Vav1 is the only family member that displays a KR region located between the C1 and the proline rich region (PPR)–NSH3 cassette (
Figure 1A and
Figure S1). Vav3 also harbors an analogous region, although, in this case, the positively charged amino acids are distributed in a more disperse manner than in Vav1 (
Figure S1). The KR region represents a late evolutionary acquisition in the Vav family, as it first becomes apparent in teleosts (
Figure S1) [
15], the first group of species exhibiting a fully developed adaptive immune system [
46]. We generated two frameshift mutations that alter the amino acid sequence of the N- (KR1) and C-terminal (KR2) parts of that region to assess its potential involvement in Vav1 signaling output (
Figure 1C). In the case of the KR1, the mutation (KR1
Mut) leads to a 50% reduction in the number of positively charged amino acids, while it does not alter the total number of negatively charged ones. This causes a change from a basic (10.28) to a more acidic (6.41) isoelectric point (pI) of the entire region (
Figure 1C). The frameshift mutation within the KR2 region (KR2
Mut) leaves the positively charged amino acids intact, while it eliminates 50% of the negatively charged residues present in that region. These changes result in an increase in the pI of the Vav1 KR region from the 10.28 WT value to 10.56 (
Figure 1C). Given the implication of Vav1 in T cell development and signaling [
16], we next tested the biological activity of the mutant proteins using luciferase reporter-based c-Jun N-terminal kinase (JNK) and NFAT activity assays in the T cell acute lymphoblastic leukemia Jurkat cell line. Vav1 activates JNK in a Rac1-dependent manner [
21,
24,
42] and, therefore, the activity of this serine/threonine kinase is traditionally used as a cell-based readout for the activation level of the Vav1 catalysis-dependent pathways (
Figure 1B). By contrast, the stimulation of NFAT by Vav1 is catalysis-independent, requiring the activation of a Vav1 CH domain-dependent effector pathway that includes the engagement of PLCγ and the production of Ca
2+ and diacylglycerol (
Figure 1B) [
22,
47,
48]. It also synergizes with other signaling pathways triggered by the T cell receptor (TCR) in order to elicit optimal NFAT activation levels [
22,
47,
48] (
Figure 1B). As comparative controls, we used Vav1
WT and versions of the protein with C- (Vav1
Δ835–845) and N-terminal deletions (Vav1
Δ1–186). Because of the loss of the inhibitory CSH3, Vav1
Δ835–845 can stimulate both JNK and NFAT activities in a phosphorylation-independent and constitutive manner [
24] (
Figure 1B). The hyperactive Vav1
Δ1–186 is also phosphorylation-independent [
21]. However, unlike Vav1
Δ835–845 and Vav1
WT, it cannot stimulate the NFAT pathway owing to the absence of the CH domain [
21,
24,
42] (
Figure 1B). Given that the activity of these two mutant proteins is phosphorylation-independent [
21,
24], their inclusion in these experiments allowed us to discriminate whether any alteration in Vav1 signaling induced by the KR region mutations could be related to either the early phosphorylation-dependent activation step mediated by the upstream kinases or to later effector signaling phases of phosphorylated Vav1 (e.g., membrane off-rates, dephosphorylation kinetics). Consistent with a potential regulatory role of the KR region, we observed that the full-length Vav1 KR1
Mut and KR2
Mut mutant proteins display reduced and enhanced activities when tested in both JNK (
Figure 1D,E) and NFAT (
Figure 1E,F) assays, respectively. This deregulated activity is observed both in untreated cells and in cells stimulated with antibodies to the CD3 subunit of the TCR (
Figure 1D–F). The impact of the mutations in Vav1 activity is much larger in the case of NFAT than in JNK assays, according to fold-change criteria (compare
Figure 1D,F). These two frameshift mutants elicit the same effects when incorporated in the context of the Vav1
Δ835–845 (
Figure 1G–H) and Vav1
Δ1–186 (
Figure 1I), indicating that the changes in activity induced by those mutations are not owing to alterations in the initial phosphorylation-dependent activation step of the protein. Immunoblot analyses confirmed that the alterations in the activity found in these experiments are not the result of spurious variations in the abundance of the ectopically expressed proteins (
Figure 1J).
To check whether the effect induced by the KR mutations is cell-type specific, we tested the activity of the Vav1 mutants in both hematopoietic (chicken DT40 B lymphocytes) and nonhematopoietic (monkey COS1 cells) cell lines. In the former case, we found that Vav1 KR1
Mut shows WT-like activity when tested in NFAT assays. By contrast, the Vav1 KR2
Mut displays an exacerbated activity when tested both in nonstimulated and B cell receptor stimulated DT40 cells (
Figure S2A,B). In the case of COS1 cells, we measured the activity of the ectopically expressed Vav1 proteins using as readouts the stimulation of serum responsive factor (SRF) and the induction of membrane ruffling in the transfected cells. These two activities require the GEF activity of Vav1 [
21,
24]. We observed that the full-length Vav1 KR1
Mut and KR2
Mut proteins display WT-like activities in those two cell-based readouts (
Figure S2C–E). The incorporation of the KR1
Mut and KR2
Mut mutations does not alter the biological activity of both Vav1
Δ835–845 (
Figure S2E–G) and Vav1
Δ1–186 (
Figure S2E,H,I) in those two assays. These results indicate that the contribution of the KR region to Vav1 activity is probably limited to lymphocytes. Given that Vav2 lacks the KR region (
Figure S1) and is uncapable of stimulating the NFAT pathway [
49], we finally assessed whether the insertion of the Vav1 KR region in the Vav2 structure could promote the stimulation of this transcriptional factor. This was not the case (
Figure S2J,K). Taken together, these results indicate that the role of the KR region is related to the modulation of Vav1 signaling in lymphocytes.