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Differential regulation of TCR-mediated gene transcription by Vav family members.

Zakaria S, Gomez TS, Savoy DN, McAdam S, Turner M, Abraham RT, Billadeau DD - J. Exp. Med. (2004)

Bottom Line: However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity.In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription.These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

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Vav3 interacts with SLP76 and 3BP2. (A) Jurkat T cells were transfected with the indicated expression vectors. Cells were stimulated as indicated, cell lysates were prepared, and FLAG-tagged protein was immunoprecipitated. Immunoprecipitates were analyzed as described above and probed for anti-pTyr (top) and anti-FLAG (bottom). The asterisk denotes an interacting protein of ∼76 kD. The arrow denotes a nonspecific band. (B) Jurkat T cells were treated as indicated, and cell lysates were prepared and subject to immunoprecipitation with NRS, anti-Vav3, or anti-SLP76 polyclonal rabbit antisera. Proteins were detected using anti-pTyr (top), anti-SLP76 (middle), or anti-Vav3 (bottom). In the anti-pTyr blot, the asterisk denotes SLP76, and the arrow identifies Vav3. (C) GST or GST fusion proteins containing the Vav1, Vav2, or Vav3 SH2 domains were used to immunoprecipitate interacting proteins from unstimulated (−) or CD3-stimulated (+) Jurkat T cells. Interacting proteins were analyzed using anti-pTyr (top) or anti-SLP76 (bottom). The arrow denotes the SLP76 band. (D) Jurkat T cells were transfected with the indicated expression vectors. Cells were treated as in B, and the membrane was subsequently probed with the GST–Vav3–SH2 (1 μg/ml) and detected with anti-GST (top), anti-pTyr (middle), and anti-FLAG (bottom).
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fig2: Vav3 interacts with SLP76 and 3BP2. (A) Jurkat T cells were transfected with the indicated expression vectors. Cells were stimulated as indicated, cell lysates were prepared, and FLAG-tagged protein was immunoprecipitated. Immunoprecipitates were analyzed as described above and probed for anti-pTyr (top) and anti-FLAG (bottom). The asterisk denotes an interacting protein of ∼76 kD. The arrow denotes a nonspecific band. (B) Jurkat T cells were treated as indicated, and cell lysates were prepared and subject to immunoprecipitation with NRS, anti-Vav3, or anti-SLP76 polyclonal rabbit antisera. Proteins were detected using anti-pTyr (top), anti-SLP76 (middle), or anti-Vav3 (bottom). In the anti-pTyr blot, the asterisk denotes SLP76, and the arrow identifies Vav3. (C) GST or GST fusion proteins containing the Vav1, Vav2, or Vav3 SH2 domains were used to immunoprecipitate interacting proteins from unstimulated (−) or CD3-stimulated (+) Jurkat T cells. Interacting proteins were analyzed using anti-pTyr (top) or anti-SLP76 (bottom). The arrow denotes the SLP76 band. (D) Jurkat T cells were transfected with the indicated expression vectors. Cells were treated as in B, and the membrane was subsequently probed with the GST–Vav3–SH2 (1 μg/ml) and detected with anti-GST (top), anti-pTyr (middle), and anti-FLAG (bottom).

Mentions: To determine if the SH2 domain of Vav3 is required for its tyrosine phosphorylation downstream of the TCR, we transfected Jurkat T cells with either a WT or SH2 domain mutant of Vav3 and analyzed TCR-induced tyrosine phosphorylation of the two proteins. As shown in Fig. 2 A, the mutant SH2 domain–containing Vav3 protein fails to undergo TCR-mediated tyrosine phosphorylation. This was not the result of different levels of protein expression since both Vav3 proteins were equivalently expressed.


Differential regulation of TCR-mediated gene transcription by Vav family members.

Zakaria S, Gomez TS, Savoy DN, McAdam S, Turner M, Abraham RT, Billadeau DD - J. Exp. Med. (2004)

Vav3 interacts with SLP76 and 3BP2. (A) Jurkat T cells were transfected with the indicated expression vectors. Cells were stimulated as indicated, cell lysates were prepared, and FLAG-tagged protein was immunoprecipitated. Immunoprecipitates were analyzed as described above and probed for anti-pTyr (top) and anti-FLAG (bottom). The asterisk denotes an interacting protein of ∼76 kD. The arrow denotes a nonspecific band. (B) Jurkat T cells were treated as indicated, and cell lysates were prepared and subject to immunoprecipitation with NRS, anti-Vav3, or anti-SLP76 polyclonal rabbit antisera. Proteins were detected using anti-pTyr (top), anti-SLP76 (middle), or anti-Vav3 (bottom). In the anti-pTyr blot, the asterisk denotes SLP76, and the arrow identifies Vav3. (C) GST or GST fusion proteins containing the Vav1, Vav2, or Vav3 SH2 domains were used to immunoprecipitate interacting proteins from unstimulated (−) or CD3-stimulated (+) Jurkat T cells. Interacting proteins were analyzed using anti-pTyr (top) or anti-SLP76 (bottom). The arrow denotes the SLP76 band. (D) Jurkat T cells were transfected with the indicated expression vectors. Cells were treated as in B, and the membrane was subsequently probed with the GST–Vav3–SH2 (1 μg/ml) and detected with anti-GST (top), anti-pTyr (middle), and anti-FLAG (bottom).
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fig2: Vav3 interacts with SLP76 and 3BP2. (A) Jurkat T cells were transfected with the indicated expression vectors. Cells were stimulated as indicated, cell lysates were prepared, and FLAG-tagged protein was immunoprecipitated. Immunoprecipitates were analyzed as described above and probed for anti-pTyr (top) and anti-FLAG (bottom). The asterisk denotes an interacting protein of ∼76 kD. The arrow denotes a nonspecific band. (B) Jurkat T cells were treated as indicated, and cell lysates were prepared and subject to immunoprecipitation with NRS, anti-Vav3, or anti-SLP76 polyclonal rabbit antisera. Proteins were detected using anti-pTyr (top), anti-SLP76 (middle), or anti-Vav3 (bottom). In the anti-pTyr blot, the asterisk denotes SLP76, and the arrow identifies Vav3. (C) GST or GST fusion proteins containing the Vav1, Vav2, or Vav3 SH2 domains were used to immunoprecipitate interacting proteins from unstimulated (−) or CD3-stimulated (+) Jurkat T cells. Interacting proteins were analyzed using anti-pTyr (top) or anti-SLP76 (bottom). The arrow denotes the SLP76 band. (D) Jurkat T cells were transfected with the indicated expression vectors. Cells were treated as in B, and the membrane was subsequently probed with the GST–Vav3–SH2 (1 μg/ml) and detected with anti-GST (top), anti-pTyr (middle), and anti-FLAG (bottom).
Mentions: To determine if the SH2 domain of Vav3 is required for its tyrosine phosphorylation downstream of the TCR, we transfected Jurkat T cells with either a WT or SH2 domain mutant of Vav3 and analyzed TCR-induced tyrosine phosphorylation of the two proteins. As shown in Fig. 2 A, the mutant SH2 domain–containing Vav3 protein fails to undergo TCR-mediated tyrosine phosphorylation. This was not the result of different levels of protein expression since both Vav3 proteins were equivalently expressed.

Bottom Line: However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity.In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription.These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

Show MeSH