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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 phosphorylation, membrane localization, and JNK1 activation depends on Syk activity. (A) Plasma membrane localization of Vav requires Syk activity. Syk-deficient cells (−Syk) were transfected with Vav-GFP (−Syk/+Vav-GFP) or with both Vav-GFP and Syk (+Syk/+Vav-GFP). Transfected cells were stimulated for 3 min with 30 ng of Ag (+Ag). Fixed and permeabilized cells were incubated with mouse antibody to Fc∈RI β chain followed by Cy-5–conjugated goat anti–mouse. (B) Vav tyrosine phosphorylation requires Syk. Syk-deficient cells were transfected with Vav-GFP or with both Vav-GFP and Syk. Fc∈RI-stimulated (Ag+) cells were lysed and incubated with anti-GFP. Proteins were resolved and transferred for immunoblots (IB) and probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti-Vav. (C) Syk and Vav synergize to activate JNK1. Syk-deficient cells were transfected with either GFP, Syk, Vav-GFP, or both Vav-GFP and Syk. Transfected cells were stimulated (Ag+) or not (Ag−) with 300 ng/ml of Ag for 8 min, lysed, and incubated with antibody to JNK1. Resolved proteins were first immunoblotted with anti-phosphoJNK1, then stripped and reprobed with anti-JNK1. Quantitation of immunoblots (IB) was by densitometry. Fold induction is the mean of two experiments where values obtained were normalized to the immunoprecipitated protein. SD were <7%.
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Figure 3: Vav phosphorylation, membrane localization, and JNK1 activation depends on Syk activity. (A) Plasma membrane localization of Vav requires Syk activity. Syk-deficient cells (−Syk) were transfected with Vav-GFP (−Syk/+Vav-GFP) or with both Vav-GFP and Syk (+Syk/+Vav-GFP). Transfected cells were stimulated for 3 min with 30 ng of Ag (+Ag). Fixed and permeabilized cells were incubated with mouse antibody to Fc∈RI β chain followed by Cy-5–conjugated goat anti–mouse. (B) Vav tyrosine phosphorylation requires Syk. Syk-deficient cells were transfected with Vav-GFP or with both Vav-GFP and Syk. Fc∈RI-stimulated (Ag+) cells were lysed and incubated with anti-GFP. Proteins were resolved and transferred for immunoblots (IB) and probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti-Vav. (C) Syk and Vav synergize to activate JNK1. Syk-deficient cells were transfected with either GFP, Syk, Vav-GFP, or both Vav-GFP and Syk. Transfected cells were stimulated (Ag+) or not (Ag−) with 300 ng/ml of Ag for 8 min, lysed, and incubated with antibody to JNK1. Resolved proteins were first immunoblotted with anti-phosphoJNK1, then stripped and reprobed with anti-JNK1. Quantitation of immunoblots (IB) was by densitometry. Fold induction is the mean of two experiments where values obtained were normalized to the immunoprecipitated protein. SD were <7%.

Mentions: Inhibition of Syk activity or a deficiency of Syk in mast cells results in the loss of Vav phosphorylation 30 41. To assess if Vav redistribution was dependent on Syk activity or on the presence of Syk, we used several approaches. Vav was overexpressed in the TB1A2 cells, a Syk− RBL-2H3 cell line 30. In the absence or presence of Fc∈RI engagement, Vav failed to redistribute to the plasma membrane and form aggregates ( Fig. 3 A). Furthermore, analogous to the mutation of the Vav SH2 domain, the endogenous and overexpressed Vav was not phosphorylated in these cells ( Fig. 3 B, and data not shown). Cotransfection of Syk and Vav in the TB1A2 cells led to the reconstitution of plasma membrane–localized Vav, its tyrosine phosphorylation, and its colocalization with Fc∈RI after the latter's engagement ( Fig. 3a and Fig. b). As summarized in Table , Vav aggregates were also observed after Fc∈RI engagement in a TB1A2-derived clone stably reconstituted with Syk (clone 3A1). In contrast, the TB1A2-derived clone reconstituted with a catalytically inactive Syk (clone 3BA12) or RBL cells pretreated with piceatannol, a Syk-selective inhibitor, failed to show Vav redistribution (Table ), clearly demonstrating the requirement for Syk activity in the tyrosine phosphorylation of Vav and its redistribution to the plasma membrane.


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 phosphorylation, membrane localization, and JNK1 activation depends on Syk activity. (A) Plasma membrane localization of Vav requires Syk activity. Syk-deficient cells (−Syk) were transfected with Vav-GFP (−Syk/+Vav-GFP) or with both Vav-GFP and Syk (+Syk/+Vav-GFP). Transfected cells were stimulated for 3 min with 30 ng of Ag (+Ag). Fixed and permeabilized cells were incubated with mouse antibody to Fc∈RI β chain followed by Cy-5–conjugated goat anti–mouse. (B) Vav tyrosine phosphorylation requires Syk. Syk-deficient cells were transfected with Vav-GFP or with both Vav-GFP and Syk. Fc∈RI-stimulated (Ag+) cells were lysed and incubated with anti-GFP. Proteins were resolved and transferred for immunoblots (IB) and probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti-Vav. (C) Syk and Vav synergize to activate JNK1. Syk-deficient cells were transfected with either GFP, Syk, Vav-GFP, or both Vav-GFP and Syk. Transfected cells were stimulated (Ag+) or not (Ag−) with 300 ng/ml of Ag for 8 min, lysed, and incubated with antibody to JNK1. Resolved proteins were first immunoblotted with anti-phosphoJNK1, then stripped and reprobed with anti-JNK1. Quantitation of immunoblots (IB) was by densitometry. Fold induction is the mean of two experiments where values obtained were normalized to the immunoprecipitated protein. SD were <7%.
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Figure 3: Vav phosphorylation, membrane localization, and JNK1 activation depends on Syk activity. (A) Plasma membrane localization of Vav requires Syk activity. Syk-deficient cells (−Syk) were transfected with Vav-GFP (−Syk/+Vav-GFP) or with both Vav-GFP and Syk (+Syk/+Vav-GFP). Transfected cells were stimulated for 3 min with 30 ng of Ag (+Ag). Fixed and permeabilized cells were incubated with mouse antibody to Fc∈RI β chain followed by Cy-5–conjugated goat anti–mouse. (B) Vav tyrosine phosphorylation requires Syk. Syk-deficient cells were transfected with Vav-GFP or with both Vav-GFP and Syk. Fc∈RI-stimulated (Ag+) cells were lysed and incubated with anti-GFP. Proteins were resolved and transferred for immunoblots (IB) and probed with antiphosphotyrosine (Anti-PY), then stripped and reprobed with anti-Vav. (C) Syk and Vav synergize to activate JNK1. Syk-deficient cells were transfected with either GFP, Syk, Vav-GFP, or both Vav-GFP and Syk. Transfected cells were stimulated (Ag+) or not (Ag−) with 300 ng/ml of Ag for 8 min, lysed, and incubated with antibody to JNK1. Resolved proteins were first immunoblotted with anti-phosphoJNK1, then stripped and reprobed with anti-JNK1. Quantitation of immunoblots (IB) was by densitometry. Fold induction is the mean of two experiments where values obtained were normalized to the immunoprecipitated protein. SD were <7%.
Mentions: Inhibition of Syk activity or a deficiency of Syk in mast cells results in the loss of Vav phosphorylation 30 41. To assess if Vav redistribution was dependent on Syk activity or on the presence of Syk, we used several approaches. Vav was overexpressed in the TB1A2 cells, a Syk− RBL-2H3 cell line 30. In the absence or presence of Fc∈RI engagement, Vav failed to redistribute to the plasma membrane and form aggregates ( Fig. 3 A). Furthermore, analogous to the mutation of the Vav SH2 domain, the endogenous and overexpressed Vav was not phosphorylated in these cells ( Fig. 3 B, and data not shown). Cotransfection of Syk and Vav in the TB1A2 cells led to the reconstitution of plasma membrane–localized Vav, its tyrosine phosphorylation, and its colocalization with Fc∈RI after the latter's engagement ( Fig. 3a and Fig. b). As summarized in Table , Vav aggregates were also observed after Fc∈RI engagement in a TB1A2-derived clone stably reconstituted with Syk (clone 3A1). In contrast, the TB1A2-derived clone reconstituted with a catalytically inactive Syk (clone 3BA12) or RBL cells pretreated with piceatannol, a Syk-selective inhibitor, failed to show Vav redistribution (Table ), clearly demonstrating the requirement for Syk activity in the tyrosine phosphorylation of Vav and its redistribution to the plasma membrane.

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
Related in: MedlinePlus