The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK, and NF-kappaB pathways

Abstract

Vav is a GTP/GDP exchange factor (GEF) for members of the Rho-family of GTPases that is rapidly tyrosine-phosphorylated after engagement of the T cell receptor (TCR), suggesting that it may transduce signals from the receptor. T cells from mice made Vav-deficient by gene targeting (Vav-/-) fail to proliferate in response to TCR stimulation because they fail to secrete IL-2. We now show that this is due at least in part to the failure to initiate IL-2 gene transcription. Furthermore, we analyze TCR-proximal signaling pathways in Vav-/- T cells and show that despite normal activation of the Lck and ZAP-70 tyrosine kinases, the mutant cells have specific defects in TCR-induced intracellular calcium fluxes, in the activation of extracellular signal-regulated mitogen-activated protein kinases and in the activation of the NF-kappaB transcription factor. Finally, we show that the greatly reduced TCR-induced calcium flux of Vav-deficient T cells is an important cause of their proliferative defect, because restoration of the calcium flux with a calcium ionophore reverses the phenotype.

Figure 1

TCR-induced proliferation and activation of IL-2 gene transcription in Vav^−/− T cells. (a) Proliferation of Vav^+/+ or Vav^−/− CD4⁺ splenic T cells in response to a range of concentrations of plate-bound anti-CD3 antibody in the presence or absence of soluble anti-CD28 antibody (10 μg/ml). Proliferation was assessed by the incorporation of ³H-thymidine during the final 4 hr of a 48 hr assay. Graph shows the mean ³H-thymidine incorporation (±SEM) of triplicate samples. (b) Induction of IL-2 gene transcription assessed by the production of luciferase by Vav^+/+ or Vav^−/−CD4⁺ T cells purified from mice carrying a luciferase transgene under the control of the IL-2 promoter. Cells were incubated for 24 hr either with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (10 μg/ml) or in the absence of any added stimulus (medium). (c) Proliferation of Vav^+/+ or Vav^−/− CD4⁺ T cells in response to plate-bound anti-CD3 antibody (5 μg/ml) and soluble anti-CD28 antibody (10 μg/ml) in the absence or presence of ionomycin (198 ng/ml). Proliferation was assessed as in Fig. 1a.

Figure 2

TCR-induced tyrosine phosphorylation. Immunoblot of cytoplasmic extracts of CD4⁺ splenic T cells purified from Vav^+/+ or Vav^−/− mice. Where indicated cells were precoated with anti-CD3 and anti-CD28 antibodies (αCD3 + αCD28) that in some samples were then crosslinked with goat anti-hamster Ig polyclonal antiserum (gαhIg; 100 μg/ml) and samples taken after the indicated time. (a) Samples were immunoprecipitated (IP) with antibodies to PLCγ1, and analyzed by immunoblotting with an antibody to phosphotyrosine (pTyr) and then stripped and reprobed with an antibody to PLCγ1. (b) Proteins binding to a GST–Grb2 fusion protein were isolated from stimulated cell extracts and analyzed by immunoblotting with an anti-pTyr antibody. The identity of individual pTyr-containing proteins was suggested by reprobing the blot with antibodies to Cbl, Vav, and SLP-76 (data not shown). The 36-kDa phosphoprotein is likely to be LAT (38). Sizes are shown in kDa.

Figure 3

Release of intracellular calcium and IP₃. (a) Intracellular calcium flux is reduced in Vav-deficient CD4⁺ splenic T cells. The two panels at the top show flow cytometric analysis of CD4 and CD8 staining of Vav-deficient (Vav^−/−) or control (Vav^+/+) splenocytes preloaded with Indo-1 and coated with anti-CD3. Panels below show intracellular calcium concentrations in the CD4⁺ T cells (gated as shown in the CD4/CD8 plots) as a ratio of Indo-1 violet/blue fluorescence versus time. Cells were stimulated with 0.1–100 μg/ml goat anti-hamster antibody to crosslink the anti-CD3 at the time indicated by the break in the calcium trace. Adequate loading of the Vav^−/− T cells with Indo-1 was demonstrated by inducing a calcium flux with ionomycin (not shown). (b) Graph showing mean levels of IP₃ (±SEM) in CD4⁺ T cells precoated with anti-CD3 and anti-CD28 antibodies and stimulated by the addition of gαhIg (300 μg/ml).

Figure 4

NF-AT and NF-κB pathways in Vav^−/− T cells. Immunoblot of cytoplasmic extracts of CD4⁺ splenic T cells stimulated as described in Fig. 2. In addition in some cases the cells were also stimulated with ionomycin (500 ng/ml) or phorbol 12,13-dibutyrate (PDBU; 10 ng/ml). (a) Immunoblot probed with an antiserum to NF-ATp. Dephosphorylation of NF-ATp results in a faster relative electrophoretic mobility and lower apparent molecular weight. (b) Immunoblot probed with an anti-IκBα antiserum and reprobed with an anti-JNK1 antibody to control for loading. Numbers below the IκBα blot represent relative amounts of IκBα normalized to JNK1 in each lane and to the amount of IκBα in the first lane (unstimulated Vav^+/+ cells). Degradation of IκBα in TCR-stimulated Vav^+/+ CD4⁺ T cells can be seen in the disappearance of the IκBα band. (c) NF-κB complexes visualized by electrophoretic mobility shift assay using ³²P-labeled NF-κB probe bound to extracts from Vav^+/+ or Vav^−/− CD4⁺ splenic T cells. Based on data in the literature (24) and on our own experiments using anti-p50 and anti-p65 antibodies to supershift these complexes (data not shown), C1 contains both p50 and p65, whereas C2 contains p50, but not p65.

Figure 5

Activation of MAPK pathways. CD4⁺ splenic T cells purified from Vav^+/+ or Vav^−/− mice were stimulated as described in Figs. 2 and 4. (a) ERK2 MAPK was immunoprecipitated from stimulated cells and used in an in vitro kinase assay with ³²P-ATP to phosphorylate myelin basic protein (MBP). (Upper) An autoradiograph of a blot showing phosphorylation of myelin basic protein. In the lower panel the same blot has been probed with an anti-ERK2 antibody to control for loading. (b) Immunoblot of stimulated cell extracts probed with an anti-phosphoERK antibody and reprobed with an anti-ERK2 antibody to control for loading. Phosphorylation of the ERK1 and ERK2 kinases is indicative of their activation by the MEK kinase. (c, Upper) an immunoblot of stimulated cell extracts probed with an anti-Lck antibody. The shift in mobility of Lck to a slower migrating form of higher apparent molecular weight is caused by serine phosphorylation, which is likely to be downstream of ERK activation (26). Tyrosine phosphorylation of Lck does not cause this mobility shift. This shift is readily seen on 7–15% acrylamide gradient gels. (c, Lower) A blot of the same samples probed with an anti-ERK2 antibody showing the phosphorylation-induced mobility shift. In this experiment ionomycin was used at 500 ng/ml. These shifts are only seen upon extended electrophoresis; they were not seen in a and b as electrophoresis times were too short. (d) p38 MAPK was immunoprecipitated from stimulated cells and used in an in vitro kinase assay with ³²P-ATP to phosphorylate a GST–ATF2 fusion protein. (Upper) An autoradiograph of the blot showing phosphorylation of GST–ATF2. In the lower panel the same blot has been probed with an anti-p38 antibody to control for loading.