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W2008756544 abstract "Rlk/Txk is a T-cell-specific member of the Btk/Tec family of tyrosine kinases, whereas SLP-76 is a lymphoid adaptor that is essential for pre-TcR and mature TcR signaling. Although Rlk deficient T-cells show partial defects in T-cell proliferation, Rlk can complement ITK−/− cells with multiple defects in TcR initiated early events and interleukin (IL)-2 production. A key question is the nature of the target of Rlk responsible for bridging the TcR with the activation of IL-2 transcription. In this study, we identify a pathway in which Rlk phosphorylates SLP-76 leading to the phosphorylation of PLCγ1, activation of ERKs, and the synergistic up-regulation of TcR-driven IL-2 NFAT/AP-1 transcription. Rlk phosphorylated the N-terminal region of SLP-76, a region that has been previously shown to serve as a target for ZAP-70. Loss of N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated cooperativity between Rlk and SLP-76. These observations support a model where the TcR can utilize Rlk (as well as ZAP-70) in the phosphorylation of key sites in SLP-76 leading to the up-regulation of Th1 preferred cytokine IL-2. Rlk/Txk is a T-cell-specific member of the Btk/Tec family of tyrosine kinases, whereas SLP-76 is a lymphoid adaptor that is essential for pre-TcR and mature TcR signaling. Although Rlk deficient T-cells show partial defects in T-cell proliferation, Rlk can complement ITK−/− cells with multiple defects in TcR initiated early events and interleukin (IL)-2 production. A key question is the nature of the target of Rlk responsible for bridging the TcR with the activation of IL-2 transcription. In this study, we identify a pathway in which Rlk phosphorylates SLP-76 leading to the phosphorylation of PLCγ1, activation of ERKs, and the synergistic up-regulation of TcR-driven IL-2 NFAT/AP-1 transcription. Rlk phosphorylated the N-terminal region of SLP-76, a region that has been previously shown to serve as a target for ZAP-70. Loss of N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated cooperativity between Rlk and SLP-76. These observations support a model where the TcR can utilize Rlk (as well as ZAP-70) in the phosphorylation of key sites in SLP-76 leading to the up-regulation of Th1 preferred cytokine IL-2. interleukin-2 monoclonal antibody phospholipase C hemagglutinin rabbit anti-mouse T-cell activation involves the ligation of CD4/CD8-p56 lck and the T-cell receptor complex leading to the activation of Src kinases p56 lck and p59 fyn (1.Rudd C.E. Janssen O. Cai Y.-C. da Silva A.J. Raab M. Prasad K.V.S. Immunol. Today. 1994; 15: 225-234Abstract Full Text PDF PubMed Scopus (181) Google Scholar) and the phosphorylation of immunoreceptor tyrosine-based activation motifs of the TcRζ/CD3 chain (2.Weiss D.R. Littman D.R. Cell. 1994; 71: 649-662Google Scholar, 3.Mustelin T. Immunity. 1994; 1: 351-356Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 4.Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). ZAP-70 catalytic activity is then up-regulated by p56 lck phosphorylation (5.Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (323) Google Scholar, 6.Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). p56 lck and ZAP-70 play pivotal roles in thymic differentiation (7.Groves T. Smiley P. Cooke M.P. Forbush K. Perlmutter R.M. Guidos C.J. Immunity. 1996; 5: 417-428Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8.Van Oers N.S.C. Lowen-Kropf B. Finlay D. Connolly K. Weiss A. Immunity. 1996; 5: 429-436Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 9.Cheng A.M. Chan A.C. Curr. Opin. Immunol. 1997; 9: 528-533Crossref PubMed Scopus (44) Google Scholar) and TcR-mediated signaling in mature T-cells (10.Straus D. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 11.Williams B.L. Schreiber K.L. Zhang W. Wange R. Samelson L. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (223) Google Scholar).In addition to Src and ZAP/SYK-related kinases, the Tec family of tyrosine kinases has recently been implicated in the regulation of immune function. Tec kinases typified by Btk are comprised of an N-terminal phosphatidylinositol phosphate binding pleckstrin homology domain, followed by an SH2 and SH3 domain and a catalytic domain. Btk mutations have been documented to cause severe immunodeficiencies (12.Vetrie D. Vorechovsky I. Sideras P. Holland J. Davies A. Flinter F. Hammarström L. Kinnon C. Levinsky R. Bobrow M. Smith C.I.E. Bentley D.R. Nature. 1993; 361: 226-233Crossref PubMed Scopus (1226) Google Scholar, 13.Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisak I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Belmont J.W. Cooper M.D. Conley M.E. Witte O.N. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1135) Google Scholar, 14.Thomas J.D. Sideras P. Smith C.I.E. Vorechovsky I. Chapman V. Paul W.E. Science. 1993; 261: 355-358Crossref PubMed Scopus (571) Google Scholar, 15.Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (775) Google Scholar) and aberrant B-cell development (16.Rawlings D.J. Witte O.N. Semin. Immunol. 1995; 7: 237Crossref PubMed Scopus (113) Google Scholar). Tec kinases are positioned downstream of Src kinases that regulate their activity by phosphorylating within the activation loop of the kinase domain (17.Heyeck S.D. Wilcox H.M. Bunnell S.C. Berg L.J. J. Biol. Chem. 1997; 272: 25401-25408Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 18.Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-53011Crossref PubMed Scopus (135) Google Scholar, 19.Rawlings D.J. Scharenberg A.M. Park H. Wahl M.I. Lin S. Kato R.M. Fluckiger A.C. Witte O.N. Kinet J.P. Science. 1996; 271: 822-825Crossref PubMed Scopus (377) Google Scholar). T-cells express three members of the Tec family of tyrosine kinases that include Rlk (resting lymphocytekinase), ITK (interleukin-2-responsiveT-cell specific kinase) (otherwise known as Tsk (T-cell-specific kinase) or EMT), and Tec (20.Desiderio S. Siliciano J.D. Chem. Immunol. 1994; 59: 191Crossref PubMed Scopus (24) Google Scholar, 21.Gibson S. August A. Kawakami Y. Kawakami T. Dupont B. Mills G.B. J. Immunol. 1996; 156: 2716-2722PubMed Google Scholar, 22.Siliciano J.D. Morrow T.A. Desiderio S.V. Proc. Natl. Acad. Sci., U. S. A. 1992; 89: 11194-11198Crossref PubMed Scopus (235) Google Scholar). Rlk resembles other Tec kinases except for the absence of a pleckstrin homology domain, which has been replaced with palmitoylated cysteine ring motif that is required for membrane localization (23.Debnath J. Chamorro M. Czar M.J. Schaeffer E.M. Lenardo M.J. Varmus H.E. Schwartzberg P.L. Mol. Cell. Biol. 1999; 19: 1498-1507Crossref PubMed Scopus (71) Google Scholar). Two forms of Rlk exist that include a full-length form and an alternately spliced version that lacks the cysteine ring motif (23.Debnath J. Chamorro M. Czar M.J. Schaeffer E.M. Lenardo M.J. Varmus H.E. Schwartzberg P.L. Mol. Cell. Biol. 1999; 19: 1498-1507Crossref PubMed Scopus (71) Google Scholar). ITK and Rlk appear to complement each other in the regulation of T-cell function. Although ITK−/− mice show mild to moderate impairments of responses to TcR ligation (24.Bachman M.F. Littman D.R. Liao X.C. J. Virol. 1997; 71: 7253-7257Crossref PubMed Google Scholar), the Rlk−/− phenotype is less pronounced with a slight decrease in numbers of mature T-cells, in particular of the CD4+ subset (25.Schaeffer E.M. Debnath J. Yap G. McVicar D. Liao C. Littman D. Sher A. Varmus H. Lenardo M. Schwartzberg P.L. Science. 1999; 284: 638-641Crossref PubMed Scopus (328) Google Scholar). By contrast, the combined loss of Rlk and ITK shows major defects in TcR signaling events such as Ca2+ mobilization and inositol 1,4,5-trisphosphate and IL-21production (25.Schaeffer E.M. Debnath J. Yap G. McVicar D. Liao C. Littman D. Sher A. Varmus H. Lenardo M. Schwartzberg P.L. Science. 1999; 284: 638-641Crossref PubMed Scopus (328) Google Scholar).Aside from kinases, recent studies have identified an array of immune cell-specific adaptor proteins in T-cells that act as substrates for upstream kinases and play important roles in T-cell function (26.Rudd C.E. Cell. 1999; 96: 1-20Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 27.Peterson E.J. Clements J.L. Fang N. Koretzky G.A. Curr. Opin. Immunol. 1998; 10: 337-344Crossref PubMed Scopus (79) Google Scholar). One of these adaptors, SLP-76, is a relatively hydrophilic protein with an acidic N-terminal region, several tyrosine consensus motifs, a central proline-rich region, and a C-terminal SH2 domain (28.Jackman 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). As with Rlk-deficient T-cells, SLP-76−/− T-cells show defects in Ca2+ mobilization and inositol 1,4,5-trisphosphate and IL-2 production (29.Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (353) Google Scholar). To date, the ZAP-70 and SYK kinases have been shown to couple receptors to SLP-76 by virtue of their ability to phosphorylate SLP-76 (30.Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). ZAP-70 phosphorylates residues within an acidic region of SLP-76 that includes several tyrosine motifs: YESP, YESP, and YEPP (31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These N-terminal residues are required for the ability of SLP-76 to potentiate IL-2 production (32.Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar).Despite its importance to T-cell proliferation, the signaling pathway that connects Rlk to the TcR complex and the subsequent up-regulation of ERK activity and IL-2 transcription is unclear. In particular, it has been uncertain whether the Rlk kinase targets any of the newly identified lymphoid adaptors in T-cells. In this study, we report that Rlk phosphorylates SLP-76 at its N-terminal YESP/YEPP sites, a region that previously was found to act as a site for ZAP-70, and further that Rlk and SLP-76 potently and cooperatively up-regulate TcR-driven IL-2 transcription in T-cells. Loss of the N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated the cooperative effect. These observations support a model where the TcR can utilize the two kinases Rlk and ZAP-70 in the phosphorylation of SLP-76 leading to the up-regulation of IL-2 production in T-cells.RESULTST-cell activation is accompanied by an increase in phosphorylation of the lymphoid adaptor protein SLP-76 (28.Jackman 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, 31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 34.Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 35.Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Recent studies have shown that SLP-76−/− and Rlk−/− × ITK−/− T-cells show similar defects in Ca2+ mobilization and inositol 1,4,5-trisphosphate and IL-2 production (29.Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (353) Google Scholar). To assess the role of Rlk in TcRζ/CD3 signaling and in particular whether the kinase might target SLP-76, Rlk was transfected into Jurkat T-cells and assessed for an effect on tyrosine phosphorylation of SLP-76. As shown in Fig.1 A (top left panel), anti-CD3 stimulation induced an increase in SLP-76 phosphorylation (lane 2 versus lane 1), whereas overexpression of Rlk caused a further increase (lane 6). Significantly, the increase was comparable with that observed with the overexpression of ZAP-70 (lane 4), a kinase previously shown to phosphorylate SLP-76 (31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Further, the levels of ZAP-70 and Rlk expression were roughly equivalent in the transfectants (lower panels). The anti-Rlk antiserum is specific for the transfected murine Rlk protein without cross-reaction with the endogenous human kinase (Txk). As a further control, similar levels of SLP-76 were found expressed in lysates from the different cells (right upper panel). These data indicate that Rlk can cooperate with the TcR complex in the generation of signals leading to SLP-76 phosphorylation.A previous study using SLP-76-deficient cells have demonstrated a requirement for SLP-76 in the phosphorylation of phospholipase Cγ1 (29.Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (353) Google Scholar). In this context, it was interesting that the overexpression of Rlk augmented the level of TcR-induced PLCγ1 phosphorylation (Fig.1 A, middle panel, lane 6). Taken together, these observations are consistent with a role for Rlk in events leading to SLP-76 phosphorylation and its connection with PLCγ1. No effect was observed on TcRζ phosphorylation or the recruitment of ZAP-70 (data not shown). Rlk-induced phosphorylation of SLP-76 could be either direct or indirect. We and others have previously shown that SLP-76 acts as a substrate for the tyrosine kinase ZAP-70 (30.Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). To assess further whether SLP-76 acts as a direct substrate for Rlk, the kinase and SLP-76 were co-expressed in COS cells and analyzed for changes in tyrosine phosphorylation. Transfection of heterologous cells such as COS cells allows for an analysis of lymphoid kinases and substrates in the absence of other lymphoid proteins. As observed in T-cells, the expression of Rlk resulted in the marked phosphorylation of SLP-76 (Fig. 1 B,top panel, lane 2 versus lane 1). By contrast, as a control, kinase inactive Rlk (dRlk) failed to induce phosphorylation (lane 3). Significantly, Rlk phosphorylated SLP-76 at levels that were roughly comparable with that mediated by ZAP-70 (lane 4). Similar amounts of SLP-76 were immunoprecipitated from the different transfectants as shown in an anti-SLP-76 immunoblot (middle panel). Immunoblotting with anti-Rlk antiserum was used as a control for the expression of Rlk (lower panel).SLP-76 possesses three key tyrosines (YESP, YESP, and YEPP) in an acidic N-terminal region that are important for the ability of the adaptor to augment IL-2 transcription in T-cells (32.Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar). ZAP-70 and SYK kinases phosphorylate SLP-76 at these tyrosines (30.Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 36.Wardenburg J.B. Pappu R. Bu J.-Y. Mayer B. Chernoff J. Straus D. Chan A.C. Immunity. 1998; 9: 607-616Abstract Full Text Full Text PDF PubMed Google Scholar). To assess the specificity of Rlk phosphorylation and in particular whether Rlk targeted the same region as ZAP-70, Rlk was co-expressed in T-cells (Fig. 2 A, top panel) and in COS cells (Fig. 2B, top panel) with a mutant form of SLP-76 in which the three key tyrosines have been mutated to phenylalanines. Significantly, the mutation of the key tyrosines resulted in a complete loss of Rlk-mediated phosphorylation in T-cells (Fig. 2 A, top panel, lane 6). An anti-SLP-76 immunoblot showed the same amounts of SLP-76 in the different transfectants (middle panel). Anti-CD3-mediated phosphorylation of SLP-76 in cells transfected with mutant SLP-76Y3F3 alone was also abolished (lane 4 versus lane 2). In COS cells, a 50–60% loss in SLP-76 phosphorylation was observed when the mutant was co-transfected with Rlk (Fig. 2 B, upper panel, lane 4 versus lane 3). As a control, phosphorylation of SLP-76 by ZAP-70 was no longer detectable with the loss of the tyrosines (lane 2 versus lane 1). The same amounts of SLP-76 were immunoprecipitated from the different COS cell transfectants as shown in an anti-SLP-76 immunoblot (middle panel). Immunoblotting with anti-Rlk antiserum was used as a control for the expression of Rlk (lower panel). These data indicate that Rlk and ZAP-70 share an ability to phosphorylate SLP-76 within its acidic N-terminal region, a region that is crucial to the function of the SLP-76 adaptor. The residual phosphorylation of SLP-76Y3F3 in COS cells could be due to the phosphorylation of tyrosines other than the YESP/YEPP sites. In T-cells, overexpressed Rlk mainly phosphorylated the three key tyrosines. The affinity for other tyrosines might be very low, which resulted in no detectable phosphorylation of transfected SLP-76Y3F3.Figure 2Rlk directly phosphorylates the N-terminal region of SLP-76 in COS and T-cells. A, overexpression of mutant SLP-76Y3F3 and Rlk in Jurkat T-cells. Top panel, Jurkat T-cells, transfected with HA-tagged wild-type SLP-76 (lanes 1 and 2), HA-tagged mutant SLP-76Y3F3 (lanes 3 and 4) or together with HA-tagged SLP-76Y3F3 and Rlk (lanes 5 and 6) were either left unstimulated (RαM) (lanes 1, 3, and5) or stimulated with OKT3 and RαM antibodies (lanes 2, 4, and 6), lysed, immunoprecipitated with anti-HA mAb, and subjected to immunoblotting with anti-pTyr mAb.Middle panel, the same blot was reprobed with anti-HA mAb.Bottom panel, Rlk expression level of transfected Jurkat cells as shown by anti-Rlk immunoblotting. B, overexpression of mutant SLP-76Y3F3 and Rlk in COS cells. Top panel, COS cells were transfected together with wild-type SLP-76 and ZAP-70 (lane 1), mutant SLP-76Y3F3 plus ZAP-70 (lane 2), SLP-76 plus Rlk (lane 3), and SLP-76Y3F3 plus Rlk (lane 4), immunoprecipitated with anti-SLP-76 mAb and assessed for SLP-76 phosphorylation by immunoblotting with an anti-pTyr mAb. Middle panel, the same blot was reprobed with anti-SLP-76 mAb. Bottom panel, Rlk expression level of transfected murine Rlk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because SLP-76 expression is needed for TcR-mediated activation of ERKs (29.Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (353) Google Scholar), the next question was whether the TcRζ/CD3 complex employed Rlk-SLP-76 in the regulation of ERK activity. For this purpose, the kinase and substrate were individually and jointly transfected into Jurkat T-cells, stimulated for 15 min with CD3 mAb, and subjected to immunoprecipitation with ERK1/2 antibodies. Myelin basic protein was then used as a substrate in an in vitro kinase assay for the assessment of ERK activity. As shown in Fig.3, anti-CD3 stimulation induced an increase in ERK activity in vector transfected cells (lane 2 versus lane 1) that was further augmented by expression of SLP-76 (lane 4) or Rlk (lane 6). The combined expression of SLP-76 and Rlk led to a further increase in ERK activity in response to TcRζ/CD3 ligation (lane 8). Similar amounts of cell lysates were subjected to the in vitro kinase assay as shown in an anti-ERK1/2 immunoblot (lower panel). These data therefore demonstrate that both Rlk and SLP-76 operate in response to TcRζ/CD3 ligation to up-regulate ERK activity.Figure 3Co-expression of SLP-76 and Rlk leads to increased ERK activity. Upper panel, Jurkat T-cells transfected with vector (mock) alone (lanes 1 and2), SLP-76 (lanes 3 and 4), Rlk (lanes 5 and 6), or SLP-76 plus Rlk (lanes 7 and 8) were either left unstimulated (RαM) (lanes 1, 3, 5, and 7) or stimulated with OKT3 and RαM antibodies (lanes 2,4, 6, and 8) for 15 min, lysed, immunoprecipitated with anti-ERK1/2 mAbs, and subjected to an in vitro kinase assay. Lower panel, similar amounts of cell lysates were subjected to the in vitro kinase assay as shown in an anti-ERK1/2 immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given these stimulatory effects, a key question was whether the TcR complex also uses Rlk and its phosphorylation of SLP-76 in the regulation of IL-2 transcription. As seen in Fig.4, Rlk or SLP-76 itself led to a slight increase in anti-CD3 induced NFAT/AP-1 transcriptional activity when compared with cells transfected with vector alone. The effect of SLP-76 on NFAT/AP-1 transcription is comparable with that reported by others (32.Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar). Interestingly, the combined expression of Rlk and SLP-76 resulted in a marked synergy in the potentiation of IL-2 transcription. In fact, the level of transcription was some 100-fold greater than in cells that were individually transfected with Rlk or SLP-76 (Fig. 4 A). Levels of SLP-76 and Rlk expression were monitored by anti-SLP-76 and anti-Rlk immunoblotting, respectively (right panels). Significantly, the importance of Rlk-mediated phosphorylation was evident with the attenuation of SLP-76/Rlk cooperativity with the kinase inactive Rlk (dRlk) (Fig. 4 A). These data indicated that Rlk kinase activity was needed for the potentiation of IL-2 gene activation. To further connect this phosphorylation with the N-terminal region of SLP-76, the mutant form of SLP-76 (lacking the key tyrosine residues) was also analyzed. In this case, it was also defective in supporting increased IL-2 transcription (Fig. 4 B). Levels of SLP-76 and Rlk expression were monitored by immunoblotting with anti-HA and anti-Rlk antibodies, respectively (right panels). Taken together, these results demonstrate that Rlk-mediated phosphorylation of SLP-76 cooperatively up-regulates TcRζ/CD3-induced IL-2 transcription in T-cells and that the kinase activity of Rlk and its phosphorylation site on SLP-76 are needed for this potentiation.Figure 4Rlk and SLP-76 cooperatively up-regulate TcRζ/CD3-induced IL-2 production. A, Jurkat T-cells were co-transfected with 3X NFAT/AP-1-LUC reporter plasmid and vector (mock), HA-tagged SLP-76, Rlk, kinase inactive Rlk (dRlk), HA-tagged SLP-76 plus Rlk, or HA-tagged SLP-76 plus dRlk. Cells were either left unstimulated (RαM) (black bars) or stimulated with OKT3 and RαM mAbs (hatched bars) and assayed for luciferase activity (left panel). Expression levels of SLP-76 and Rlk are shown in an anti-SLP-76 and anti-Rlk immunoblot. B, Jurkat T-cells were co-transfected with 3X NFAT/AP-1 and vector, HA-tagged wild-type SLP-76 plus Rlk, or HA-tagged mutant SLP-76Y3F3 plus Rlk. Cells were either left unstimulated (RαM) (black bars) or stimulated with OKT3 and RαM mAbs (hatched bars) and assayed for luciferase activity (left panel). Expression levels of SLP-76 and Rlk are shown in an anti-HA and anti-Rlk immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONPrevious studies have demonstrated the importance of SLP-76 and the Tec kinases Rlk and ITK in the control of T-cell proliferation (25.Schaeffer E.M. Debnath J. Yap G. McVicar D. Liao C. Littman D. Sher A. Varmus H. Lenardo M. Schwartzberg P.L. Science. 1999; 284: 638-641Crossref PubMed Scopus (328) Google Scholar,29.Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (353) Google Scholar, 37.Liao X.C. Fournier S. Killeen N. Weiss A. Allison J.P. Littman D.R. J. Exp. Med. 1997; 186: 221-22838Crossref PubMed Scopus (45) Google Scholar). However, despite the importance of SLP-76, the identity of kinases that connect the TcR complex to the adaptor and the subsequent up-regulation of ERK activity and IL-2 transcription has been unclear. In this study, we provide the first evidence of a link between Rlk and the SLP-76 adaptor. We found that Rlk phosphorylates SLP-76 at its N-terminal YESP/YEPP sites, a region that previously was found to act as a site for ZAP-70 phosphorylation (31.Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 1-11Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These N-terminal residues are required for the ability of SLP-76 to up-regulate IL-2 transcription (32.Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar). Further, Rlk and SLP-76 potently and cooperatively up-regulate TcR-driven IL-2 transcription in T-cells. This synergy depended on Rlk phosphorylation of SLP-76 tyrosine sites. Loss of the N-terminal YESP/YEPP sites of SLP-76 attenuated the cooperative effect, as did the loss of Rlk kinase activity. These observations support a model where the TcR can utilize the two kinases Rlk and ZAP-70 in the phosphorylation of SLP-76 leading to the up-regulation of IL-2 production in T-cells (Fig. 5). Whether these two kinases operate in synergy at some other juncture in the cascade (i.e. PLCγ1 phosphorylation) awaits further studies.Figure 5Model of Rlk targeting of SLP-76 in TcR signaling. TcR complex can use Rlk and its ability to phosphorylate SLP-76 to up-regulate IL-2 transcription. As with ZAP-70, Rlk can phosphorylate SLP-76 in its acidic N-terminal region, the region needed for the SLP-76 potentiated IL-2 transcription. Both TcR-driven ERK activation and NFAT transcription were increased by co-expression of SLP-76 and Rlk in T-cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to placing Rlk in a TcR-mediated signaling network, our data emphasize the central role of SLP-76 as an integrator of signals from both the ZAP-70 and Rlk kinases. It is also consistent with the observation that T-cells from Rlk−/− × ITK−/− and SLP-76−/− mice show a similar phenotype with defects in Ca2+ mobilization and inositol 1,4,5-trisphosphate and IL-2 production (25.Schaeffer E.M. Debnath J. Yap G. McVicar D. Liao C. Littman D. Sher A. Varmus H. Lenardo M. Schwartzberg P.L. Science. 1999; 284: 638-641Crossref PubMed Scopus (328) Google Scholar, 29.Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 218: 413-416Crossref Scopus (353) Google Scholar). The ability of co-transfected SLP-76 and Rlk to up-regulate ERK activity is also in agreement with the delayed onset of ERK activation in T-cells from Rlk−/− × ITK−/− mice (25.Schaeffer E.M. Debnath J. Yap G. McVicar D. Liao C. Littman D. Sher A. Varmus H. Lenardo M. Schwartzberg P.L. Science. 1999; 284: 638-641Crossref PubMed Scopus (328) Google Scholar). In addition, the knock-out mice showed alterations in Ca2+ mobilization, which was also observed in our transfection studies (data not shown). Our observation that Rlk transfection led to increased PLCγ1 phosphorylation suggests that Rlk could be one of the bridging protein tyrosine kinases between SLP-76 and PLCγ1. Taken together, our results provide an alternate pathway from the TcR that phosphorylates SLP-76 and potentiates PLCγ1 phosphorylation.The overlapping roles of Rlk and ZAP-70 in the phosphorylation of SLP-76 do not exclude additional functions of ZAP-70 and SLP-76 unconnected with Rlk. This is suggested by the difference in the phenotypes of the Rlk−/− × ITK−/− and ZAP-70−/− mice, where ZAP-70−/− mice show a block in thymic development that is not evident in RLK−/− × ITK−/− mice (38.Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. Parslow T.G. Weiss A. Science. 1994; 264: 1599-1601Crossref PubMed Scopus (428) Google Scholar). This suggests that ZAP-70 mediates additional functions other than SLP-76 phosphorylation that are essential for thymic development. The phosphorylation of the adaptor LAT is one possibility, an adaptor needed for differentiation (39.Zhang W. Sommers C.L. Burshtyn D.N. Stebbins C.C. DeJarnette J.B. Trible R.P. Grinberg A. Tsay H.C. Jacobs H.M. Kessler C.M. Long E.O. Love P.E. Samelson L.E. Immunity. 1999; 10: 323-332Abstract Ful" @default.
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W2008756544 title "Resting Lymphocyte Kinase (Rlk/Txk) Targets Lymphoid Adaptor SLP-76 in the Cooperative Activation of Interleukin-2 Transcription in T-cells" @default.
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