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W2014177218 abstract "Membrane-associated adaptors play an important role in coupling antigen receptor engagement to downstream signaling events, such as Ras-MAPK activation, Ca2+ flux, and nuclear factor of activated T cells (NFAT) activation. Here we identified a novel membrane-associated adaptor protein, LAX. LAX is mainly expressed in B cells, T cells, and other lymphoid-specific cell types. It shares no overall sequence homology with LAT and is not localized to lipid rafts. However, like LAT, LAX has tyrosine motifs for binding Grb2, Gads, and the p85 subunit of phosphatidylinositol 3-kinase. Upon stimulation via the B or T cell receptors, LAX is rapidly phosphorylated by Src and Syk family tyrosine kinases and interacts with Grb2, Gads, and p85. Overexpression of LAX in Jurkat cells specifically inhibits T cell receptor-mediated p38 MAPK activation and NFAT/AP-1 transcriptional activation. Our data suggested that LAX functions to negatively regulate signaling in lymphocytes. Membrane-associated adaptors play an important role in coupling antigen receptor engagement to downstream signaling events, such as Ras-MAPK activation, Ca2+ flux, and nuclear factor of activated T cells (NFAT) activation. Here we identified a novel membrane-associated adaptor protein, LAX. LAX is mainly expressed in B cells, T cells, and other lymphoid-specific cell types. It shares no overall sequence homology with LAT and is not localized to lipid rafts. However, like LAT, LAX has tyrosine motifs for binding Grb2, Gads, and the p85 subunit of phosphatidylinositol 3-kinase. Upon stimulation via the B or T cell receptors, LAX is rapidly phosphorylated by Src and Syk family tyrosine kinases and interacts with Grb2, Gads, and p85. Overexpression of LAX in Jurkat cells specifically inhibits T cell receptor-mediated p38 MAPK activation and NFAT/AP-1 transcriptional activation. Our data suggested that LAX functions to negatively regulate signaling in lymphocytes. Recognition of antigens by antigen receptors, the B cell receptor (BCR) 1The abbreviations used for: BCR, B cell receptor; TCR, T cell receptor; ITAMs, immunoreceptor tyrosine-based activation motifs; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PI-3 kinase, phosphatidylinositol 3-kinase; ITAM, immunoreceptor tyrosine-based activation motifs; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; WT, wild type; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SH, Src homology; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T cells; TK, thymidine kinase. 1The abbreviations used for: BCR, B cell receptor; TCR, T cell receptor; ITAMs, immunoreceptor tyrosine-based activation motifs; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PI-3 kinase, phosphatidylinositol 3-kinase; ITAM, immunoreceptor tyrosine-based activation motifs; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; WT, wild type; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SH, Src homology; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T cells; TK, thymidine kinase. and the T cell receptor (TCR), initiates a series of biochemical events involving a variety of distinct signaling pathways that eventually lead to gene transcription, clonal expansion, and cellular differentiation. Although the BCR and TCR have different structures and recognize different forms of antigens, signaling pathways activated via these two receptors are strikingly similar. Both the BCR and TCR utilize receptor-encoded signaling motifs termed ITAMs (immunoreceptor tyrosine-based activation motifs) to activate non-receptor tyrosine kinases (1Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1154) Google Scholar). Following receptor engagement, Src family tyrosine kinases, activated by the CD45 phosphatase, phosphorylate the paired tyrosine residues within ITAMs. Syk family kinases are then recruited to the receptor by binding phosphorylated ITAMs via their tandem SH2 domains and are subsequently activated by Src family kinases (2Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (876) Google Scholar). These activated tyrosine kinases further phosphorylate multiple cellular proteins, including enzymes and adaptor proteins (3Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1943) Google Scholar, 4Chan A.C. Shaw A.S. Curr. Opin. Immunol. 1996; 8: 394-401Crossref PubMed Scopus (170) Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 6Clements J.L. Boerth N.J. Lee J.R. Koretzky G.A. Annu. Rev. Immunol. 1999; 17: 89-108Crossref PubMed Scopus (173) Google Scholar, 7Kurosaki T. Curr. Opin. Immunol. 2000; 12: 276-281Crossref PubMed Scopus (106) Google Scholar, 8Tamir I. Cambier J.C. Oncogene. 1998; 17: 1353-1364Crossref PubMed Scopus (104) Google Scholar), leading to activation of the Ras-MAPK pathway and Ca2+ flux.Several studies using deficient cell lines have shown that adaptor proteins are essential for lymphocyte activation by coupling receptor engagement to activation of the Ras-MAPK pathway, Ca2+mobilization, and cytokine production (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 10Kelly M.E. Chan A.C. Curr. Opin. Immunol. 2000; 12: 267-275Crossref PubMed Scopus (33) Google Scholar, 11Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (353) Google Scholar). In T cells, LAT and SLP-76 have been intensively studied in recent years (6Clements J.L. Boerth N.J. Lee J.R. Koretzky G.A. Annu. Rev. Immunol. 1999; 17: 89-108Crossref PubMed Scopus (173) Google Scholar, 12Zhang W. Samelson L.E. Semin. Immunol. 2000; 12: 35-41Crossref PubMed Scopus (110) Google Scholar, 13Rudd C.E. Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 14Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 15Boerth N.J. Sadler J.J. Bauer D.E. Clements J.L. Gheith S.M. Koretzky G.A. J. Exp. Med. 2000; 192: 1047-1058Crossref PubMed Scopus (103) Google Scholar). LAT is a membrane-associated adaptor protein. Upon phosphorylation, LAT interacts with Grb2, Gads, and PLC-γ1 (16Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). The binding of Grb2 to LAT is postulated to recruit Sos to the membrane to activate Ras. The association of LAT with PLC-γ1 recruits PLC-γ1 to the membrane so it can be phosphorylated and activated. Activation of PLC-γ1 is essential for TCR-mediated Ca2+ flux and activation of Ras-GRP, a molecule that functions to activate Ras (17Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (545) Google Scholar, 18Ebinu J.O. Stang S.L. Teixeira C. Bottorff D.A. Hooton J. Blumberg P.M. Barry M. Bleakley R.C. Ostergaard H.L. Stone J.C. Blood. 2000; 95: 3199-3203Crossref PubMed Google Scholar). TCR-mediated Ras-MAPK activation and Ca2+ flux are defective in LAT-deficient Jurkat cells, indicating that LAT is essential for TCR-mediated signaling (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 19Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (226) Google Scholar). Binding of Gads to LAT recruits SLP-76 indirectly to the membrane (20Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 21Yoder J. Pham C. Iizuka Y.M. Kanagawa O. Liu S.K. McGlade J. Cheng A.M. Science. 2001; 291: 1987-1991Crossref PubMed Scopus (124) Google Scholar). SLP-76 is also essential in TCR-mediated signaling as indicated in SLP-76-deficient cells (11Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (353) Google Scholar, 22Yablonski D. Kadlecek T. Weiss A. Mol. Cell. Biol. 2001; 21: 4208-4218Crossref PubMed Scopus (168) Google Scholar). TCR-mediated MAPK activation and Ca2+ flux are severely compromised in these cells.As a membrane-associated adaptor protein, LAT plays a critical role in signaling in T cells. Similar molecules might also exist in other cell types with immune receptors. A similar molecule has not been found in B cells. It has been proposed that BLNK functions as both LAT and SLP-76 to link the BCR engagement to MAPK activation and Ca2+ flux (23Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). BCR-induced PLC-γ2 activation and Ca2+ flux are defective in BLNK-deficient cells (24Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). BCR-mediated JNK and Erk activation are also compromised. However, in contrast to LAT, BLNK is not constitutively localized to the membrane. Furthermore, LAT deficiency in Jurkat cells cannot be complemented by BLNK (25Wong J. Ishiai M. Kurosaki T. Chan A.C. J. Biol. Chem. 2000; 275: 33116-33122Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Therefore, it is less likely that BLNK functions as both LAT and SLP-76 in B cells. It is possible that B cells or other lymphoid cells use a LAT-like molecule to link the receptor engagement to Ras-MAPK activation and Ca2+ flux. Due to the near completion of the human genome sequencing, it might be possible to find a LAT homolog in the human genome data base.To look for a LAT-like molecule in B cells and other cell types, we searched the human genome data base with the tyrosine motifs in LAT and identified a novel gene. We named it LAX. Like LAT, LAX is a membrane-associated adaptor protein. It is expressed in T cells, B cells, and other cell types of lymphoid origin. It associates with Grb2, Gads, and the p85 subunit of PI-3 kinase. However, it is unlikely that LAX functions as a B cell LAT-like molecule. Our data show that as opposed to LAT, LAX functions to negatively regulate antigen-receptor signaling in T cells by inhibiting TCR-mediated p38 MAPK activation.DISCUSSIONAccumulating evidence indicates that adaptor proteins are important in antigen receptor-mediated signaling pathways. In this paper, we report identification of a novel membrane-associated adaptor molecule, LAX. LAX was exclusively expressed in lymphoid tissues. Of the several cell lines we tested, LAX was found in B, T, NK, and monocyte cell lines. In the cytoplasmic domain of LAX, there are multiple tyrosines. These tyrosines are within the Grb2- or p85-binding motifs. Upon stimulation via the TCR or BCR, LAX was tyrosine-phosphorylated and interacted with Grb2, Gads, and p85. By coexpressing LAX with Src and/or Syk tyrosine kinases, we showed that LAX could be phosphorylated by Lck, Syk, and ZAP-70. Phosphorylation of LAX was reduced in ZAP-70-deficient cells and was abolished in Lck-deficient cells. By overexpressing wild-type LAX and a mutant LAX with mutations at four critical tyrosines, we showed that overexpression of wild-type LAX inhibited p38 MAPK activation and NFAT/AP-1-mediated transcription in Jurkat cells, whereas overexpression of the mutant LAX had no effect. Our data indicated that LAX is an adaptor molecule that potentially functions to negatively regulate TCR signaling.LAX and LAT are membrane-associated adaptor proteins. Both of them have a short extracellular domain, a transmembrane domain, and a cytoplasmic domain. Whereas LAT is expressed in T cells, NK cells, mast cells, and platelets (31Facchetti F. Chan J.K. Zhang W. Tironi A. Chilosi M. Parolini S. Notarangelo L.D. Samelson L.E. Am. J. Pathol. 1999; 154: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), LAX is expressed in T cells, B cells, NK cells, and monocytes. We have not tested whether LAX is present in mast cells, platelets, or other cell types. Although LAT and LAX have no overall homology in amino acid sequences, the tyrosine motifs in their cytoplasmic domains are very similar (Fig. 1 B). These motifs are responsible for binding the SH2 domain-containing proteins. LAX has five Grb2-binding motifs (YXN) in its cytoplasmic tail. It also has a Gads motif (YVNV) identical to those in LAT. In addition, LAX has a p85-binding motif (YXXM), which is not present in LAT, although LAT is able to associate with p85. Upon antigen receptor stimulation, LAX also interacted with Grb2, Gads, and p85 like LAT. However, we have not been able to detect any significant interaction between LAX and PLC-γ1/2.Overexpression of LAX-WT inhibited NFAT activation, whereas overexpression of a LAX mutant with mutations at four tyrosines had no effect. It is possible that overexpression of LAX could sequester other signaling proteins from LAT and further inhibit TCR-mediated signaling. Biochemical analysis of Jurkat cells stably transfected with WT-LAX and LAX-4YF showed that overexpression of LAX had no significant effect on tyrosine phosphorylation of proteins, Ca2+, Erk, or Jnk activation, suggesting that overexpression of LAX did not inhibit LAT-mediated signaling by sequestering Grb2, Gads, and p85 from LAT. Interestingly, overexpression of LAX specifically inhibited TCR-mediated p38 MAPK activation. This suggested that LAX likely functions in the pathway of p38 MAPK.Erk, Jnk, and p38 MAPKs are three subgroups of the MAPK superfamily. These three kinases are all activated following T cell activation. These MAPKs phosphorylate different subsets of substrates (32Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar). The substrates for p38 MAPK include transcription factors (Elk-1, ATF2, CHOP, MEF2C, and SAP-1) and downstream protein kinases (Mnk1, Mnk2, PRAK, MSK1, etc). It is not clear how p38 MAPK activation is coupled to TCR engagement and how p38 contributes to IL-2 production in T cells. Pretreatment of Jurkat cells with a specific p38 MAPK inhibitor, SB203580, or expression of a dominant negative form of MKK6, one of the upstream kinase of p38, can suppress the transcriptional activation of the IL-2 promoter (29Matsuda S. Moriguchi T. Koyasu S. Nishida E. J. Biol. Chem. 1998; 273: 12378-12382Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). It has been shown in mice that p38 MAPK activation can modulate T cell development and is required for the activation of Th1 cells but not for activation of Th2 cells (32Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar, 33Mulroy T. Sen J. Eur. J. Immunol. 2001; 31: 3056-3063Crossref PubMed Scopus (18) Google Scholar). Our data placed LAX in the p38 pathway. However, how LAX suppresses p38 activation remains to be determined. Because the LAX mutant with mutations of four tyrosine residues failed to inhibit p38 activation, the interaction between LAX with Grb2, Gads, or p85 might be required for LAX function. It is likely that LAX might recruit a negative regulator, such as phosphatase, to the membrane to turn off p38 MAPK activation. However, we have not been able to detect any interaction between LAX and phosphatases.Previous studies (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 19Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (226) Google Scholar) showed that LAT is critical in T cell activation. LAT-deficient cells are defective in TCR-mediated Ras-MAPK activation and Ca2+ flux. Our data here suggested that LAX functions differently from LAT as follows. 1) In contrast to LAT, LAX is not localized in lipid rafts. 2) Defective signaling in LAT-deficient Jurkat cells could not be rescued by expression of LAX. 3) LAX does not interact with PLC-γ1 or -γ2. Thus, LAX is less likely to function in linking receptor engagement to Ca2+flux.Signaling via the BCR shares many similar features as signaling via the TCR. BLNK associates with Grb2, Vav, PLC-γ1, and Nck in B cells similar to LAT and SLP-76 in T cells. BLNK is not constitutively localized in the membrane, which is different from LAT. Therefore, there might exist a LAT-like molecule in B cells. This molecule functions to recruit BLNK and its associated proteins to the membrane. As opposed to LAT, LAX is expressed in B cells. Upon activation via the BCR, LAX became tyrosine-phosphorylated. We have attempted to perform similar experiments to determine whether overexpression of LAX affects BCR-mediated NFAT activation. We failed to obtain any conclusive results due to a low efficiency of transfection with these B cells. The function of LAX in B cells needs to be further studied. Because LAX is not localized in lipid rafts and does not associate with PLC-γ1/2, it is less likely that LAX functions like LAT in B cells.In summary, we identified a novel membrane-associated adaptor protein, and we showed that it functions to negatively regulate TCR signaling. This inhibitory signal delivered by LAX may be critical for terminating IL-2 production in the late stage of immune responses. Interestingly, consistent with this notion, the amount of LAX protein extracted by Brij detergent was increased dramatically upon stimulation of Jurkat cells with anti-TCR antibody or PMA. 2M. Zhu, E. Janssen, K. Leung, and W. Zhang, unpublished data. Increased LAX protein at the membrane might bring more inhibitory molecules to the membrane to turn off a T cell response. The mechanism by which LAX inhibited p38 MAPK and NFAT/AP-1 transcriptional activation remains to be determined. The precise function of LAX in lymphocyte signaling and immune response will be revealed by analysis of LAX-deficient mice. Recognition of antigens by antigen receptors, the B cell receptor (BCR) 1The abbreviations used for: BCR, B cell receptor; TCR, T cell receptor; ITAMs, immunoreceptor tyrosine-based activation motifs; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PI-3 kinase, phosphatidylinositol 3-kinase; ITAM, immunoreceptor tyrosine-based activation motifs; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; WT, wild type; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SH, Src homology; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T cells; TK, thymidine kinase. 1The abbreviations used for: BCR, B cell receptor; TCR, T cell receptor; ITAMs, immunoreceptor tyrosine-based activation motifs; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PI-3 kinase, phosphatidylinositol 3-kinase; ITAM, immunoreceptor tyrosine-based activation motifs; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; WT, wild type; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SH, Src homology; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T cells; TK, thymidine kinase. and the T cell receptor (TCR), initiates a series of biochemical events involving a variety of distinct signaling pathways that eventually lead to gene transcription, clonal expansion, and cellular differentiation. Although the BCR and TCR have different structures and recognize different forms of antigens, signaling pathways activated via these two receptors are strikingly similar. Both the BCR and TCR utilize receptor-encoded signaling motifs termed ITAMs (immunoreceptor tyrosine-based activation motifs) to activate non-receptor tyrosine kinases (1Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1154) Google Scholar). Following receptor engagement, Src family tyrosine kinases, activated by the CD45 phosphatase, phosphorylate the paired tyrosine residues within ITAMs. Syk family kinases are then recruited to the receptor by binding phosphorylated ITAMs via their tandem SH2 domains and are subsequently activated by Src family kinases (2Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (876) Google Scholar). These activated tyrosine kinases further phosphorylate multiple cellular proteins, including enzymes and adaptor proteins (3Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1943) Google Scholar, 4Chan A.C. Shaw A.S. Curr. Opin. Immunol. 1996; 8: 394-401Crossref PubMed Scopus (170) Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 6Clements J.L. Boerth N.J. Lee J.R. Koretzky G.A. Annu. Rev. Immunol. 1999; 17: 89-108Crossref PubMed Scopus (173) Google Scholar, 7Kurosaki T. Curr. Opin. Immunol. 2000; 12: 276-281Crossref PubMed Scopus (106) Google Scholar, 8Tamir I. Cambier J.C. Oncogene. 1998; 17: 1353-1364Crossref PubMed Scopus (104) Google Scholar), leading to activation of the Ras-MAPK pathway and Ca2+ flux. Several studies using deficient cell lines have shown that adaptor proteins are essential for lymphocyte activation by coupling receptor engagement to activation of the Ras-MAPK pathway, Ca2+mobilization, and cytokine production (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 10Kelly M.E. Chan A.C. Curr. Opin. Immunol. 2000; 12: 267-275Crossref PubMed Scopus (33) Google Scholar, 11Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (353) Google Scholar). In T cells, LAT and SLP-76 have been intensively studied in recent years (6Clements J.L. Boerth N.J. Lee J.R. Koretzky G.A. Annu. Rev. Immunol. 1999; 17: 89-108Crossref PubMed Scopus (173) Google Scholar, 12Zhang W. Samelson L.E. Semin. Immunol. 2000; 12: 35-41Crossref PubMed Scopus (110) Google Scholar, 13Rudd C.E. Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 14Jackman J.K. Motto D.G. Sun Q. Tanemoto M. Turck C.W. Peltz G.A. Koretzky G.A. Findell P.R. J. Biol. Chem. 1995; 270: 7029-7032Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 15Boerth N.J. Sadler J.J. Bauer D.E. Clements J.L. Gheith S.M. Koretzky G.A. J. Exp. Med. 2000; 192: 1047-1058Crossref PubMed Scopus (103) Google Scholar). LAT is a membrane-associated adaptor protein. Upon phosphorylation, LAT interacts with Grb2, Gads, and PLC-γ1 (16Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). The binding of Grb2 to LAT is postulated to recruit Sos to the membrane to activate Ras. The association of LAT with PLC-γ1 recruits PLC-γ1 to the membrane so it can be phosphorylated and activated. Activation of PLC-γ1 is essential for TCR-mediated Ca2+ flux and activation of Ras-GRP, a molecule that functions to activate Ras (17Ebinu J.O. Bottorff D.A. Chan E.Y. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (545) Google Scholar, 18Ebinu J.O. Stang S.L. Teixeira C. Bottorff D.A. Hooton J. Blumberg P.M. Barry M. Bleakley R.C. Ostergaard H.L. Stone J.C. Blood. 2000; 95: 3199-3203Crossref PubMed Google Scholar). TCR-mediated Ras-MAPK activation and Ca2+ flux are defective in LAT-deficient Jurkat cells, indicating that LAT is essential for TCR-mediated signaling (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 19Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (226) Google Scholar). Binding of Gads to LAT recruits SLP-76 indirectly to the membrane (20Liu S.K. Fang N. Koretzky G.A. McGlade C.J. Curr. Biol. 1999; 9: 67-75Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 21Yoder J. Pham C. Iizuka Y.M. Kanagawa O. Liu S.K. McGlade J. Cheng A.M. Science. 2001; 291: 1987-1991Crossref PubMed Scopus (124) Google Scholar). SLP-76 is also essential in TCR-mediated signaling as indicated in SLP-76-deficient cells (11Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (353) Google Scholar, 22Yablonski D. Kadlecek T. Weiss A. Mol. Cell. Biol. 2001; 21: 4208-4218Crossref PubMed Scopus (168) Google Scholar). TCR-mediated MAPK activation and Ca2+ flux are severely compromised in these cells. As a membrane-associated adaptor protein, LAT plays a critical role in signaling in T cells. Similar molecules might also exist in other cell types with immune receptors. A similar molecule has not been found in B cells. It has been proposed that BLNK functions as both LAT and SLP-76 to link the BCR engagement to MAPK activation and Ca2+ flux (23Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). BCR-induced PLC-γ2 activation and Ca2+ flux are defective in BLNK-deficient cells (24Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). BCR-mediated JNK and Erk activation are also compromised. However, in contrast to LAT, BLNK is not constitutively localized to the membrane. Furthermore, LAT deficiency in Jurkat cells cannot be complemented by BLNK (25Wong J. Ishiai M. Kurosaki T. Chan A.C. J. Biol. Chem. 2000; 275: 33116-33122Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Therefore, it is less likely that BLNK functions as both LAT and SLP-76 in B cells. It is possible that B cells or other lymphoid cells use a LAT-like molecule to link the receptor engagement to Ras-MAPK activation and Ca2+ flux. Due to the near completion of the human genome sequencing, it might be possible to find a LAT homolog in the human genome data base. To look for a LAT-like molecule in B cells and other cell types, we searched the human genome data base with the tyrosine motifs in LAT and identified a novel gene. We named it LAX. Like LAT, LAX is a membrane-associated adaptor protein. It is expressed in T cells, B cells, and other cell types of lymphoid origin. It associates with Grb2, Gads, and the p85 subunit of PI-3 kinase. However, it is unlikely that LAX functions as a B cell LAT-like molecule. Our data show that as opposed to LAT, LAX functions to negatively regulate antigen-receptor signaling in T cells by inhibiting TCR-mediated p38 MAPK activation. DISCUSSIONAccumulating evidence indicates that adaptor proteins are important in antigen receptor-mediated signaling pathways. In this paper, we report identification of a novel membrane-associated adaptor molecule, LAX. LAX was exclusively expressed in lymphoid tissues. Of the several cell lines we tested, LAX was found in B, T, NK, and monocyte cell lines. In the cytoplasmic domain of LAX, there are multiple tyrosines. These tyrosines are within the Grb2- or p85-binding motifs. Upon stimulation via the TCR or BCR, LAX was tyrosine-phosphorylated and interacted with Grb2, Gads, and p85. By coexpressing LAX with Src and/or Syk tyrosine kinases, we showed that LAX could be phosphorylated by Lck, Syk, and ZAP-70. Phosphorylation of LAX was reduced in ZAP-70-deficient cells and was abolished in Lck-deficient cells. By overexpressing wild-type LAX and a mutant LAX with mutations at four critical tyrosines, we showed that overexpression of wild-type LAX inhibited p38 MAPK activation and NFAT/AP-1-mediated transcription in Jurkat cells, whereas overexpression of the mutant LAX had no effect. Our data indicated that LAX is an adaptor molecule that potentially functions to negatively regulate TCR signaling.LAX and LAT are membrane-associated adaptor proteins. Both of them have a short extracellular domain, a transmembrane domain, and a cytoplasmic domain. Whereas LAT is expressed in T cells, NK cells, mast cells, and platelets (31Facchetti F. Chan J.K. Zhang W. Tironi A. Chilosi M. Parolini S. Notarangelo L.D. Samelson L.E. Am. J. Pathol. 1999; 154: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), LAX is expressed in T cells, B cells, NK cells, and monocytes. We have not tested whether LAX is present in mast cells, platelets, or other cell types. Although LAT and LAX have no overall homology in amino acid sequences, the tyrosine motifs in their cytoplasmic domains are very similar (Fig. 1 B). These motifs are responsible for binding the SH2 domain-containing proteins. LAX has five Grb2-binding motifs (YXN) in its cytoplasmic tail. It also has a Gads motif (YVNV) identical to those in LAT. In addition, LAX has a p85-binding motif (YXXM), which is not present in LAT, although LAT is able to associate with p85. Upon antigen receptor stimulation, LAX also interacted with Grb2, Gads, and p85 like LAT. However, we have not been able to detect any significant interaction between LAX and PLC-γ1/2.Overexpression of LAX-WT inhibited NFAT activation, whereas overexpression of a LAX mutant with mutations at four tyrosines had no effect. It is possible that overexpression of LAX could sequester other signaling proteins from LAT and further inhibit TCR-mediated signaling. Biochemical analysis of Jurkat cells stably transfected with WT-LAX and LAX-4YF showed that overexpression of LAX had no significant effect on tyrosine phosphorylation of proteins, Ca2+, Erk, or Jnk activation, suggesting that overexpression of LAX did not inhibit LAT-mediated signaling by sequestering Grb2, Gads, and p85 from LAT. Interestingly, overexpression of LAX specifically inhibited TCR-mediated p38 MAPK activation. This suggested that LAX likely functions in the pathway of p38 MAPK.Erk, Jnk, and p38 MAPKs are three subgroups of the MAPK superfamily. These three kinases are all activated following T cell activation. These MAPKs phosphorylate different subsets of substrates (32Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar). The substrates for p38 MAPK include transcription factors (Elk-1, ATF2, CHOP, MEF2C, and SAP-1) and downstream protein kinases (Mnk1, Mnk2, PRAK, MSK1, etc). It is not clear how p38 MAPK activation is coupled to TCR engagement and how p38 contributes to IL-2 production in T cells. Pretreatment of Jurkat cells with a specific p38 MAPK inhibitor, SB203580, or expression of a dominant negative form of MKK6, one of the upstream kinase of p38, can suppress the transcriptional activation of the IL-2 promoter (29Matsuda S. Moriguchi T. Koyasu S. Nishida E. J. Biol. Chem. 1998; 273: 12378-12382Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). It has been shown in mice that p38 MAPK activation can modulate T cell development and is required for the activation of Th1 cells but not for activation of Th2 cells (32Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar, 33Mulroy T. Sen J. Eur. J. Immunol. 2001; 31: 3056-3063Crossref PubMed Scopus (18) Google Scholar). Our data placed LAX in the p38 pathway. However, how LAX suppresses p38 activation remains to be determined. Because the LAX mutant with mutations of four tyrosine residues failed to inhibit p38 activation, the interaction between LAX with Grb2, Gads, or p85 might be required for LAX function. It is likely that LAX might recruit a negative regulator, such as phosphatase, to the membrane to turn off p38 MAPK activation. However, we have not been able to detect any interaction between LAX and phosphatases.Previous studies (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 19Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (226) Google Scholar) showed that LAT is critical in T cell activation. LAT-deficient cells are defective in TCR-mediated Ras-MAPK activation and Ca2+ flux. Our data here suggested that LAX functions differently from LAT as follows. 1) In contrast to LAT, LAX is not localized in lipid rafts. 2) Defective signaling in LAT-deficient Jurkat cells could not be rescued by expression of LAX. 3) LAX does not interact with PLC-γ1 or -γ2. Thus, LAX is less likely to function in linking receptor engagement to Ca2+flux.Signaling via the BCR shares many similar features as signaling via the TCR. BLNK associates with Grb2, Vav, PLC-γ1, and Nck in B cells similar to LAT and SLP-76 in T cells. BLNK is not constitutively localized in the membrane, which is different from LAT. Therefore, there might exist a LAT-like molecule in B cells. This molecule functions to recruit BLNK and its associated proteins to the membrane. As opposed to LAT, LAX is expressed in B cells. Upon activation via the BCR, LAX became tyrosine-phosphorylated. We have attempted to perform similar experiments to determine whether overexpression of LAX affects BCR-mediated NFAT activation. We failed to obtain any conclusive results due to a low efficiency of transfection with these B cells. The function of LAX in B cells needs to be further studied. Because LAX is not localized in lipid rafts and does not associate with PLC-γ1/2, it is less likely that LAX functions like LAT in B cells.In summary, we identified a novel membrane-associated adaptor protein, and we showed that it functions to negatively regulate TCR signaling. This inhibitory signal delivered by LAX may be critical for terminating IL-2 production in the late stage of immune responses. Interestingly, consistent with this notion, the amount of LAX protein extracted by Brij detergent was increased dramatically upon stimulation of Jurkat cells with anti-TCR antibody or PMA. 2M. Zhu, E. Janssen, K. Leung, and W. Zhang, unpublished data. Increased LAX protein at the membrane might bring more inhibitory molecules to the membrane to turn off a T cell response. The mechanism by which LAX inhibited p38 MAPK and NFAT/AP-1 transcriptional activation remains to be determined. The precise function of LAX in lymphocyte signaling and immune response will be revealed by analysis of LAX-deficient mice. Accumulating evidence indicates that adaptor proteins are important in antigen receptor-mediated signaling pathways. In this paper, we report identification of a novel membrane-associated adaptor molecule, LAX. LAX was exclusively expressed in lymphoid tissues. Of the several cell lines we tested, LAX was found in B, T, NK, and monocyte cell lines. In the cytoplasmic domain of LAX, there are multiple tyrosines. These tyrosines are within the Grb2- or p85-binding motifs. Upon stimulation via the TCR or BCR, LAX was tyrosine-phosphorylated and interacted with Grb2, Gads, and p85. By coexpressing LAX with Src and/or Syk tyrosine kinases, we showed that LAX could be phosphorylated by Lck, Syk, and ZAP-70. Phosphorylation of LAX was reduced in ZAP-70-deficient cells and was abolished in Lck-deficient cells. By overexpressing wild-type LAX and a mutant LAX with mutations at four critical tyrosines, we showed that overexpression of wild-type LAX inhibited p38 MAPK activation and NFAT/AP-1-mediated transcription in Jurkat cells, whereas overexpression of the mutant LAX had no effect. Our data indicated that LAX is an adaptor molecule that potentially functions to negatively regulate TCR signaling. LAX and LAT are membrane-associated adaptor proteins. Both of them have a short extracellular domain, a transmembrane domain, and a cytoplasmic domain. Whereas LAT is expressed in T cells, NK cells, mast cells, and platelets (31Facchetti F. Chan J.K. Zhang W. Tironi A. Chilosi M. Parolini S. Notarangelo L.D. Samelson L.E. Am. J. Pathol. 1999; 154: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), LAX is expressed in T cells, B cells, NK cells, and monocytes. We have not tested whether LAX is present in mast cells, platelets, or other cell types. Although LAT and LAX have no overall homology in amino acid sequences, the tyrosine motifs in their cytoplasmic domains are very similar (Fig. 1 B). These motifs are responsible for binding the SH2 domain-containing proteins. LAX has five Grb2-binding motifs (YXN) in its cytoplasmic tail. It also has a Gads motif (YVNV) identical to those in LAT. In addition, LAX has a p85-binding motif (YXXM), which is not present in LAT, although LAT is able to associate with p85. Upon antigen receptor stimulation, LAX also interacted with Grb2, Gads, and p85 like LAT. However, we have not been able to detect any significant interaction between LAX and PLC-γ1/2. Overexpression of LAX-WT inhibited NFAT activation, whereas overexpression of a LAX mutant with mutations at four tyrosines had no effect. It is possible that overexpression of LAX could sequester other signaling proteins from LAT and further inhibit TCR-mediated signaling. Biochemical analysis of Jurkat cells stably transfected with WT-LAX and LAX-4YF showed that overexpression of LAX had no significant effect on tyrosine phosphorylation of proteins, Ca2+, Erk, or Jnk activation, suggesting that overexpression of LAX did not inhibit LAT-mediated signaling by sequestering Grb2, Gads, and p85 from LAT. Interestingly, overexpression of LAX specifically inhibited TCR-mediated p38 MAPK activation. This suggested that LAX likely functions in the pathway of p38 MAPK. Erk, Jnk, and p38 MAPKs are three subgroups of the MAPK superfamily. These three kinases are all activated following T cell activation. These MAPKs phosphorylate different subsets of substrates (32Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar). The substrates for p38 MAPK include transcription factors (Elk-1, ATF2, CHOP, MEF2C, and SAP-1) and downstream protein kinases (Mnk1, Mnk2, PRAK, MSK1, etc). It is not clear how p38 MAPK activation is coupled to TCR engagement and how p38 contributes to IL-2 production in T cells. Pretreatment of Jurkat cells with a specific p38 MAPK inhibitor, SB203580, or expression of a dominant negative form of MKK6, one of the upstream kinase of p38, can suppress the transcriptional activation of the IL-2 promoter (29Matsuda S. Moriguchi T. Koyasu S. Nishida E. J. Biol. Chem. 1998; 273: 12378-12382Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). It has been shown in mice that p38 MAPK activation can modulate T cell development and is required for the activation of Th1 cells but not for activation of Th2 cells (32Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar, 33Mulroy T. Sen J. Eur. J. Immunol. 2001; 31: 3056-3063Crossref PubMed Scopus (18) Google Scholar). Our data placed LAX in the p38 pathway. However, how LAX suppresses p38 activation remains to be determined. Because the LAX mutant with mutations of four tyrosine residues failed to inhibit p38 activation, the interaction between LAX with Grb2, Gads, or p85 might be required for LAX function. It is likely that LAX might recruit a negative regulator, such as phosphatase, to the membrane to turn off p38 MAPK activation. However, we have not been able to detect any interaction between LAX and phosphatases. Previous studies (9Finco T.S. Kadlecek T. Zhang W. Samelson L.E. Weiss A. Immunity. 1998; 9: 617-626Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 19Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (226) Google Scholar) showed that LAT is critical in T cell activation. LAT-deficient cells are defective in TCR-mediated Ras-MAPK activation and Ca2+ flux. Our data here suggested that LAX functions differently from LAT as follows. 1) In contrast to LAT, LAX is not localized in lipid rafts. 2) Defective signaling in LAT-deficient Jurkat cells could not be rescued by expression of LAX. 3) LAX does not interact with PLC-γ1 or -γ2. Thus, LAX is less likely to function in linking receptor engagement to Ca2+flux. Signaling via the BCR shares many similar features as signaling via the TCR. BLNK associates with Grb2, Vav, PLC-γ1, and Nck in B cells similar to LAT and SLP-76 in T cells. BLNK is not constitutively localized in the membrane, which is different from LAT. Therefore, there might exist a LAT-like molecule in B cells. This molecule functions to recruit BLNK and its associated proteins to the membrane. As opposed to LAT, LAX is expressed in B cells. Upon activation via the BCR, LAX became tyrosine-phosphorylated. We have attempted to perform similar experiments to determine whether overexpression of LAX affects BCR-mediated NFAT activation. We failed to obtain any conclusive results due to a low efficiency of transfection with these B cells. The function of LAX in B cells needs to be further studied. Because LAX is not localized in lipid rafts and does not associate with PLC-γ1/2, it is less likely that LAX functions like LAT in B cells. In summary, we identified a novel membrane-associated adaptor protein, and we showed that it functions to negatively regulate TCR signaling. This inhibitory signal delivered by LAX may be critical for terminating IL-2 production in the late stage of immune responses. Interestingly, consistent with this notion, the amount of LAX protein extracted by Brij detergent was increased dramatically upon stimulation of Jurkat cells with anti-TCR antibody or PMA. 2M. Zhu, E. Janssen, K. Leung, and W. Zhang, unpublished data. Increased LAX protein at the membrane might bring more inhibitory molecules to the membrane to turn off a T cell response. The mechanism by which LAX inhibited p38 MAPK and NFAT/AP-1 transcriptional activation remains to be determined. The precise function of LAX in lymphocyte signaling and immune response will be revealed by analysis of LAX-deficient mice. We thank Drs. Arthur Weiss and Robert Abraham for kindly providing deficient Jurkat cell lines and Dr. Mike Cook for fluorescence-activated cell sorter analysis." @default.
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W2014177218 title "Molecular Cloning of a Novel Gene Encoding a Membrane-associated Adaptor Protein (LAX) in Lymphocyte Signaling" @default.
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