Vavl a Rac/Rho guanine nucleotide exchange factor and a critical component

Vavl a Rac/Rho guanine nucleotide exchange factor and a critical component of the T cell receptor (TCR) signaling cascade is rapidly tyrosine phosphorylated in response to T cell activation. chains CD3 δ ε γ chains as well as activation sites around the crucial T cell tyrosine kinases Itk Lck and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways. Keywords: Phosphoproteomics T cell receptor signaling mass spectrometry Vav1 Introduction Engagement of the TCR by a cognate peptide-major histocompatibility complex (MHC) molecule activates intricate signaling cascades involving multiple enzymes adaptors and other cellular proteins that result in T WZ8040 cell activation. The Src tyrosine kinases Lck and Fyn are the first molecules recruited to the activated TCR complex where they phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) of the ζ and CD3 chains (1). Phosphorylation of ITAMs leads to recruitment of the Syk family tyrosine kinase ζ-chain-associated protein kinase 70 (ZAP-70) via its tandem Src homology 2 (SH2) domains (2 3 Subsequent activation of ZAP-70 facilitates phosphorylation of downstream adaptor proteins resulting in the formation of a signalosome WZ8040 complex nucleated by linker for activation of T cells (LAT) and SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) (4 5 This signalosome recruits a variety of effector proteins which in turn activate a number of signaling pathways including Ca2+ mobilization activation of mitogen-activated protein kinase (MAPK) cascades activation of transcription factors and cytoskeletal reorganization (6 7 Vav1 is usually a member of the Dbl family of guanine nucleotide exchange factors (GEFs) exclusively expressed in hematopoietic cells (8). In T cells Vav1 is usually rapidly tyrosine phosphorylated upon TCR stimulation which activates its GEF activity towards Rac and Rho and initiates various pathways downstream of these GTPases (9-14). In addition to its function as a GEF Vav1 has been implicated in GEF-independent functions which is usually evidenced by its complex domain name structure. In addition to the Dbl homology (DH) domain name which confers GEF activity Vav1 contains a calponin homology (CH) domain name an acidic motif a pleckstrin homology (PH) domain name a cysteine-rich domain name (CRD) and a SH3-SH2-SH3 domain name (15). Vav proteins are the only known Rho GEFs that combine in the same protein the DH and PH motifs as well as the structural hallmark of signal transducer proteins the SH2 and Src homology 3 (SH3) domains (16) suggesting that Vav1 can interact with multiple components of signal transduction pathways. The functional importance of Vav1 has been exhibited in thymocyte development and mature T cell activation. Mice deficient in Vav1 have a partial block at the pre-TCR checkpoint in the thymus and T cell development is strongly blocked in both positive and negative T cell selection (17-20). In mature T cells Vav1 deficiency reduces TCR-induced proliferation intracellular Ca2+ flux upregulation of activation markers and cytokine secretion (18 20 Vav1 is also required to transduce TCR signals that lead to actin polymerization and TCR clustering (21 25 Consistent with a role for linking TCR signaling to the actin cytoskeleton the TCR-induced recruitment of the actin cytoskeleton to ζ chain ITAMs is usually impaired in Vav1-deficient T cells (21). Vav1 is also thought to play a role in the early molecular mechanisms that synergize TCR and CD28 mediating signaling (26). Interestingly there have been contradictory observations on whether AIbZIP Vav1 regulates the activation of the ERK and JNK MAPKs which requires further investigation (21 24 25 27 Although great progress has been made in understanding the role of Vav1 in TCR signaling our understanding of the WZ8040 molecular mechanisms by which Vav1 regulates TCR signaling pathways downstream of TCR triggering is usually far from complete. The current paradigm for the role of Vav1 in TCR signaling has been developed primarily through studies investigating whether specific TCR effector functions are altered in Vav1-deficient T cells (21 23 27 Although these studies have been invaluable to the understanding of Vav1’s role in TCR signaling they provide little insight into the specific biochemical events that are WZ8040 regulated by Vav1 upstream of effector responses. Protein phosphorylation constitutes.