The Vav1 SH2-domain can be functionally replaced by SH2-domains of other

Having established that Vav1 can directly interact with the BCR, it remained unknown if this interaction is functionally relevant for BCR signaling. Inactivation of the Vav1-interacting ITAM-tyrosines would corrupt the entire BCR signaling cascade. To circumvent this problem, I generated Vav1 constructs that contain either the SH2-domain of SLP-65 or the tandem SH2-domains of Syk instead of the Vav1 SH2-domain (figure 4.10 A). Both, the SLP-65 and Syk SH2-domains are well known to interact primarily with the Igα/Igβ heterodimer of the BCR (Fütterer et al., 1998; Engels et al., 2001), so that the resulting chimeric Vav1 proteins should bind the BCR. Constructs encoding Vav1, Vav1-Syk [SH2]₂, Vav1-SLP-65 SH2 or EGFP were expressed in Vav1-deficient DG75 cells and Ca²⁺-mobilization was measured. The results show that Vav1-deficient DG75 cells expressing wild-type Vav1 or either of the chimeric proteins had an increased Ca²⁺ -mobilization compared to control cells (figure 4.10 B), even though cells expressing the Vav1-SLP-65 SH2 chimera showed a somewhat reduced Ca²⁺-mobilization compared to cells reconstituted with wild-type Vav1. In contrast, Vav1-deficient DG75 cells expressing the Vav1-Syk [SH2]₂ chimera showed a dramatically enhanced Ca²⁺-mobilization.

Collectively, both chimeric proteins could restore Ca²⁺-mobilization in Vav1-deficient DG75 cells albeit with different efficiencies.

To prove that the chimeric proteins can indeed bind the Igα/Igβ heterodimer, I tested their binding capability in affinity purification experiments. Therefore, I generated GST-fusion proteins consisting of the C-terminal adaptor region of Vav1 (hereafter referred to as C2) containing either the wild-type Vav1 SH2 or the SH2 domains of SLP-65 or Syk (figure 4.10 D). In addition, GST-only was used as negative control. Purified proteins were subjected to western blot analysis with antibodies to Igα, Igβ or GST, showing that all chimeric proteins could bind Igα as well as Igβ, except the negative controls (figure 4.10 E).

Taken together, I could show that Vav1 chimeric proteins containing other BCR-binding SH2-domains can functionally replace the Vav1 SH2-domain in the context of BCR-induced Ca²⁺-mobilization. This indicates that Vav1 needs to be localized close to the BCR for efficient Ca²⁺-mobilization.

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Figure 4.10: binding SH2-domains can replace the Vav1 SH2-domain in the context of BCR-induced Ca²⁺-mobilization. A) Schematic drawing of the domain architecture of Vav1 and the chimeric Vav1-SLP-65 SH2 and Vav1-Syk [SH2]2 proteins. In addition, the binding capabilities of the individual SH2-domains are depicted, which can bind different phosphorylated tyrosines (P) in the cytoplasmic tails of Igα/Igβ. B) Vav1-deficient DG75 cells expressing Citrine-tagged wild-type Vav1, Vav1-SLP-65 SH2, Vav1-Syk [SH2]2 or EGFP were analyzed for Ca²⁺-mobilization upon BCR stimulation as before. The graph shows Ca²⁺ -mobilization of gated cells (positive gate (C)). Data show one representative example of three independent experiments. C) Expression levels of analyzed proteins were measured by flow cytometry. D) Domain architecture of the used GST-fusion proteins in affinity purifications. E) Affinity purifications with C2 regions of chimeric Vav1 proteins. DG75 cells were stimulated with 20 μg/ml α-IgM F(ab’)2 fragments for 3 min or left untreated. Cleared cellular lysates were used for affinity purification with GST-C2 Vav1, GST-C2 Vav1 SH2 R696A, GST-C2 Vav1-Syk [SH2]2, GST-C2 Vav1-SLP-65 SH2 or GST-only. Samples were subjected to western blot analysis with anti-Igα, anti-Igβ and anti-GST antibodies as indicated. The molecular weight of marker proteins (in kDa) is indicated on the left. Data show one representative example of three independent experiments.

To further characterize the functional relevance of the Vav1-BCR interaction for Ca²⁺ -mobilization upon BCR stimulation, I generated an experimental system that combines the Vav1-SLP-65 SH2-chimera and a chimeric receptor consisting of the extracellular part of CD8 and the intracellular part of Igα (figure 4.11 A). The intracellular part of Igα contains the non-ITAM tyrosine (Y204), which is described as exclusive binding site for SLP-65 (Engels et al., 2001). Vice versa, The SLP-65 SH2-domain is described to bind primarily to the phosphorylated non-ITAM Y204 of Igα, so that binding of the Vav1-SLP-65 SH2 chimera to the CD8/Igα chimera can be selectively investigated and is supposed to resemble the interaction between the Vav1 SH2-domain and the BCR.

I first expressed CD8/Igα or CD8/Igα Y204F chimeric proteins in Vav1-deficient DG75 cells. Next, the resulting two cell lines were retrovirally transfected with constructs coding for wild-type Vav1, Vav1-SLP-65 SH2, Vav1-SLP-65 SH2 R372L or EGFP and Ca²⁺ -mobilization was measured upon stimulation of the CD8 chimeras with anti-CD8 antibodies. Figure 4.11 B shows that expression of Vav1 and SLP-65 SH2 in Vav1-deficient DG75 cells led to Ca²⁺-mobilization, which resembles the Ca²⁺-mobilization signal of DG75 cells stimulated via the endogenous BCR (figure 4.10). This indicates that the chimeric receptor can mimic the endogenous BCR. The function of the chimeric Vav1-SLP-65 domain fusion protein relies on its domain since inactivation of the SH2-domain (Vav1-SLP-65 SH2 R372L) led to a Ca²⁺-mobilization defect similar to Vav1-deficient DG75 cells (figure 4.11 B).

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Figure 4.11: Interaction of Vav1 with the BCR is sufficient for Vav1 function in Ca²⁺-mobilization. A) Schematic drawing of the domain architecture of Vav1 and the chimeric Vav1-SLP-65 SH2 and Vav1-SLP-65 SH2 R372L proteins. In addition, the binding capability of the individual SH2-domains is depicted, which can bind different phosphorylated tyrosines (P) in the cytoplasmic tail of CD8/Igα or CD8/Igα Y204F. B, E) Vav1-deficient DG75 cells expressing CD8/Igα or CD8/Igα Y204 were retrovirally transfected with constructs coding for Citrine-tagged wild-type Vav1, Vav1-SLP-65 SH2, Vav1-SLP-65 SH2 R372L or EGFP and analyzed for the Vav1-SLP-65 SH2 chimera could not restore Ca²⁺-mobilization, when its docking site, Y204, was inactivated. Collectively, I could show by mutational analysis that recruitment of Vav1 to phosphorylated BCR tyrosine residues enables BCR-induced Ca²⁺-mobilization.

4.3 The interaction between Vav1 and SLP-65 permits