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Mechanism Of Action Of SAP And PIX In Immune Cells

Posted on:2008-06-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:C P GuFull Text:PDF
GTID:1114360242476072Subject:Biochemistry and Molecular Biology
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The X-linked lymphoproliferative disease is an inherited severe or fatal immune dysfunction generally triggered by infection with Epstein-Barr virus. Boys with XLP often develop a lethal fulminant infectious mononucleosis following EBV infection. Some patients also develop progressive dysgammaglobulinemia or aggressive lymphoproliferative disorders, such as malignant lymphomas. Less commonly, the XLP patients may also develop autoimmunity, such as vasculitis, colitis or aplastic anemia with or without EBV exposure. The pleiotropic defects of XLP demonstrate a fundamental immune dysregulation associated with this disease.In 1998, three groups identified the gene aberrant in XLP, named as SAP/SH2D1A/DSHP, which is expressed in natural killer (NK) and T cells. The human SAP protein is comprised of 128 amino acids (A.A.), of which residues 6-102 contribute to an amino-terminal SH2 domain and the remaining carboxyl-terminus does not resemble any recognizable structural motif. Many experiments have shown that the SH2 domain of SAP preferentially recognize specific tyrosine-based motifs conforming to the consensus TxYxxV/I that are present within the cytoplasmic tails of SLAM as well as several other SLAM-related receptors, including 2B4 (CD244), CD84, Ly9 (CD229), NTB-A (Ly108), and a CRACC. Thus, signaling through these receptors may be regulated by the recruitment of SAP. As XLP patients lack functional SAP, it has been speculated that the phenotypes of XLP likely reflect perturbed signaling downstream of one or more of these receptors, ultimately resulting in abnormal immune responses.Several immune cell types were shown to exhibit functional alterations in XLP, including CD4+ T cells, CD8+ T cells, NK cells and B cells. In addition, the critical importance of SAP in immune regulation was also demonstrated in the SAP-deficient mice. These animals manifest increased susceptibility to murine lymphocytic choriomeningitis virus (LCMV) and gammaherpevirus-68. Alterations of antiviral CD8+ T cells responses were found in these animals. Moreover, CD4+ T cells isolated from the SAP-deficient mice revealed diminished IL-4 production in response to activation with anti-CD3 antibodies, suggesting additional dysregulations in TCR-mediated responses.Given the severe clinical manifestations in XLP patients, recent efforts have been focused on elucidating the signaling mechanism by which SAP regulates in T and NK cells. It has been shown that SAP recruits a Src kinase Fyn to the SLAM related receptors via the Fyn SH3 domain, and such recruitment and downstream signaling may be responsible for the SLAM receptors-mediated T cell activation. However, given the different phenotypes of SLAM-deficient and Fyn-deficient mice and the pleiotropic defects of XLP patients, it is reasonable to suggest that other important molecules might also be involved in SLAM-SAP signaling pathway.We used a yeast two-hybrid system to screen a human lymphoid cDNA library with full length human SAP. Interestingly, we have identified multiple clones containing various lengths of the cDNA sequence corresponding toβPIX, a guanine nucleotide exchange factor for Rac/Cdc42 small GTPases.βPIX, also named as COOL-1, is a 646 A.A. protein, which is coded by gene ARHGEF7.βPIX is composed of an N-terminus SH3 domain, a DH domain, a PH domain and a C-terminus coiled-coiled domain. Another PIX family member,αPIX, also named as COOL-2, is also identified in the same screening.We show that SAP interacts with PIX directly by Co-IP assays. Moreover, PIX is recruited to SLAM family receptor by SAP, suggesting it might be involved in the downstream signal pathway. EAT-2, the homology of SAP, fails to interact with PIX and recruit PIX to SLAM family receptors. Taken together, our data demonstrate a specific interaction between SAP and PIX in T cells. By Co-IP studies on key amino acid mutation of SAP, we confirm that similar to the interaction of Fyn and SAP, the interaction of PIX and SAP involves a second binding surface in the SH2 domain of SAP, distinct from the receptor binding surface. In mammalian cell lines, PIX competes with Fyn for the binding of SAP as demonstrated by our Co-IP assays.By Co-IP studies on different truncations and key amino acid mutations of PIX, we identify the SH3 domain of PIX is not only necessary, but also sufficient for SAP-binding. Moreover, SAP, PIX and an activated form of Cdc42 form a tri-molecular complex, suggesting SAP and PIX might link the signal from SLAM family receptors to Cdc42-mediated signaling pathway.In order to elucidate the possible PIX and SAP-mediated signaling pathway, we study the potential function of SAP in NFκB, NFAT, AP-1 and p53 pathway by our established reporter gene screening assays. SAP overexpression causes no obvious effect on NFκB, AP-1 and p53 pathway. However, we find that overexpression of SAP activates NFAT synergetically with a calcium signal in T cells. So far, only an activated form of Ras has such dramatic effect on NFAT activation. In contrast, the SAP homology EAT-2 fails to have any effect on NFAT activation, suggesting that the SAP effect is specific. Moreover, overexpression of either SAP mutant, SAP-R32Q or SAP-R78A, causes no effect on NFAT activation, indicating that the stimulation might depend on the SAP binding to the upstream receptors as well as the downstream effectors such as PIX or Fyn. In addition, the synergetic effect of SAP with calcium signal on NFAT activation was completely abolished by the dominant negative form of Ras, suggesting that Ras or Ras-related pathway might be involved in SAP function in T cells.In order to test whether PIX is involved in mediating the SAP effect, we construct the SH3 domain of PIX as a dominant negative form of PIX and test its effect on SAP-mediated NFAT activation. Interestingly, the SH3 domain of PIX dramatically inhibits the SAP-mediated NFAT activation in the presence of ionomycin. In contrast, the PIX SH3 domain mutation (PIX-SH3 W43P/W44G), which fails to bind to SAP, has no effect on the SAP-stimulated effect. Moreover, the SH3 domain of Fyn, when expressed at a level comparable with PIX SH3 domain, did not have any significant inhibitory effect on the SAP-induced NFAT activation. These data suggest that SAP might use PIX, rather than Fyn, to mediate such function in T cells. Taken together, our data indicate the SAP-PIX interaction might have specific role in T cell activation.To date, this is the first study describing the physical and potential functional interaction between SAP and a Rac/Cdc42 GEF, which may provide insight into the pathophysiology of XLP and the basis for XLP therapy.
Keywords/Search Tags:nuclear factor of activating T cell (NFAT), Cdc42, 2B4, Fyn
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