Phosphatidylinositol Monophosphates Regulate Optimal Vav1 Signaling Output

Phosphatidylinositol–5 phosphate (PI5P) and other mono-phosphoinositides (mono-PIs) play second messenger roles in both physiological and pathological conditions. Despite this, their intracellular targets and mechanisms of action remain poorly characterized. Here, we show that Vav1, a protein that exhibits both Rac1 GDP/GTP exchange and adaptor activities, is positively modulated by PI5P and, possibly, other mono-PIs. Unlike other phospholipid–protein complexes, the affinity and specificity of the Vav1–lipid interaction entail a new structural solution that involves the synergistic action of the Vav1 C1 domain and an adjacent polybasic tail. This new regulatory layer, which is not conserved in the Vav family paralogs, favors the engagement of optimal Vav1 signaling outputs in lymphocytes.


FIGURE S1. Evolution of the KR in Vav family proteins
Amino acid sequence and charge features of the Vav1 KR region were analyzed in the indicated species. Positively charged residues are shown in red. FIGURE S2. The Vav1 KR regulatory mechanism is specific for lymphocytes (A) Activation of NFAT triggered by indicated Vav1 proteins in nonstimulated and BCR-stimulated DT40 cells. Data represent the mean ± SEM. Statistical values were obtained using the Mann-Whitney U test. Blue and salmon asterisks indicate the significance level compared with nonstimulated and BCR-stimulated Vav1 WT -expressing cells, respectively. Black asterisks refer to the P values obtained between the indicated experimental pairs (in brackets). n = 3 independent experiments. (B) Representative example of the abundance of the indicated Vav1 proteins and tubulin α (loading control) in the assays performed in A.
(C) Activation of SRF by the indicated Vav1 constructs in COS1 cells. Data represent the mean ± SEM. Statistical values were obtained using the Mann-Whitney U test. All the comparisons are referred to Vav1 WT . n = 3 independent experiments, each performed in triplicate.

(D)
Representative example of the abundance of the indicated Vav1 proteins and tubulin α (loading control) in the assays performed in C.
(E) Effect of the indicated proteins (top) in the F-actin cytoskeleton of COS1 cells. EGFPs and F-actin are shown in green and red, respectively. Areas of colocalization of Vav1 proteins and F-actin are shown in yellow (bottom panels). Scale bar, 20 µm. n = 3 independent experiments.
(F and H) Activation of SRF by the indicated Vav1 proteins in COS1 cells. Data represent the mean ± SEM. Statistical values were generated applying the Mann-Whitney U test using as comparative control the values obtained in Vav1 D835-845 -(F) and Vav1 D1-186 -expressing (H) cells. n =3 independent experiments, each performed in triplicate.
(G and I) Representative example of the abundance of the indicated Vav1 proteins and tubulin α (loading control) in the assays performed in F (G) and H (I).
(J) Activation of NFAT triggered by Vav1 WT and indicated polyhistidine-tagged Vav2 (His-Vav2) proteins in nonstimulated and TCR-stimulated Jurkat cells. Data represent the mean ± SEM. Statistical values were obtained using the Mann-Whitney U test. Blue and salmon asterisks indicate the significance level compared with nonstimulated and TCR-stimulated Vav1 WT -expressing cells, respectively. Black asterisks refer to the P values obtained between the indicated experimental pairs (in brackets). n = 3 independent experiments, each performed in duplicate.

(K)
Representative example of the abundance of the indicated Vav proteins and endogenous tubulin α (loading control) in the assays performed in J. Vav2 was detected using an antibody to the polyhistidine tag. The asterisk pinpoints the residual signal from the previous blotting carried out with the antibody to polyhistidine residues. (E) Example (top panels) and quantification (bottom) of the effect of EGFP and indicated EGFP-tagged Vav1 versions (top) in the polymerization of actin inside the contact area and outside or at the peripheral area of the immune synapse. This data is part of the same experiment performed in Figure 3G. As in that case, values were obtained comparing the F-actin signal in these areas to the signal in other regions of both the T and B cell as detailed in the methods. Histograms represent the mean ± SEM and statistics were performed using two-way ANOVA and Dunnett's multiple comparison tests using as reference control the detection of Factin inside (blue) and outside (salmon) the contact area in Jurkat cells expressing the indicated EGFPprotein. n = 3 independent experiments. (C) Representative experiment showing the association of the specified MBP proteins (top) with the liposomes of the indicated composition (left). S, soluble (unbound) fraction; P, pelleted (bound) fraction. PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine. Proteins were stained with Coomassie.
(D) Quantification of the experiments shown in C. Data represent the mean ± SEM. Statistical values were obtained using two-way ANOVA followed by Dunnett's test for multiple comparisons. n = 5 independent experiments.
(E) Stained SDS-PAGE gel showing the purified Vav1 WT protein used in Figure 5E. The migration of the molecular weight markers is shown on the right.
(F) Immunoprecipitation of indicated Vav1 proteins (top) with antibodies to epitopes located in the Vav1 DH domain (top panel on the left) and KR (top panel on the right). As control, we show the expression of each of the protein versions used aliquots of the total cellular lysates used in the immunoprecipitation experiments (bottom panel).
(G) Stained SDS-PAGE gels showing the purified MBPs used in Figure 6. The migration of the molecular weight markers is shown on the right of each panel. WT and VAV1 knockdown (shVav1) Jurkat cells were transfected with EGFP-tagged domains that specifically recognize PI3P (NAPD oxidase PX domain), PI4P (four phosphate adaptor protein 1 PH domain) and PI5P (Ing2 PHD domain). Upon synapse formation, cells were fixed, stained with phalloidin, and subjected to confocal microscopy. The bioreporters and F-actin are seen as green and red signals. Areas of colocalization between the bioreporters and F-actin are seen in yellow.

FIGURE S6. Comparison of the Vav1 C1-KR with the PHD-KR cassette of nuclear proteins
The residues involved in the coordination of Zn 2+ are shown in green and shaded in dark gray. Basic residues present in the KR regions of the indicated proteins are shown in red. The basic residues present in the Ing2 PHD that contribute to binding to phosphatidylinositol monophosphates are also indicated in red.  *F, forward primer (in the case of a two-step mutagenesis protocol, the primers are referred to as F1 and F2 for the first and second step, respectively); R, reverse primer (in the case of a two-step mutagenesis protocol, the primers are referred to as R1 and R2 for the first and second step, respectively); [p]-, phosphorylated primer. Nucleotides used for the generation of the indicated mutations are shown in red. Nucleotides that have been inserted or deleted in the WT sequence are indicated in red parenthesis and with a red line, respectively. *Basic amino acid residues are shaded in red.