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• W2077290438 abstract "MIST (also termed Clnk) is an adaptor protein structurally related to SLP-76 and BLNK/BASH/SLP-65 hematopoietic cell-specific adaptor proteins. By using the BLNK-deficient DT40 chicken B cell system, we demonstrated MIST functions through distinct intramolecular domains in immunoreceptor signaling depending on the availability of linker for activation of T cells (LAT). MIST can partially restore the B cell antigen receptor (BCR) signaling in the BLNK-deficient cells, which requires phosphorylation of the two N-terminal tyrosine residues. Co-expression of LAT with MIST fully restored the BCR signaling and dispenses with the requirement of the two tyrosines in MIST for BCR signaling. However, some other tyrosine(s), as well as the Src homology (SH) 2 domain and the two proline-rich regions in MIST, is still required for full reconstitution of the BCR signaling, in cooperation with LAT. The C-terminal proline-rich region of MIST is dispensable for the LAT-aided full restoration of MAP kinase activation, although it is responsible for the interaction with LAT and for the localization in glycolipid-enriched microdomains. On the other hand, the N-terminal proline-rich region, which is a binding site of the SH3 domain of phospholipase Cγ, is essential for BCR signaling. These results revealed a marked plasticity of MIST function as an adaptor in the cell contexts with or without LAT. MIST (also termed Clnk) is an adaptor protein structurally related to SLP-76 and BLNK/BASH/SLP-65 hematopoietic cell-specific adaptor proteins. By using the BLNK-deficient DT40 chicken B cell system, we demonstrated MIST functions through distinct intramolecular domains in immunoreceptor signaling depending on the availability of linker for activation of T cells (LAT). MIST can partially restore the B cell antigen receptor (BCR) signaling in the BLNK-deficient cells, which requires phosphorylation of the two N-terminal tyrosine residues. Co-expression of LAT with MIST fully restored the BCR signaling and dispenses with the requirement of the two tyrosines in MIST for BCR signaling. However, some other tyrosine(s), as well as the Src homology (SH) 2 domain and the two proline-rich regions in MIST, is still required for full reconstitution of the BCR signaling, in cooperation with LAT. The C-terminal proline-rich region of MIST is dispensable for the LAT-aided full restoration of MAP kinase activation, although it is responsible for the interaction with LAT and for the localization in glycolipid-enriched microdomains. On the other hand, the N-terminal proline-rich region, which is a binding site of the SH3 domain of phospholipase Cγ, is essential for BCR signaling. These results revealed a marked plasticity of MIST function as an adaptor in the cell contexts with or without LAT. T cell antigen receptor B cell antigen receptor phospholipase C extracellular signal-regulated kinase Fc receptor type I for IgE protein-tyrosine kinase glutathioneS-transferase antibody c-Jun N-terminal kinase proline-rich glycolipid-enriched microdomain tyrosine to phenylalanine mutation Src homology deletion mutant of proline-rich regions Adaptor/linker proteins that lack enzymatic activities exert their function as a scaffold molecule to generate active signaling complexes, which are essential for transducing receptor signals to downstream effectors (1Clements J.L. Boerth N.J. Lee J.R. Koretzky G.A. Annu. Rev. Immunol. 1999; 17: 89-108Crossref PubMed Scopus (173) Google Scholar, 2Kurosaki T. Annu. Rev. Immunol. 1999; 17: 555-592Crossref PubMed Scopus (365) Google Scholar). Among these adaptor/linker proteins are SLP-76 and its close relative BLNK (also known as BASH or SLP-65), which are pivotal for T and B cell development and for T cell antigen receptor (TCR)1 and B cell antigen receptor (BCR) signaling, respectively. Common structural features of these proteins are the presence of N-terminal tyrosine phosphorylation sites, central proline-rich (PR) regions, and a C-terminal SH2 domain (3Jackman 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, 4Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 5Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar, 6Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielsen P.J. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (231) Google Scholar). Upon TCR and BCR cross-linking, SLP-76 and BLNK are phosphorylated by ZAP70 and Syk protein-tyrosine kinases (PTKs), respectively, and then associate with signaling proteins, including phospholipase Cγ (PLCγ), Vav, and Grb2 (3Jackman 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, 4Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 7Wardenburg 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 (268) Google Scholar, 8Tuosto L. Michel F. Acuto O. J. Exp. Med. 1996; 184: 1161-1166Crossref PubMed Scopus (173) Google Scholar, 9Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In a SLP-76-deficient Jurkat T cell line, elevation of intracellular Ca2+ concentration ([Ca2+]i) and the activation of the Ras pathway following TCR cross-linking are severely impaired (10Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (353) Google Scholar). Moreover, a profound block in thymocyte development in SLP-76-deficient mice indicates an essential role for SLP-76 in pre-TCR signaling (11Clements J.L. Yang B. Ross-Barta S.E. Eliason S.L. Hrstka R.F. Williamson R.A. Koretzky G.A. Science. 1998; 281: 416-419Crossref PubMed Scopus (359) Google Scholar, 12Pivniouk V. Tsitsikov E. Swinton P. Rathbun G. Alt F.W. Geha R.S. Cell. 1998; 94: 229-238Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Similarly, a BLNK-deficient DT40 cell line, a chicken B lymphoma, displayed severe defects in tyrosine phosphorylation of PLCγ, [Ca2+]i increase, and c-Jun N-terminal kinase (JNK) and p38 MAP kinase activation following BCR stimulation (13Ishiai 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 (288) Google Scholar). Extracellular signal-regulated kinase (ERK) 2 activation was also moderately affected by the absence of PLCγ activation in BLNK-deficient DT40 cells. In addition, we and others have reported abnormal B cell differentiation and functions in BLNK-deficient mice (14Hayashi K. Nittono R. Okamoto N. Tsuji S. Hara Y. Goitsuka R. Kitamura D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2755-2760Crossref PubMed Scopus (124) Google Scholar, 15Jumaa H. Wollscheid B. Mitterer M. Wienands J. Reth M. Nielsen P.J. Immunity. 1999; 11: 547-554Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 16Pappu R. Cheng A.M. Li B. Gong Q. Chiu C. Griffin N. White M. Sleckman B.P. Chan A.C. Science. 1999; 286: 1949-1954Crossref PubMed Scopus (249) Google Scholar, 17Xu S. Tan J.E. Wong E.P. Manickam A. Ponniah S. Lam K.P. Int. Immunol. 2000; 12: 397-404Crossref PubMed Scopus (127) Google Scholar). The third member of SLP-76/BLNK adaptor family was recently isolated and called either MIST (for mast cell immunoreceptor signal transducer) (18Goitsuka R. Kanazashi H. Sasanuma H. Fujimura Y. Hidaka Y. Tatsuno A. Ra C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: 573-580Crossref PubMed Scopus (40) Google Scholar) or Clnk (for cytokine-dependent hemopoietic cell linker) (19Cao M.Y. Davidson D., Yu, J. Latour S. Veillette A. J. Exp. Med. 1999; 190: 1527-1534Crossref PubMed Scopus (52) Google Scholar). MIST/Clnk is constitutively expressed in several mast cell lines, and its expression was induced by cytokines in T cells and a variety of cytokine-dependent cell lines of myeloid and lymphoid origins (18Goitsuka R. Kanazashi H. Sasanuma H. Fujimura Y. Hidaka Y. Tatsuno A. Ra C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: 573-580Crossref PubMed Scopus (40) Google Scholar, 19Cao M.Y. Davidson D., Yu, J. Latour S. Veillette A. J. Exp. Med. 1999; 190: 1527-1534Crossref PubMed Scopus (52) Google Scholar). MIST shares structural features with SLP-76 and BLNK and is tyrosine-phosphorylated upon either the high affinity IgE receptor (FcεRI) or TCR stimulation. MIST can associate with PLCγ, Vav, Grb2, and LAT either constitutively or inducibly. Overexpression of a phosphorylation-deficient form of MIST in the rat mast cell line RBL-2H3 inhibited FcεRI-mediated mast cell degranulation, [Ca2+]i increase, and phosphorylation of LAT (18Goitsuka R. Kanazashi H. Sasanuma H. Fujimura Y. Hidaka Y. Tatsuno A. Ra C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: 573-580Crossref PubMed Scopus (40) Google Scholar). Furthermore, the transient expression of Clnk in Jurkat T cells augmented TCR-induced NF-AT activation (19Cao M.Y. Davidson D., Yu, J. Latour S. Veillette A. J. Exp. Med. 1999; 190: 1527-1534Crossref PubMed Scopus (52) Google Scholar). These findings indicate that, similarly to SLP-76 and BLNK, MIST/Clnk functions as a signaling molecule downstream of the FcεRI in mast cells and the TCR in T cells. To clarify domains/motifs required for MIST function in immunoreceptor signal transduction, we utilized BLNK-deficient DT40 cells as a signal reconstitution system. This B cell line expresses none of the SLP-76 family members and thus can be the simplest system currently available to test the function of various mutant forms of MIST. By this reconstitution system, we studied a structure-function relationship of MIST in linking immunoreceptor signal to the downstream biochemical events. Wild-type and mutant DT40 cells deficient for Lyn, Syk, Btk, or BLNK (13Ishiai 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 (288) Google Scholar, 20Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar, 21Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (423) Google Scholar) were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 1% chicken serum, 50 μm 2-mercaptoethanol, 2 mml-glutamine, and antibiotics. COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. Glutathione S-transferase (GST) protein fused to the C-terminal SH2 domain or the SH3 domain of PLCγ1 was purchased from Santa Cruz Biotechnology. The monoclonal Ab against chicken IgM (M4) was provided from Dr. C-L. H. Chen (University of Alabama at Birmingham). The rabbit Abs against chicken PLCγ2 and mouse MIST were described previously (18Goitsuka R. Kanazashi H. Sasanuma H. Fujimura Y. Hidaka Y. Tatsuno A. Ra C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: 573-580Crossref PubMed Scopus (40) Google Scholar, 22Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (182) Google Scholar). The following antibodies were purchased: anti-ERK2 and anti-Grb2 Abs from Santa Cruz Biotechnology, anti-phosphotyrosine PY20 Ab conjugated with horseradish peroxidase from Transduction Laboratories, anti-LAT Ab from Upstate Biotechnology, anti-JNK Ab from PharMingen, and anti-T7 Ab from Novagen. The mutant mouse MIST containing tyrosine to phenylalanine substitutions (YF) or arginine to lysine substitution at the SH2 domain (R335K) were generated using a QuickChange site-directed mutagenesis kit (Stratagene). The MIST mutants lacking PR regions (160PLPPPR165 or/and178PPAPP182) were also generated by PCR-mediated mutagenesis as described previously (23Imai Y. Matsushima Y. Sugimura T. Terda M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (314) Google Scholar). Their sequences were verified by automated sequence analysis. Wild-type and the mutant MIST cDNAs were subcloned into pApuro2 and pCAT7neo expression vectors (5Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar, 21Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (423) Google Scholar). Human LAT cDNA was also inserted in pAzeo expression vector (24Ishiai M. Kurosaki M. Inabe K. Chan A.C. Sugamura K. Kurosaki T. J. Exp. Med. 2000; 192: 847-856Crossref PubMed Scopus (68) Google Scholar). Human Grb2 expression vector was provided by Dr. M. Tanaka (Hamamatsu Medical College). COS-7 cells were transfected with plasmid vectors by using a TransIT-LT1 reagent (Pan Vera) as described previously (5Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar). The MIST or LAT expression vectors were transfected into wild-type and mutant DT40 cell lines by electroporation, and selected in the presence of 0.5 μg/ml puromycin (Sigma) for pApuro-based constructs or 0.4 mg/ml Zeocin (Invitrogen) for pAzeo-LAT. The BLNK-deficient DT40 cell clone stably expressing human LAT (2H12) was further transfected with expression vectors containing various forms of MIST. Cell surface expression of BCR on each transfectants was analyzed by FACSsort (Becton Dickinson) using fluorescein isothiocyanate-conjugated anti-chicken IgM Ab (Bethyl Laboratories), and transfectants expressing similar levels of MIST/LAT proteins as well as surface IgM were used for the analysis. Cells were lysed with 1% Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, and precipitated with indicated Abs or GST fusion proteins bound to glutathione-Sepharose (Amersham Pharmacia Biotech). The precipitates and aliquots of total cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were immunoblotted with Abs described above, and the secondary Ab was conjugated with horseradish peroxidase and then developed with the ECL system (Amersham Pharmacia Biotech). DT40 cell clones (5 × 106 cells) were loaded with 5 μm Fura-2/AM (Molecular Probes) at 37 °C for 30 min, washed twice with RPMI 1640 medium containing 0.1% bovine serum albumin and adjusted to 106 cells/ml. Cells were then stimulated with 10 μg/ml of anti-IgM Ab at 37 °C. [Ca2+]i was monitored at a 510 nm emission wavelength excited by 340 nm and 360 nm using a fluorescence spectrophotometer (F-2000, Hitachi). The assay conditions were described previously (25Hashimoto A. Okada H. Jiang A. Kurosaki M. Greenberg S. Clark E.A. Kurosaki T. J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar). In brief, lysates from 5 × 106 cells were immunoprecipitated with 1 μg of anti-ERK2 Ab or anti-JNK Ab and protein-G Sepharose. Half of the immunoprecipitates was suspended in 30 μl of kinase assay buffer containing [γ-32P]ATP, 5 μm cold ATP, and 5 μg of GST-Elk or GST-Jun fusion proteins as a substrate. After 20 min of incubation at 30 °C, the reaction was terminated by the addition of SDS sample buffer, followed by boiling for 5 min. The samples were separated on SDS-polyacrylamide gel electrophoresis gels, and then the gels were dried and subjected to autoradiography. The other half of the immunoprecipitates were used for Western blot analysis to quantify immunoprecipitated proteins. BLNK-deficient DT40 cells were transfected with 10 μg of a luciferase reporter plasmid driven by NF-AT and AP-1 response element from the mouse interleukin 2 gene promoter (a gift from Dr. K. Arai, Institute of Medical Science, University of Tokyo), together with 15 μg of either empty vector or an expression vector harboring various forms of MIST, BLNK, or LAT, in serum-free RPMI at a density of 107 cells/400 μl with a Gene Pulser (Bio-Rad Laboratories) set at 250 V and 975 μF. After electroporation, the cells were transferred to complete RPMI and incubated at 40 °C for 24 h. Triplicates of 5 × 105 viable cells were then left unstimulated or stimulated with anti-IgM Ab for 6 h and subsequently assayed for luciferase activity, as described previously (5Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar). Light emission was measured in a Lumat LB9501 luminometer (Berthold). GEM fractions were prepared as described previously (24Ishiai M. Kurosaki M. Inabe K. Chan A.C. Sugamura K. Kurosaki T. J. Exp. Med. 2000; 192: 847-856Crossref PubMed Scopus (68) Google Scholar). In brief, cells (2.5 × 108) were either unstimulated or stimulated with anti-IgM Ab for 2 min and then lysed in 1 ml of 0.5% Triton X lysis buffer. Cell lysates were mixed with 1 ml of 80% sucrose in lysis buffer, overlaid with 6.5 ml of 30% sucrose and 2.5 ml of 5% sucrose in lysis buffer, and then subjected to ultracentrifugation at 35,000 rpm for 16 h at 4 °C. Eleven aliquots (1 ml each) were collected from the top of the gradient fraction and subjected to Western blot analysis. Mouse MIST contains eight tyrosine residues potentially phosphorylated by PTKs, two PR regions, and a C-terminal SH2 domain (Fig.1 A). Of these tyrosine residues, five are conserved with those in human homologue (18Goitsuka R. Kanazashi H. Sasanuma H. Fujimura Y. Hidaka Y. Tatsuno A. Ra C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: 573-580Crossref PubMed Scopus (40) Google Scholar), and two of them are located in the sequence context (DY69EDP and EY96ADT) similar to those in mouse SLP-76 (DY113ESP, DY128ESP, and DY145EPP) and BLNK (DY72ENP and EY119VDN). These tyrosine residues in SLP-76 are essential for TCR-induced tyrosine phosphorylation of whole molecule and for its function in TCR-induced NF-AT transcriptional activation (26Fang N. Motto D.G. Ross S.E. Koretzky G.A. J. Immunol. 1996; 157: 3769-3773PubMed Google Scholar). We first determined PTKs responsible for MIST phosphorylation in the context of BCR signaling by introducing MIST into wild-type and PTK-deficient DT40 cell lines. A low level of tyrosine phosphorylation of MIST was detected before stimulation, and it was markedly increased within 1 min after BCR stimulation in wild-type DT40 cells (Fig. 1 B). By contrast, the level of BCR-induced tyrosine phosphorylation of MIST was profoundly decreased in Lyn-deficient DT40 cells, modestly decreased in Syk-deficient DT40 cells, and not decreased at all in Btk-deficient DT40 cells (Fig. 1 A). This result indicates that both Lyn and Syk, but not Btk, mainly mediate tyrosine phosphorylation of MIST in the context of BCR signaling. In order to determine whether the two conserved tyrosine residues of MIST are the targets for BCR-associated PTKs, we generated mutant forms of MIST that contain either single (Y69F or Y96F) or double (YF2) tyrosine to phenylalanine substitutions at positions 69 and 96 and examined their tyrosine phosphorylation in wild-type DT40 cells. As shown in Fig. 1 C, Y69F and Y96F were poorly tyrosine-phosphorylated upon BCR stimulation, whereas YF2 was not, indicating that tyrosines 69 and 96 of MIST are the main targets of BCR-associated PTKs and required for the phosphorylation of MIST in the BCR-signaling context. To clarify the role of MIST as a component of the immunoreceptor signaling complex and to verify the significance of the tyrosine phosphorylation of MIST, we introduced wild-type and mutant forms of MIST into BLNK-deficient DT40 cells (13Ishiai 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 (288) Google Scholar). All transfectants expressed comparative levels of MIST protein (Fig.2 A) and surface IgM (data not shown). The level of BCR-mediated tyrosine phosphorylation of Y69F was markedly lower, whereas that of Y96F was slightly lower, compared with that of wild-type MIST (Fig. 2 A). Tyrosine phosphorylation of YF2 was undetectable. As reported previously, no [Ca2+]i increase was induced by BCR stimulation in parental BLNK-deficient cells. Remarkably, the introduction of wild-type MIST into BLNK-deficient DT40 cells partially restored the calcium response, whereas the response was not restored in BLNK-deficient cells expressing Y69F, Y96F, or YF2 (Fig.2 B). This result indicates that both Tyr-69 and Tyr-96 of MIST are required for the BCR-induced calcium response. Because PLCγ2 is required for BCR-induced [Ca2+]i increase in DT40 cells (22Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (182) Google Scholar) and the activation of PLCγ2 is accompanied with its tyrosine phosphorylation (27Nishibe S. Wahl M.I. Hernandez-Sotomayor S.M. Tonks N.K. Rhee S.G. Carpenter G. Science. 1990; 250: 1253-1256Crossref PubMed Scopus (499) Google Scholar), we next examined tyrosine phosphorylation of PLCγ2 upon BCR stimulation in BLNK-deficient DT40 cells expressing various forms of MIST. Tyrosine phosphorylation of PLCγ2 was restored in BLNK-deficient cells expressing wild-type MIST but not in transfectants expressing Y69F, Y96F, and YF2 mutants (Fig. 2 C). BLNK has been reported to interact with PLCγ2 and thus recruit PLCγ2 to the membrane proximal compartment, where PLCγ2 is phosphorylated by Syk (13Ishiai 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 (288) Google Scholar). To test whether MIST can play such a role, we assessed the interaction of MIST with PLCγ by an in vitro binding assay using a GST fusion protein containing the C-terminal SH2 domain of PLCγ1 (GST-PLCγ-SH2). COS-7 cells were transfected with plasmids encoding wild-type or tyrosine mutants of MIST, in combination with Lyn, and cell lysates were precipitated with GST-PLCγ-SH2 fusion protein. As shown in Fig. 2 D, GST-PLCγ-SH2 interacted with Lyn-phosphorylated wild-type MIST but not with unphosphorylated MIST. Of five single tyrosine mutants that were equivalently phosphorylated by Lyn, only Y96F mutant did not interact with GST-PLCγ-SH2. A MIST mutant (YF6) containing six tyrosine to phenylalanine substitutions at positions 69, 96, 101, 153, 174, and 188, which showed no detectable tyrosine phosphorylation by Lyn, also failed to associate with GST-PLCγ-SH2. These results indicate that MIST interacts with the SH2 domain of PLCγ via its phosphorylated tyrosine 96, which may lead to the recruitment of PLCγ to the site of PTKs for its activation and the subsequent [Ca2+]i increase. It remains to be examined, however, how tyrosine 69 is involved in the PLCγ activation. As Y69F mutation more severely affected BCR-induced tyrosine phosphorylation of MIST than Y96F, phosphotyrosine 69 may serve as a binding site for a PTK, which subsequently phosphorylates tyrosine 96. Alternatively, the PTK bound to the phosphotyrosine 69 may be responsible for the phosphorylation of PLCγ2. BCR-induced activation of ERK2 has been shown to require both PLCγ2 and Ras pathways in DT40 cells (25Hashimoto A. Okada H. Jiang A. Kurosaki M. Greenberg S. Clark E.A. Kurosaki T. J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar), and reduced ERK2 activity in BLNK-deficient DT40 cells has principally been ascribed to a defect of the PLCγ2-mediated pathway (13Ishiai 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 (288) Google Scholar). ERK activity after the BCR cross-linking was partially restored in BLNK-deficient DT40 cells expressing wild-type MIST. However Y69F, Y96F, and YF2 mutants failed to restore BCR-induced ERK2 activation in BLNK-deficient cells (Fig.2 E). These results indicate that MIST can restore the PLCγ2-mediated pathway of BCR signaling in BLNK-deficient cells by molecular interactions through tyrosines 69 and 96. It is of note that the restoration of the PLCγ2 pathway by MIST is partial in any biochemical events examined here. A possible explanation for the incomplete reconstitution of BCR signaling by MIST in the BLNK-deficient DT40 cells is that the B cells lack some signaling molecules required for full activity of MIST. One molecule that is known to be expressed in T cells and mast cells in which MIST is also expressed, but not in B cells, is LAT (28Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar). LAT is a membrane-anchored adaptor protein that localizes to specific plasma membrane compartments known as GEMs (29Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar) and has been demonstrated to be required for SLP-76-mediated NF-AT activation upon TCR stimulation (30Finco 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, 31Zhang W. Irvin B.J. Trible R.P. Abraham R.T. Samelson L.E. Int. Immunol. 1999; 11: 943-950Crossref PubMed Scopus (226) Google Scholar). Because LAT is one of the signaling proteins associated with MIST in FcεRI-signaling (18Goitsuka R. Kanazashi H. Sasanuma H. Fujimura Y. Hidaka Y. Tatsuno A. Ra C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: 573-580Crossref PubMed Scopus (40) Google Scholar), LAT seems to be required for MIST to fully exhibit its function in BCR signaling. To assess this possibility, we transiently expressed various forms of MIST in combination with LAT in BLNK-deficient cells and examined BCR-mediated NF-AT activation by luciferase-reporter assay. As shown in Fig. 3, transfection of wild-type MIST or LAT alone showed weak restoration of the BCR-induced NF-AT activation as compared with chicken BLNK. Combined expression of wild-type MIST and LAT resulted in a marked synergy in reconstituting BCR-induced NF-AT activation in BLNK-deficient cells. Surprisingly, Y69F, Y96F, and YF2 mutants, which were not functional without LAT (data not shown), restored BCR-mediated NF-AT activation when co-expressed with LAT, to a similar level that was achieved by wild-type MIST with LAT (Fig. 3). Similarly, MIST co-expressed with LAT fully reconstituted BCR-mediated calcium response in BLNK-deficient cells, independently of the two tyrosines 69 and 96 (see below). These findings indicate that LAT cooperatively functions with MIST and dispenses with the requirement of two tyrosines 69 and 96 of MIST to restore BCR-mediated [Ca2+]i increase and the following NF-AT activation in BLNK-deficient cells. To dissect the mechanism for the LAT-MIST cooperation in the immunoreceptor signaling, we generated BLNK-deficient DT40 cells stably expressing LAT together with various forms of MIST, including mutants with a deletion of PR regions (dPR1, dPR2, and dPR1/2), a mutant containing the nonfunctional SH2 domain (R335K), and the tyrosine mutants (YF2 and YF6) (Fig.4 A). Introduction of LAT alone into BLNK-deficient DT40 cells partially restored the calcium response to a level similar to that observed in transfectants expressing wild-type MIST alone (Figs. 2 B and4 B). Consistent with the restoration of BCR-induced NF-AT activation, BCR-induced [Ca2+]i increase in cells expressing both LAT and wild-type MIST was restored to an equivalent level to that observed in wild-type DT40 cells (Figs. 2 B and4 B). In the presence of LAT, YF2 restored the BCR-induced calcium response to a level comparable to that achieved by wild-type MIST. By contrast, YF6 and the SH2 domain mutant, as well as mutants lacking either of the two PR regions, rather weakly restored calcium response. The mutant lacking both PR regions failed to restore the response. We next assessed BCR-induced activation of MAP kinases, ERK2 and JNK, in the BLNK-deficient DT40 cells reconstituted with LAT and the mutant forms of MIST using in vitro kinase assay. Wild-type DT40 cells exhibited sustained ERK2 activation until 10 min, with a peak response at 3 min following BCR stimulation, whereas in BLNK-deficient cells expressing either wild-type MIST or LAT alone, ERK2 activation was transient and decreased at 10 min (Figs.2 E and 4 C). Co-expression of wild-type MIST and LAT restored both peak and sustained responses of BCR-induced ERK2 activation. The SH2 mutant, YF6, or dPR2 co-expressed with LAT showed the reconstitution activity comparable to the wild-type MIST. By contrast, dPR1 lacking the N-terminal PR region of MIST failed to augment ERK activation above the level that was achieved by LAT alone, suggesting that the N-terminal PR region of MIST is responsible for cooperation with LAT in BCR-induced ERK2 activation. As with the calcium response, the cells expressing dPR1/2 and LAT showed even weaker ERK2 response than the cells expressing LAT alone. The reason for this is currently unclear, but it is possible that the dPR1/2 lacking two PR regions behaves as dominant-negative mutant in BCR signaling through LAT. Maximal JNK activation was observed 10 min after BCR stimulation in wild-type DT40 cells, whereas no BCR-induced JNK activation was detected in the absence of BLNK, as previously reported (13Ishiai M. Kurosaki M. Pa" @default.
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• W2077290438 title "MIST Functions through Distinct Domains in Immunoreceptor Signaling in the Presence and Absence of LAT" @default.
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