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The Src homology 2 domain of Vav is required for its compartmentation to the plasma membrane and activation of c-Jun NH(2)-terminal kinase 1.

Arudchandran R, Brown MJ, Peirce MJ, Song JS, Zhang J, Siraganian RP, Blank U, Rivera J - J. Exp. Med. (2000)

Bottom Line: We found that Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement.The FcinRI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs).Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.

View Article: PubMed Central - PubMed

Affiliation: Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Vav is a hematopoietic cell-specific guanine nucleotide exchange factor (GEF) whose activation is mediated by receptor engagement. The relationship of Vav localization to its function is presently unclear. We found that Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement. The redistribution of Vav was mediated by its Src homology 2 (SH2) domain and required Syk activity. The FcinRI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs). The scaffold protein, linker for activation of T cells (LAT), and Rac1 (a target of Vav activity) were constitutively present in GEMs. Expression of an SH2 domain-containing COOH-terminal fragment of Vav inhibited Vav phosphorylation and movement to the GEMs but had no effect on the tyrosine phosphorylation of the adaptor protein, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD), and LAT. However, assembly of the multiprotein complex containing these proteins was inhibited. In addition, FcinRI-dependent activation of c-Jun NH(2)-terminal kinase 1 (JNK1) was also inhibited. Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.

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Vav-C inhibits Vav phosphorylation and association with but not phosphorylation of Syk, SLP-76, and LAT. (A) Vav-C (SH3-SH2-SH3) is membrane localized upon Fc∈RI engagement. RBL-2H3 cells were transfected with Vav-C-GFP or an SH2 domain–mutated Vav-C (R694K)-GFP. Cells were stimulated with 30 ng/ml of Ag for 3 min (+Ag) or left untreated (−Ag) and examined by confocal laser microscopy. (B) Expression levels of GFP alone, Vav-C, or Vav-C (R694K) tagged with GFP. Cells transfected as in A were lysed, and 5 × 104 cell equivalents per lane of protein were resolved by SDS-PAGE and transferred for immunoblotting (IB). For immunoprecipitation experiments, 1.0–2.0 × 107 cells were used. Expression levels were determined by probing with an antibody to GFP (shown) or in some cases with a mouse mAb to Vav. (C) Vav-C inhibits phosphorylation of Vav and its association with Syk but not Syk phosphorylation. RBL-2H3 cells transfected with Vav, Vav-C, and Syk or Vav, Vav-C (R694K), and Syk were stimulated as in the legend to  Fig. 2 D, lysed, and incubated with antibody to Syk. Recovered proteins were immunoblotted (IB) with antiphosphotyrosine (Anti-PY), then stripped and reprobed sequentially with anti-Vav and anti-Syk. (D) Nontransfected RBL-2H3 cells were stimulated (Ag+) or not (Ag−) as in the legend to  Fig. 2 D. Cells were lysed as described in Materials and Methods, and LAT or Vav was immunoprecipitated. Proteins were resolved and immunoblotted with antibody to LAT, SLP-76, and Vav. (E) Vav-C inhibits the association of Vav and LAT with SLP-76 without affecting the SLP-76 phosphorylation. RBL-2H3 cells were transfected with Vav and GFP or Vav and Vav-C-GFP. Cells were stimulated (Ag+) or not (Ag−) as in the legend to  Fig. 2 D, then lysed and incubated with antibody to SLP-76. Immunoblots were first probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti–SLP-76, anti-Vav, anti-LAT, and anti-Grb2. (F) Vav-C has no effect on the tyrosine phosphorylation of LAT. Experiments were done as in E, but cell lysates were incubated with antibody to LAT (Anti-LAT). Immunoblots were probed with antiphosphotyrosine and subsequently with anti-LAT.
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Figure 4: Vav-C inhibits Vav phosphorylation and association with but not phosphorylation of Syk, SLP-76, and LAT. (A) Vav-C (SH3-SH2-SH3) is membrane localized upon Fc∈RI engagement. RBL-2H3 cells were transfected with Vav-C-GFP or an SH2 domain–mutated Vav-C (R694K)-GFP. Cells were stimulated with 30 ng/ml of Ag for 3 min (+Ag) or left untreated (−Ag) and examined by confocal laser microscopy. (B) Expression levels of GFP alone, Vav-C, or Vav-C (R694K) tagged with GFP. Cells transfected as in A were lysed, and 5 × 104 cell equivalents per lane of protein were resolved by SDS-PAGE and transferred for immunoblotting (IB). For immunoprecipitation experiments, 1.0–2.0 × 107 cells were used. Expression levels were determined by probing with an antibody to GFP (shown) or in some cases with a mouse mAb to Vav. (C) Vav-C inhibits phosphorylation of Vav and its association with Syk but not Syk phosphorylation. RBL-2H3 cells transfected with Vav, Vav-C, and Syk or Vav, Vav-C (R694K), and Syk were stimulated as in the legend to Fig. 2 D, lysed, and incubated with antibody to Syk. Recovered proteins were immunoblotted (IB) with antiphosphotyrosine (Anti-PY), then stripped and reprobed sequentially with anti-Vav and anti-Syk. (D) Nontransfected RBL-2H3 cells were stimulated (Ag+) or not (Ag−) as in the legend to Fig. 2 D. Cells were lysed as described in Materials and Methods, and LAT or Vav was immunoprecipitated. Proteins were resolved and immunoblotted with antibody to LAT, SLP-76, and Vav. (E) Vav-C inhibits the association of Vav and LAT with SLP-76 without affecting the SLP-76 phosphorylation. RBL-2H3 cells were transfected with Vav and GFP or Vav and Vav-C-GFP. Cells were stimulated (Ag+) or not (Ag−) as in the legend to Fig. 2 D, then lysed and incubated with antibody to SLP-76. Immunoblots were first probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti–SLP-76, anti-Vav, anti-LAT, and anti-Grb2. (F) Vav-C has no effect on the tyrosine phosphorylation of LAT. Experiments were done as in E, but cell lysates were incubated with antibody to LAT (Anti-LAT). Immunoblots were probed with antiphosphotyrosine and subsequently with anti-LAT.

Mentions: We investigated whether Syk mediates the interaction of Vav with the plasma membrane. The following evidence suggested that Vav aggregate formation is not directly mediated by Vav interaction with Syk. First, we did not detect colocalization of Vav with Syk under conditions, described previously 39, where Syk is maximally localized to the plasma membrane (data not shown). Second, other adaptor proteins that could facilitate Vav interaction with the plasma membrane were localized with Vav aggregates (see Fig. 4d and Fig. e, and Fig. 5 A). Third, preliminary experiments in Fc∈RI- and Lyn-transfected Chinese hamster ovary (CHO) cells 42 showed no redistribution of transfected Vav to the plasma membrane under conditions where Syk was membrane localized and both Syk and Vav were tyrosine phosphorylated (data not shown). Although we cannot formally exclude a plasma membrane–localized interaction of Syk and Vav, the preponderance of evidence suggests a different mechanism for Vav plasma membrane association.


The Src homology 2 domain of Vav is required for its compartmentation to the plasma membrane and activation of c-Jun NH(2)-terminal kinase 1.

Arudchandran R, Brown MJ, Peirce MJ, Song JS, Zhang J, Siraganian RP, Blank U, Rivera J - J. Exp. Med. (2000)

Vav-C inhibits Vav phosphorylation and association with but not phosphorylation of Syk, SLP-76, and LAT. (A) Vav-C (SH3-SH2-SH3) is membrane localized upon Fc∈RI engagement. RBL-2H3 cells were transfected with Vav-C-GFP or an SH2 domain–mutated Vav-C (R694K)-GFP. Cells were stimulated with 30 ng/ml of Ag for 3 min (+Ag) or left untreated (−Ag) and examined by confocal laser microscopy. (B) Expression levels of GFP alone, Vav-C, or Vav-C (R694K) tagged with GFP. Cells transfected as in A were lysed, and 5 × 104 cell equivalents per lane of protein were resolved by SDS-PAGE and transferred for immunoblotting (IB). For immunoprecipitation experiments, 1.0–2.0 × 107 cells were used. Expression levels were determined by probing with an antibody to GFP (shown) or in some cases with a mouse mAb to Vav. (C) Vav-C inhibits phosphorylation of Vav and its association with Syk but not Syk phosphorylation. RBL-2H3 cells transfected with Vav, Vav-C, and Syk or Vav, Vav-C (R694K), and Syk were stimulated as in the legend to  Fig. 2 D, lysed, and incubated with antibody to Syk. Recovered proteins were immunoblotted (IB) with antiphosphotyrosine (Anti-PY), then stripped and reprobed sequentially with anti-Vav and anti-Syk. (D) Nontransfected RBL-2H3 cells were stimulated (Ag+) or not (Ag−) as in the legend to  Fig. 2 D. Cells were lysed as described in Materials and Methods, and LAT or Vav was immunoprecipitated. Proteins were resolved and immunoblotted with antibody to LAT, SLP-76, and Vav. (E) Vav-C inhibits the association of Vav and LAT with SLP-76 without affecting the SLP-76 phosphorylation. RBL-2H3 cells were transfected with Vav and GFP or Vav and Vav-C-GFP. Cells were stimulated (Ag+) or not (Ag−) as in the legend to  Fig. 2 D, then lysed and incubated with antibody to SLP-76. Immunoblots were first probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti–SLP-76, anti-Vav, anti-LAT, and anti-Grb2. (F) Vav-C has no effect on the tyrosine phosphorylation of LAT. Experiments were done as in E, but cell lysates were incubated with antibody to LAT (Anti-LAT). Immunoblots were probed with antiphosphotyrosine and subsequently with anti-LAT.
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Figure 4: Vav-C inhibits Vav phosphorylation and association with but not phosphorylation of Syk, SLP-76, and LAT. (A) Vav-C (SH3-SH2-SH3) is membrane localized upon Fc∈RI engagement. RBL-2H3 cells were transfected with Vav-C-GFP or an SH2 domain–mutated Vav-C (R694K)-GFP. Cells were stimulated with 30 ng/ml of Ag for 3 min (+Ag) or left untreated (−Ag) and examined by confocal laser microscopy. (B) Expression levels of GFP alone, Vav-C, or Vav-C (R694K) tagged with GFP. Cells transfected as in A were lysed, and 5 × 104 cell equivalents per lane of protein were resolved by SDS-PAGE and transferred for immunoblotting (IB). For immunoprecipitation experiments, 1.0–2.0 × 107 cells were used. Expression levels were determined by probing with an antibody to GFP (shown) or in some cases with a mouse mAb to Vav. (C) Vav-C inhibits phosphorylation of Vav and its association with Syk but not Syk phosphorylation. RBL-2H3 cells transfected with Vav, Vav-C, and Syk or Vav, Vav-C (R694K), and Syk were stimulated as in the legend to Fig. 2 D, lysed, and incubated with antibody to Syk. Recovered proteins were immunoblotted (IB) with antiphosphotyrosine (Anti-PY), then stripped and reprobed sequentially with anti-Vav and anti-Syk. (D) Nontransfected RBL-2H3 cells were stimulated (Ag+) or not (Ag−) as in the legend to Fig. 2 D. Cells were lysed as described in Materials and Methods, and LAT or Vav was immunoprecipitated. Proteins were resolved and immunoblotted with antibody to LAT, SLP-76, and Vav. (E) Vav-C inhibits the association of Vav and LAT with SLP-76 without affecting the SLP-76 phosphorylation. RBL-2H3 cells were transfected with Vav and GFP or Vav and Vav-C-GFP. Cells were stimulated (Ag+) or not (Ag−) as in the legend to Fig. 2 D, then lysed and incubated with antibody to SLP-76. Immunoblots were first probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti–SLP-76, anti-Vav, anti-LAT, and anti-Grb2. (F) Vav-C has no effect on the tyrosine phosphorylation of LAT. Experiments were done as in E, but cell lysates were incubated with antibody to LAT (Anti-LAT). Immunoblots were probed with antiphosphotyrosine and subsequently with anti-LAT.
Mentions: We investigated whether Syk mediates the interaction of Vav with the plasma membrane. The following evidence suggested that Vav aggregate formation is not directly mediated by Vav interaction with Syk. First, we did not detect colocalization of Vav with Syk under conditions, described previously 39, where Syk is maximally localized to the plasma membrane (data not shown). Second, other adaptor proteins that could facilitate Vav interaction with the plasma membrane were localized with Vav aggregates (see Fig. 4d and Fig. e, and Fig. 5 A). Third, preliminary experiments in Fc∈RI- and Lyn-transfected Chinese hamster ovary (CHO) cells 42 showed no redistribution of transfected Vav to the plasma membrane under conditions where Syk was membrane localized and both Syk and Vav were tyrosine phosphorylated (data not shown). Although we cannot formally exclude a plasma membrane–localized interaction of Syk and Vav, the preponderance of evidence suggests a different mechanism for Vav plasma membrane association.

Bottom Line: We found that Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement.The FcinRI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs).Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.

View Article: PubMed Central - PubMed

Affiliation: Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Vav is a hematopoietic cell-specific guanine nucleotide exchange factor (GEF) whose activation is mediated by receptor engagement. The relationship of Vav localization to its function is presently unclear. We found that Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement. The redistribution of Vav was mediated by its Src homology 2 (SH2) domain and required Syk activity. The FcinRI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs). The scaffold protein, linker for activation of T cells (LAT), and Rac1 (a target of Vav activity) were constitutively present in GEMs. Expression of an SH2 domain-containing COOH-terminal fragment of Vav inhibited Vav phosphorylation and movement to the GEMs but had no effect on the tyrosine phosphorylation of the adaptor protein, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD), and LAT. However, assembly of the multiprotein complex containing these proteins was inhibited. In addition, FcinRI-dependent activation of c-Jun NH(2)-terminal kinase 1 (JNK1) was also inhibited. Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.

Show MeSH