1 Division of Molecular Biology, Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278, Japan
2 Inheritance and Variation Group, PRESTO, Japan Science and Technology Corp., 2669 Yamazaki, Noda, Chiba 278, Japan
3 Division of Molecular Biology, Allergy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
Correspondence to: R. Goitsuka, Division of Molecular Biology, Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278, Japan
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Abstract |
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Keywords: FcRI, RBL, signal transduction
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Introduction |
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Recently much attention has been focused on a critical role of the adaptor proteins in the immunoreceptor signaling by the identification of novel hematopoietic linker proteins, including SLP-76 (4) and BASH/SLP-65/BLNK (57). SLP-76 and BASH/SLP-65/BLNK are adaptor family members whose expression is restricted to hematopoietic lineages; SLP-76 is expressed in T cells, macrophages, NK cells and mast cells, while BASH/SLP-65/BLNK is expressed in B cells. Common structural features of this adaptor family are the presence of N-terminal tyrosine phosphorylation sites, a central proline-rich region and a C-terminal SH2 domain. Upon TCR and BCR activation, SLP-76 and BASH/SLP-65/BLNK are phosphorylated by Zap70 and Syk respectively, and then associate with a set of signaling proteins, including phospholipase C (PLC), Vav and Grb2 (510). Recent studies using genetically engineered cell lines and mice have demonstrated that SLP-76 and BASH/SLP-65/BLNK are essential components in intracellular signaling pathways downstream of TCR and BCR respectively. In a SLP-76-deficient Jurkat T cell line, increase in intracellular Ca2+ concentration ([Ca2+]i) and activation of the Ras pathway following TCR cross-linking were severely impaired (11). Moreover, a profound block in thymocyte development in SLP-76-deficient mice indicated an essential role of SLP-76 for pre-TCR signaling (12,13). On the other hand, a BLNK-deficient DT40 cell line displayed severe defects in tyrosine phosphorylation of PLC
, [Ca2+]i increase and c-Jun N-terminal kinase activation following BCR stimulation (14).
Given the close similarities of the signaling pathways transduced from FcRI and BCR/TCR, we speculated that BASH/SLP-76-related adaptor proteins may be involved in Fc
RI signaling in mast cells. By the homology search in a database and the following cDNA cloning, we have identified the third member of the BASH/SLP-76 family expressed in mast cells, termed MIST (mast cell immunoreceptor signal transducer), which has later been found to be identical to a recently reported cytokine-dependent hemopoietic cell linker, Clnk (15). Here we show that MIST/Clnk functions as a crucial adaptor molecule in the Fc
RI signaling pathway leading to mast cell degranulation.
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Methods |
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cDNA cloning and expression constructs
The mouse MIST cDNA was isolated from a PT18 cDNA library by 5'- and 3'-RACE (Marathon cDNA amplification kit; Clontech, Palo Alto, CA), using the sequence information from the unidentified expressed sequence tag (EST) cDNA clone (GenBank accession no. AA166259) that had been found to show a homology to BASH. The partial human MIST cDNA was also amplified by PCR from mRNA prepared from HCMC using oligonucleotide primers: 5'-GTGATGATGACTATGATGACCCTGAGCTTC-3' and 5'-GGGAAAATTCTTGTAGTGTTCGATGATGTC-3'. The coding region of mouse MIST cDNA was amplified by PCR, and then inserted between the EcoRI and SalI sites of the pCAT7neo expression vector (6). The MIST mutant (MIST-YF) containing phenylalanine for tyrosine substitutions at positions 69, 96, 101, 153, 174 and 188 was generated by sequential PCR-based mutagenesis using a Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and then subcloned into the pCAT7neo expression vector.
Antibodies
Rabbit anti-MIST antibody was generated by immunization with a fusion protein composed of amino acids 193435 of mouse MIST and glutathione-S-transferase (GST). Antisera were precleared with Sepharose beads coupled with GST alone and then purified with an affinity column coupled with GSTMIST fusion protein. Specificity of the affinity-purified antibody was confirmed by immunoblot analysis on cell lysates from COS7 cells transfected with mouse MIST cDNA. Other antibodies used were anti-PLC2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Vav (Upstate Biotechnology, Lake Placid, NY), anti-Grb2 (Santa Cruz Biotechnology), anti-linker for activation of T cells (LAT) (Upstate Biotechnology), anti-T7 antibody (Novagen, Madison, WI) and PY20 (Transduction Laboratories, Lexington, KY).
Northern blot, RT-PCR and immunohistochemistry
Total RNA (10 µg) was separated in a denaturing agarose gel, as described previously (6). After blotting, hybridization was performed with either mouse MIST, a full-length mouse BASH or mouse SLP-76 cDNA probes (provided from Dr H. Gu). A mouse ß-actin cDNA probe was also used to normalize the amount of RNA loaded in each lane. For RT-PCR, 2 µg total RNA was reverse-transcribed using an oligo(dT) primer and 1/20th of the resultant cDNA was subjected to PCR amplification. Oligonucleotide primers used were 5'-TATGACCAGCCAGGGCAATAAAAGGACAAC-3' and 5'-CTTACTCATGAAGTGCCTGGCTGGAGTAC-3' for mouse MIST, and primers described above for human MIST cDNA respectively. Normalization was done using primers specific for G3PDH mRNA. The PCR was carried out as follows: 30 cycles of 94°C, 30 s and 68°C, 2 min. The tyramid-based signal amplification method (NEN Life Science, Boston, MA) was used for immunohistochemical analysis. Frozen sections of skin were first incubated with the affinity-purified anti-MIST antibody, rinsed and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit IgG antibody, then with biotinylated tyramid. The reactions were developed by streptavidinHRP and 3-amino-9-ethylcarbazole. The serial sections were also stained with toluidine blue (pH 4.0) for detecting mast cells.
Immunoprecipitation and immunoblotting
RBL-2H3 cells were transfected by electroporation with either pCAT7neo-MIST-WT, MIST-YF or empty vector and selected in medium containing Geneticin (Gibco, Grand Island, NY) at 1 mg/ml. Stable expression of the transfected MIST was verified by Western blotting with anti-T7 antibody. These clones were incubated with 10 µg/ml of anti-DNP mouse IgE (Sigma, St Louis, MO) for 1 h on ice and then stimulated with 100 ng/ml of DNP-HSA at 37°C for the indicated period of time. Cells were lysed with 1% NP-40 lysis buffer containing protease and phosphatase inhibitors, and immunoprecipitated with indicated antibodies. The immunoprecipitates and aliquots of total cell lysates were resolved on SDSPAGE and transferred to PVDF membranes. The membranes were immunoblotted with antibodies described above and the secondary antibodies conjugated with HRP, and then developed with the ECL system (Amersham, Arlington Heights, IL). COS7 cells were co-transfected with 1 µg of pCAT7neo-MIST with the indicated combination of 1 µg of pME-Lyn, pME-Syk, or pEF-BOS-Btk plasmids (18,19) using a TransIT-LT1 transfection reagent (Pan Vera, Madison, WI). After incubation for 48 h, cell lysates were prepared and analyzed for tyrosine phosphorylation described above.
Degranulation assay
RBL-2H3 cells (2x105 cells in 0.4 ml, 24-well plates) stably transfected with a mock vector or with a vector encoding MIST-WT or MIST-YF were sensitized overnight with 1 µg/ml of DNP-specific IgE. Cells were subsequently washed twice with PBS and stimulated with various amounts of DNP-HSA at 37°C for 30 min. Degranulation was monitored by the release of ß-hexosaminidase, as previously described (16). Percent degranulation was calculated as activity in the culture medium/activity in the culture medium + activity in the cell lysate.
Measurement of [Ca2+]i concentration
RBL-2H3 cells (5x106) expressing MIST-WT and MIST-YF were loaded with 5 µM Fura-2/AM (Molecular Probes, Eugene, OR) in the presence of 10 µg/ml of DNP-specific IgE at 37°C for 30 min, washed twice with Tyrode buffer containing 0.1% BSA and then stimulated with 100 ng/ml of DNP-HSA or ionomycin (1 µM) at 37°C. [Ca2+]i was monitored at a 510 nm emission wavelength excited by 340 and 360 nm using a fluorescence spectrophotometer (F-4500; Hitachi, Tokyo, Japan) (16).
Luciferase assay
RBL-2H3 cell clones were transiently transfected with 10 µg of a luciferase reporter plasmid driven by seven tandem copies of the NF-AT response element from the mouse IL-2 gene promoter (a gift from Dr K. Arai) in serum-free RPMI at a density of 6x106 cells/400 µl per cuvette with a gene pulser (BioRad, Hercules, CA) set at 250 V and 975 µF. After electroporation, the cells were transferred to complete RPMI and incubated at 37°C for 24 h. Cells (2x106) were then sensitized overnight with 1 µg/ml of DNP-specific IgE and then either left unstimulated or stimulated with DNP-HSA (50 ng/ml) or phorbol myristate acetate (50 ng/ml) plus ionomycin (1 µM) for 6 h. Luciferase activity was monitored, as described previously (6).
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Results |
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MIST is a novel member of the BASH/SLP-76 adaptor protein family
The mouse MIST cDNA is predicted to encode a 435 amino acid protein, with a calculated mol. wt of 49 kDa (Fig. 2). From the N-terminus to the central region of the molecule, MIST protein contains eight tyrosine residues at amino acid positions 69, 96, 101, 153, 174, 188, 249 and 286, all of which display consensus motifs that could mediate the interaction with other proteins containing an SH2 domain (21,22). The C-terminal part of the protein contains an SH2 domain that is most homologous to that of mouse SLP-76 and BASH/SLP-65/BLNK (41 and 38% identity respectively). The central region of the protein is rich in prolines and contains one potential Grb2 SH3 domain-binding sequence (PXXPXR) (23). Comparison of a partial human MIST sequence with mouse MIST revealed that five tyrosine residues are conserved between species, two of which are also found in SLP-76 and BASH/SLP-65/BLNK. Human MIST has one additional PXXPXR SH3-binding motif between amino acid positions 246 and 251 (Fig. 2
). During the analysis of RT-PCR products of MIST mRNA, potential alternatively spliced variants of mouse and human MIST were also identified. The shorter form of mouse MIST lacks 24 amino acids (amino acid positions from 5 to 28) located proximal to N-terminus of the protein, while 37 amino acids (amino acid positions from 147 to 183) in the proline-rich region are missing in the shorter form of human MIST. MIST does not contain a stretch of acidic or basic amino acid residues in its N-terminus, but apart from that the overall structure of MIST is very similar to that of BASH/SLP-65/BLNK and SLP-76. We thus concluded that MIST is a third member of the BASH/SLP-76 adaptor family.
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MIST/Clnk functions as an adaptor protein in FcRI signaling
We next examined whether MIST/Clnk is a substrate of FcRI-associated PTK. For this purpose, we used a rat mast cell line, RBL-2H3, which has been extensively studied for Fc
RI-signaling. Expression vectors encoding a wild-type (MIST-WT) or a mutant (MIST-YF; see below) form of mouse MIST tagged with the T7 epitope at the N-terminus were transfected into RBL-2H3 cells. Clones stably expressing high levels of MIST proteins were established and the cloned cells were sensitized with anti-DNP-IgE, followed by challenge with DNP-HSA. MIST was tyrosine phosphorylated even before stimulation, and the level of phosphorylation rapidly increased as early as 10 s and reached a maximum at 30 s after the Fc
RI cross-linking (Fig. 3A
). In addition, several tyrosine phosphorylated proteins were co-precipitated with MIST, including a 55 kDa unidentified phosphoprotein (Fig. 3A
). By using a panel of antibodies against known PTK substrates, we identified the association of MIST with PLC
2 and Vav (Fig 3 B
). Grb2 was also found to be co-precipitated with MIST. By contrast, MIST-YF, which has phenylalanines substituted for tyrosines at positions 69, 96, 101, 153, 174 and 188, failed to be tyrosine phosphorylated after Fc
RI cross-linking. MIST-YF was also unable to associate with PLC
2 and Vav, but still retained the ability to associate with Grb2. The reciprocal experiment using anti-Vav, anti-PLC
2 and anti-Grb2 antibodies for immunoprecipitation confirmed these results (data not shown). These data suggest that MIST/Clnk functions as a crucial adaptor protein downstream of Fc
RI, in a manner similar to the function of BASH/SLP-65/BLNK and SLP-76 in BCR and TCR signalings.
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Overexpression of a mutant form of MIST/Clnk abolished FcRI-mediated mast cell degranulation
We next examined whether MIST/Clnk is functionally involved in FcRI-mediated degranulation using the RBL-2H3 cell stable transfectants. Although overexpression of wild-type MIST had no effect on Fc
RI-induced degranulation, overexpression of MIST-YF significantly suppressed this response (Fig. 4A
). Overexpression of either MIST-WT and MIST-YF did not affect degranulation response induced by ionomycin, which by-pass the receptor-proximal signal activation (data not shown). Since elevation in [Ca2+]i is believed to be required for the mast cell degranulation response, we examined [Ca2+]i increase, NF-AT transcriptional activation and phosphorylation of signaling molecules involved in the [Ca2+]i increase. Upon Fc
RI cross-linking, [Ca2+]i increase in RBL-2H3 clones expressing MIST-YF was diminished to almost half the level of that in clones expressing MIST-WT (Fig. 4B
). Furthermore, the Fc
RI-mediated NF-AT activation, which is believed to be dependent on both Ras and calcium pathways (24), was substantially reduced in a MIST-YF-expressing clone, as compared with a mock-transfected clone (Fig. 4C
). However, there was no significant difference in tyrosine phosphorylation of PLC
2, Vav (Fig. 4D
) and PLC
1 (data not shown). In the clones expressing MIST-YF, we observed reduced tyrosine phosphorylation of LAT, which is a critical linker molecule in TCR signaling (2527). LAT was found to associate with MIST-WT, but not with MIST-YF (Fig. 4D
). Although the mechanism by which the mutant form of MIST interferes with tyrosine phosphorylation of LAT remains unclear, it is likely that MIST is situated upstream of LAT in the Fc
RI signaling pathway and regulates the interaction of Syk with its potential substrate, LAT (26).
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Discussion |
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The observed calcium defect in MIST-YF-expressing RBL cell clones is most likely due to reduced activity of PLC, although the tyrosine phosphorylation of PLC
appears unaffected. Several lines of evidence support the notion that net tyrosine phosphorylation of PLC
may not correctly reflect the functional activation of PLC
(28,29). PLC
is primarily phosphorylated by Syk, but additional phosphorylation by Btk is believed to be required for full activation of PLC
(30), although no definitive evidence has been provided in the context of Fc
RI signaling in mast cells. For activation of Btk, membrane targeting of Btk through interaction of its PH domain and phosphatidylinositol-3,4,5-triphosphate generated by phosphatidylinositol-3-kinase appears to be necessary (31). Thus, one possible mechanism by which a mutant form of MIST suppresses Fc
RI-mediated [Ca2+]i increase could be an interference with the activation or function of either phosphatidylinositol-3-kinase or Btk, in which an endogenous MIST is involved. In this regard, tyrosine-phosphorylated BASH/SLP-65/BLNK, a B cell analogue of MIST/Clnk, was recently reported to bind to the SH2 domain of Btk and to facilitate Btk to target PLC
as a substrate (32), suggesting a possible interplay between MIST/Clnk, Btk and PLC
.
Alternatively, the reduced tyrosine phosphorylation of LAT caused by overexpression of a dominant negative MIST may imply that MIST/Clnk is involved in LAT-mediated assembly of signaling molecules including PLC at a detergent-resistant membrane raft, which is a specialized membrane domain serving as the center for receptor-mediated activation (33). The apparent discrepancy between impaired calcium elevation and preserved PLC
2 tyrosine phosphorylation in MIST-YF-expressing RBL-2H3 cells might be partly ascribed to the potential lack of PLC
2-LAT assembly, because the raft is enriched with PLC
substrate, phosphatidylinositol-4,5-biphosphate (34). Furthermore, it has been noted that mast cells from LAT-deficient mice display an impaired Fc
RI-mediated degranulation (35). Therefore, it is possible to think that MIST/Clnk may bring LAT in the vicinity of Syk to allow it to be phosphorylated and to potentiate it to be a scaffold protein which brings together the signaling proteins essential for degranulation.
SLP-76, a structural relative of MIST/Clnk, has been reported to be expressed in mast cells and be tyrosine phosphorylated upon the FcRI engagement in RBL-2H3 cells (36). Because both MIST/Clnk and SLP-76 can associate with a similar set of signaling proteins, including PLC
, Vav and Grb2 (4,9,10), the observed tyrosine phosphorylation of PLC
in MIST-YF-expressing clones may be mediated by SLP-76. Similar to the MIST-YF-expressing RBL-2H3 cell clones we described here, Fc
RI-mediated degranulation and cytokine production of BMMC from SLP-76-deficient mice were almost completely abrogated, whereas phosphorylation of PLC
and Vav and the [Ca2+]i response were not drastically reduced (37). Unlike SLP-76, MIST/Clnk is phosphorylated by Lyn and is involved in phosphorylation of LAT. Thus, SLP-76 and MIST are likely to be functionally non-redundant in Fc
RI signaling of mast cells. A careful dissection of functions of MIST/Clnk and SLP-76 may shed new light on the complexity of Fc
RI-mediated signaling that triggers diverse sets of mast cell functions.
Cao et al. reported that expression of Clnk was restricted to cytokine-dependent cell lines with a few exceptions of cytokine-independent cell lines such as P815 mast cell line (15). Cao et al. speculated that activated mutation of c-Kit in P815 mastocytoma cells resulted in the induction of Clnk mRNA in this cytokine-independent cell line. However, we have evidence that MIST/Clnk mRNA is up-regulated by IL-3 and IL-4, but not by c-Kit ligand in a IL-3-starved mast cell line, although c-Kit receptor was expressed in the same cell line (data not shown). Therefore, c-Kit activation may not account for the constitutive expression of MIST/Clnk mRNA in all mast cell lines. Furthermore, we observed the mast cell-specific expression of MIST/Clnk in skin from NC/Nga as well as normal mice. Together, it is possible that mast cells express MIST/Clnk constitutively.
We could not detect the significant MIST/Clnk expression in cells except for mast cells in allergic skin from NC/Nga mice. This might be due to the fact that local concentration of cytokines at the allergic sites is not high enough to induce MIST/Clnk expression in T or NK cells, compared to the in vitro experimental conditions described by Cao et al. (15). Thus, the cytokine-induced expression of MIST/Clnk in vivo remains to be tested.
If the expression of MIST/Clnk is up-regulated by cytokines in vivo, it would be feasible that, at sites of allergic reaction, MIST/Clnk mediates the cross-talk between the signals from FcRI and cytokine receptors: cytokines released by T cells and also by mast cells themselves will up-regulate expression of MIST/Clnk in mast cells, which may in turn render Fc
RI hypersensitive to the antigenIgE complex. As such, MIST/Clnk may be an ideal target for potential therapeutic intervention in allergy and mast cell malignancies.
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Acknowledgments |
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Abbreviations |
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BCR B cell receptor |
BMMC bone marrow-derived mast cells |
[Ca2+]i intracellular Ca2+ concentration |
EST expressed sequence tag |
Fc![]() |
GST glutathione-S-transferase |
HCMC human cord blood-derived mast cells |
HRP horseradish peroxidase |
LAT linker for activation of T cells |
PLC phospholipase C |
PTK protein tyrosine kinase |
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Notes |
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Received 8 January 2000, accepted 20 January 2000.
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References |
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