipitated with Grf40 in Jurkat cells after stimulation by TCR cross-linking with OKT3 (Fig. 2 A). We then confirmed that the 76- and 36/38-kD tyrosine-phosphorylated proteins were SLP-76 and LAT, respectively, by stimulating Jurkat cells with OKT3. Their lysates were immunoprecipitated with anti-Grf40 Ab, and the immunoprecipitates were then immunoblotted with anti-LAT, anti–SLP-76, or anti-Grf40 Ab. Grf40 precipitated SLP-76 irrespective of TCR stimulation, but precipitated LAT only after TCR stimulation (Fig. 2 B). These results indicate the association of Grf40 with SLP-76 and LAT in Jurkat cells. To determine the association site of Grf40 for SLP-76, we carried out further coimmunoprecipitation assays between the various deletion mutants of Grf40 and SLP-76. COS7 cells were transiently transfected with Myc-tagged wild-type Grf40 and four Grf40 mutants deleted of the NH2-terminal SH3 domain (Grf40-dSH3N), the COOH-terminal SH3 domain (Grf40-dSH3C), both the NH2- and COOH-terminal SH3 domains (Grf40-dSH3NC), or the SH2 domain (Grf40-dSH2). The transfected COS7 cells were immunoprecipitated with anti–SLP-76 Ab or anti-Myc mAb, and then immunoblotted with anti-Myc mAb or anti–SLP-76 Ab. Wild-type Grf40 and the Grf40-dSH2 and Grf40-dSH3N mutants were coimmunoprecipitated with SLP-76, but the Grf40-dSH3C and Grf40-dSH3NC mutants were not tested (Fig. 2 C). Conversely, SLP-76 was coimmunoprecipitated with Grf40-dSH2, Grf40-dSH3N, and wild-type Grf40, but not with Grf40-dSH3C and Grf40-dSH3NC mutants (data not shown). These results indicate that the COOH-terminal SH3 domain of Grf40 is an association site for SLP-76. We next determined the association site of SLP-76 for Grf40 by using various SLP-76 mutants. Flag-tagged wild-type and four mutants of SLP-76 were introduced into COS7 cells together with Myc-tagged Grf40, and then immunoprecipitated and immunoblotted with anti-Flag and anti-Myc Abs. Myc-tagged Grf40 was coimmunoprecipitated with the SLP-76 mutants consisting of and containing the amino acid position Glu217–Pro533, but not with the SLP-76 mutant consisting of the amino acid position Pro241–Pro533. These results indicate that the Grf40 binding site is located in the amino acid position Glu217–Lys240 of SLP-76 (Fig. 2 D). This Grf40 binding site of SLP-76 almost overlaps the amino acid position Asn224–Asp244, which has been shown to be the Grb2 binding site (15). On the other hand, the binding site of Grb2 for LAT has been shown to be the SH2 domain of Grb2, which is thought to bind to the phosphorylated tyrosine residue (18). Together with this notion, we showed that LAT is tyrosine phosphorylated and subsequently coimmunoprecipitated with Grf40 after TCR stimulation, suggesting that the SH2 domain of Grf40 is possibly the binding site for LAT.


Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT.

Asada H, Ishii N, Sasaki Y, Endo K, Kasai H, Tanaka N, Takeshita T, Tsuchiya S, Konno T, Sugamura K - J. Exp. Med. (1999)

Coimmunoprecipitation of  SLP-76 and LAT with Grf40 in Jurkat cells.  Jurkat cells were stimulated with (+) or  without (−) OKT3 for 3 min, and their lysates were immunoprecipitated (IP) with  anti-Grf40 or preimmune (control) serum.  The immunoprecipitates were separated by  SDS-PAGE and then immunoblotted (IB)  with antiphosphotyrosine mAbs (A), and  with anti–SLP-76 antiserum or anti-LAT  Ab (B). COS7 cells were transiently transfected with 10 μg expression plasmids for  Myc-tagged wild-type Grf40 (wild) or four  Myc-tagged Grf40 mutants (dSH3C, dSH2,  dSH3N, and dSH3NC), together with 10  μg expression plasmids for SLP-76 by electroporation, and then incubated for 48 h.  Their lysates were immunoprecipitated with  anti–SLP-76 or preimmune (control) serum, and then immunoblotted with anti-Myc mAb or anti–SLP-76 antiserum (C).  COS7 cells were transiently transfected with  10 μg expression plasmids for Flag-tagged  wild-type SLP-76 (wild), four Flag-tagged  SLP-76 mutants (157-533, 217-533, 241-533, and 281-533), or an empty vector  (control), together with 10 μg expression  plasmids for Myc-tagged Grf40 by electroporation, and then incubated for 48 h.  Their lysates were immunoprecipitated with  anti-Flag mAb, and then immunoblotted  with anti-Myc or anti-Flag mAb (D).
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Figure 2: Coimmunoprecipitation of SLP-76 and LAT with Grf40 in Jurkat cells. Jurkat cells were stimulated with (+) or without (−) OKT3 for 3 min, and their lysates were immunoprecipitated (IP) with anti-Grf40 or preimmune (control) serum. The immunoprecipitates were separated by SDS-PAGE and then immunoblotted (IB) with antiphosphotyrosine mAbs (A), and with anti–SLP-76 antiserum or anti-LAT Ab (B). COS7 cells were transiently transfected with 10 μg expression plasmids for Myc-tagged wild-type Grf40 (wild) or four Myc-tagged Grf40 mutants (dSH3C, dSH2, dSH3N, and dSH3NC), together with 10 μg expression plasmids for SLP-76 by electroporation, and then incubated for 48 h. Their lysates were immunoprecipitated with anti–SLP-76 or preimmune (control) serum, and then immunoblotted with anti-Myc mAb or anti–SLP-76 antiserum (C). COS7 cells were transiently transfected with 10 μg expression plasmids for Flag-tagged wild-type SLP-76 (wild), four Flag-tagged SLP-76 mutants (157-533, 217-533, 241-533, and 281-533), or an empty vector (control), together with 10 μg expression plasmids for Myc-tagged Grf40 by electroporation, and then incubated for 48 h. Their lysates were immunoprecipitated with anti-Flag mAb, and then immunoblotted with anti-Myc or anti-Flag mAb (D).
Mentions: Since Grb2 has been shown to bind to SLP-76 and LAT, which are 76- and 36/38-kD tyrosine-phosphorylated proteins essential for TCR-mediated signaling, respectively (10, 11, 18), we asked ourselves whether or not Grf40 is also associated with SLP-76 and LAT. We detected 76- and 36/38-kD tyrosine-phosphorylated proteins that coimmunoprecipitated with Grf40 in Jurkat cells after stimulation by TCR cross-linking with OKT3 (Fig. 2 A). We then confirmed that the 76- and 36/38-kD tyrosine-phosphorylated proteins were SLP-76 and LAT, respectively, by stimulating Jurkat cells with OKT3. Their lysates were immunoprecipitated with anti-Grf40 Ab, and the immunoprecipitates were then immunoblotted with anti-LAT, anti–SLP-76, or anti-Grf40 Ab. Grf40 precipitated SLP-76 irrespective of TCR stimulation, but precipitated LAT only after TCR stimulation (Fig. 2 B). These results indicate the association of Grf40 with SLP-76 and LAT in Jurkat cells. To determine the association site of Grf40 for SLP-76, we carried out further coimmunoprecipitation assays between the various deletion mutants of Grf40 and SLP-76. COS7 cells were transiently transfected with Myc-tagged wild-type Grf40 and four Grf40 mutants deleted of the NH2-terminal SH3 domain (Grf40-dSH3N), the COOH-terminal SH3 domain (Grf40-dSH3C), both the NH2- and COOH-terminal SH3 domains (Grf40-dSH3NC), or the SH2 domain (Grf40-dSH2). The transfected COS7 cells were immunoprecipitated with anti–SLP-76 Ab or anti-Myc mAb, and then immunoblotted with anti-Myc mAb or anti–SLP-76 Ab. Wild-type Grf40 and the Grf40-dSH2 and Grf40-dSH3N mutants were coimmunoprecipitated with SLP-76, but the Grf40-dSH3C and Grf40-dSH3NC mutants were not tested (Fig. 2 C). Conversely, SLP-76 was coimmunoprecipitated with Grf40-dSH2, Grf40-dSH3N, and wild-type Grf40, but not with Grf40-dSH3C and Grf40-dSH3NC mutants (data not shown). These results indicate that the COOH-terminal SH3 domain of Grf40 is an association site for SLP-76. We next determined the association site of SLP-76 for Grf40 by using various SLP-76 mutants. Flag-tagged wild-type and four mutants of SLP-76 were introduced into COS7 cells together with Myc-tagged Grf40, and then immunoprecipitated and immunoblotted with anti-Flag and anti-Myc Abs. Myc-tagged Grf40 was coimmunoprecipitated with the SLP-76 mutants consisting of and containing the amino acid position Glu217–Pro533, but not with the SLP-76 mutant consisting of the amino acid position Pro241–Pro533. These results indicate that the Grf40 binding site is located in the amino acid position Glu217–Lys240 of SLP-76 (Fig. 2 D). This Grf40 binding site of SLP-76 almost overlaps the amino acid position Asn224–Asp244, which has been shown to be the Grb2 binding site (15). On the other hand, the binding site of Grb2 for LAT has been shown to be the SH2 domain of Grb2, which is thought to bind to the phosphorylated tyrosine residue (18). Together with this notion, we showed that LAT is tyrosine phosphorylated and subsequently coimmunoprecipitated with Grf40 after TCR stimulation, suggesting that the SH2 domain of Grf40 is possibly the binding site for LAT.

Bottom Line: Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity.Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant.Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Tohoku University School of Medicine, Sendai 980-8575, Japan.

ABSTRACT
We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

Show MeSH