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. 2006 Dec 15;281(50):38257-65.
doi: 10.1074/jbc.M608913200. Epub 2006 Oct 18.

Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation

Ana V Miletic  1 , Kumiko Sakata-Sogawa, Michio Hiroshima, Michael J Hamann, Timothy S Gomez, Naruhisa Ota, Tracie Kloeppel, Osami Kanagawa, Makio Tokunaga, Daniel D Billadeau, Wojciech Swat
Affiliations

Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation

Ana V Miletic et al. J Biol Chem. .

Abstract

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.

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Figures

FIGURE 1
FIGURE 1. Vav1 Tyr160 and Tyr174 are phosphorylated in activated T cells
A, shown is the alignment of Vav1 from human, cow, dog, mouse, rat, oppossum, frog, zebrafish, fruit fly, and nematode. Spaces (marked by dashes) have been introduced for optimal comparison. Boxed sequences are tyrosine-based signaling motifs conserved among the different species. Tyrosines are numbered relative to the human sequence. B, tyrosines 160 and 174 are phosphorylated in response to T cell activation as determined by immunoblotting with anti-phospho-Tyr160 antiserum (top panel) or anti-phospho-Tyr174 antiserum (bottom panel). C, Jurkat cells or J.Vav cells stably expressing Vav1WT or tyrosine-to-phenylalanine mutants of the Vav1 AC tyrosines were treated with orthovanadate (ov) for 5 min followed by immunoprecipitation of Vav1. Immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with total anti-phosphotyrosine antibodies (4G10). In B and C, protein loading was verified by reprobing of blots with anti-Vav1 antibodies.
FIGURE 2
FIGURE 2. Vav1 acidic region tyrosines associate with several TCR-signaling proteins
A, biotinylated Vav1 Tyr142-, Tyr160-, and Tyr174-containing peptides coupled to streptavidin-agarose beads bind directly to SH2-GST proteins containing SH2 domains of Lck, PLCγ1, and PI3K p85α but not to the N-terminal SH2 domain of PLCγ1 or to Grb2 as indicated by immunoblotting with anti-GST antibodies. B, peptides containing phosphorylated Tyr142, Tyr160, and Tyr174 precipitated native Lck, PLCγ1, and PI3K p85α proteins from Jurkat cell lysates but not Grb2. TCL, total cell lysate; unP, unphosphorylated; P, phosphorylated.
FIGURE 3
FIGURE 3. Tyrosine 174 is required for Vav1 microcluster maintenance
A, J.Vav1WT, J.Vav1Y142F, J.Vav1Y160F, and J.Vav1Y174F cells expressing GFP-tagged Vav proteins were plated on anti-CD3-coated coverslips. Images were obtained in real time using TIRFM (times, above images). Shown is representative of n > 10 cells. B, diagonal lines indicate sections of a representative J.Vav1WT and J.Vav1Y174F cells used for kymographic analysis. Fluorescence of individual Vav1WT and Vav1Y174F microclusters over time are presented as horizontal streaks in kymographs for sections shown. C, relative fluorescence intensity over time of individual Vav1WT and Vav1Y174F clusters. Shown are two representative of n = 5 clusters examined.
FIGURE 4
FIGURE 4. Tyrosine 174 is required for T cell development and function
A, flow cytometric analyses of thymocytes and lymph nodes from Vav1WT or Vav1Y174F mice (see “Experimental Procedures” for generation of mice). The bottom thymocyte panel is gated on GFP+. Shown is one representative of n > 5 mice. B, proliferation of T cells as indicated, with [3H]thymidine incorporation at 48 h. C, expression level of Vav1Y174F in lymph node T cells, determined by immunoblotting with anti-Vav1 antibodies. Protein loading was verified by reblotting blots with antibodies to Erk-2. D, purified T cells from Vav1WT or Vav1Y174F mice were stimulated with anti-CD3 antibodies followed by precipitation of active, GTP-loaded Rac1 with a GST fusion protein containing the PAK1 binding domain (GST-PBD). Eluates were resolved by SDS-PAGE followed by immunoblotting with anti-Rac1 antibodies. TCL, total cell lysate.
FIGURE 5
FIGURE 5. Vav1Y174F harboring a GEF-inactivating mutation forms stable microclusters
J.Vav1WT, J.Vav1Y174F, and J.Vav1Y174F/GEF− cells were activated on anti-CD3-coated coverslips. Images were obtained in real time using TIRFM (times, above images). Shown is representative of n > 5 cells.
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