The N-terminal 20-amino acid region of guanine nucleotide exchange factor Vav1 plays a distinguished role in T cell receptor-mediated calcium signaling

Abstract

Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1-20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.

FIGURE 1.

Effect of individual Vav proteins on TCR-induced calcium mobilization. A, Jurkat cells were infected with lentivirus particles that express shRNA specifically targeting Vav1, Vav2, or Vav3, respectively. Viral particles encoding a random sequence were used as control (shCtrl). The transduced cells were selected by puromycin resistance marker and subjected to calcium analysis as described under “Experimental Procedures.” The intracellular calcium concentrations were monitored for 5 min after stimulation with OKT3. B, the expressions of individual Vav isoforms were examined by Western blot with indicated antibodies.

FIGURE 2.

Sequence alignment of the CH domains of Vav members and the association with calmodulin. A, amino acid sequences of the CH domains from three Vav isoforms were aligned using ClustalX, and the alignment result was drawn with the online BoxShade Server. The identical and conserved residues are highlighted in dark and gray boxes, respectively. The percentages in parentheses represente the CH domain similarities of Vav2/3 to the sequence of Vav1. B, Jurkat cell lysates were incubated with CaM-conjugated agarose (CaM-Agr) or nonconjugated agarose (Agrose), respectively, at 4 °C. The immobilized proteins were eluted and resolved by SDS-PAGE and Western blot analysis with antibodies against Vav1 (upper panel), Vav2 (middle panel), and Vav3 (bottom panel). The lane labeled Lysate presented 1/30 of the protein loaded for pulldown assay. The CaM-Agr lane presented binding of the indicated Vav proteins with CaM, and the Agarose lane as negative control of each pulldown assay.

FIGURE 3.

Identification of the CaM-binding region within the CH domain of Vav1. A, schematic depiction of Vav1 and its truncated mutations at CH domain. The numbers of deleted amino acids are listed in the right column, and the designated names on the left column. The CH domain was boxed in black. B, the indicated Vav1 proteins were eukaryotically expressed in 293T cells. Cell lysates were incubated with CaM-conjugated agarose at 4 °C for 3 h in the presence of 4 mm Ca²⁺. After washing, the immobilized contents were analyzed by SDS-PAGE and Western blot with anti-Vav1 antibody (Pulldown panel). The expressions of Vav1 and its mutants in whole cell lysates (WCL) were resolved by Western blot (WCL panel). C, BiFC assay. HeLa cells were cotransfected with the indicated combination of plasmids, YN-Vav1 + YC, YN-Vav1 + YC-CaM, YN-Vav1(ΔN20) + YC, and YN-Vav1(ΔN20) + YC-CaM, respectively. Then the cells were subjected to fluorescence microscopy (left column) and FACS analysis (center column), with the mean fluorescence intensity (MFI) listed in the right column. D, protein expression of each transfected component was verified by Western blot with the indicated antibodies.

FIGURE 4.

Identification of the amino acids of Vav1 responsible for TCR-induced calcium mobilization. A, lentivirus particles were produced with lentiviral vectors encoding Vav1, Vav1(ΔCH), or Vav1(ΔN20), and vector backbone as control. J.Vav1 cells were transduced and selected with antibiotic markers. The expressions of Vav1 and Vav1 mutations were verified by Western blot with anti-Vav1 antibody. For calcium profile analyses, the reconstituted J.Vav1 cells with the indicated Vav1 or Vav1 variants (labeled as the inlets) were loaded with Fura-2 AM and stimulated with OKT3 in the presence of extracellular Ca²⁺ (B) or chelating agent EGTA (C); or treated with TG in the presence of chelating agent EGTA (D). The intracellular calcium were monitored for 5 min post-stimulation and plotted with time (horizontal axis) versus the fluorescence emission ratio of the Ca²⁺ bounded (340 nm) to free form (380 nm) of Fura-2.

FIGURE 5.

The requirement of the N-terminal 20 aa of Vav1 for TCR-induced NFAT transcriptional activity. A, J.Vav1 cells were transfected with reporter plasmids, pNFAT-Luc and TK-Renilla, together with plasmid backbone (pcDNA4), or plasmids encoding Vav1, Vav1(ΔCH), or Vav1(ΔN20) as indicated. At 24 h post-transfection, the cells were left unstimulated (NS, open bars), or stimulated with OKT3 (dark bars) for 6 h. The luciferase reporter assay was performed, and NFAT activity was presented as the firefly luciferase activity normalized to Renilla luciferase activity in each sample. Histograms represente the mean values of three independent experiments with S.D. B, cell lysates were subjected to Western blot analysis to verify the protein expression. The displayed Western blot is from one of the three experiments.

FIGURE 6.

Schematic depiction of Vav mutations at the N termini and their association with CaM. A, the N-terminal 20 aa of Vav1 (black bar) was substituted by that of Vav2 (hatched bar) or Vav3 (dotted bar), and the designated names are listed in the right column. C, the FLAG-tagged Vav family members, Vav2 (hatched bar) or Vav3 (dotted bar) and their mutants in which the 1–20 aa of Vav2 and Vav3 were replaced by 1–20 aa of Vav1 (black bar). The designated names of the substituted mutations are listed in the right column. Plasmids encoding the mutations were transfected into 293T cells and the lysates were incubated with CaM-agarose at 4 °C for 3 h in the presence of 4 mm Ca²⁺. The immobilized fractions were analyzed by SDS-PAGE and Western blot with anti-Vav1 antibody (upper panel), and the expressions of Vav1 and its mutants in the whole cell lysates (WCL) were resolved by Western blot (WCL panel). The pulldown analysis for proteins listed in A are presented in panel B; and for mutations listed in panel C are presented in D.

FIGURE 7.

The effect of 1–20 aa of Vav1 on T cell activation. A, J.Vav1 cells were infected with lentiviral particles bearing vector backbone only, or Vav1, Vav1(ΔN20), and Vav1-substituted mutations as indicated, respectively. The expressions of the Vav1 variants were verified by Western blot with antibody against Vav1, or tubulin as an internal control. B, J.Vav1 cells reconstituted with the indicated Vav1 proteins were loaded with Fura-2 AM and stimulated with OKT3. The intracellular calcium concentrations were monitored for 5 min for each sample and plotted with the fluorescence emission ratio of the Ca²⁺ bound to the free forms of Fura-2 as the vertical axis, and time as the horizontal axis. C, J.Vav1 cells were transfected with reporter plasmids, pNFAT-Luc and TK-Renilla, together with plasmid backbone (pcDNA4), or plasmids encoding Vav1, Vav1(ΔN20), Vav1(Vav2-N20), or Vav1(Vav3-N20) as indicated. At 24 h post-transfection, the cells were left unstimulated (NS) or stimulated with OKT3 for 6 h. The luciferase reporter assay was performed as described under ”Experimental Procedures.“ The NFAT activity was presented as the ratio of the firefly luciferase activity to Renilla luciferase activity for each transfectant. Histograms represent the mean values from three independent experiments with S.D. D, cell lysates were analyzed by Western blot and one set of experiments was shown to indicate expression of the various Vav1 proteins for reporter assays. E, J.Vav1 cells were transduced with lentiviruses bearing various Vav1 proteins or vector backbone, respectively, and seeded at the same concentrations. Then the cells remained non-stimulated (NS) or stimulated with OKT3 for 24 h. The cell culture media were collected and IL-2 content was determined by ELISA. Three independent experiments were performed and are represented by histograms with the mean ± S.D. from triplicate samples.