CAST, a Novel CD3ε-binding Protein Transducing Activation Signal for Interleukin-2 Production in T Cells*

Antigen recognition through T cell receptor (TCR)-CD3 complex transduces signals into T cells, which regulate activation, function, and differentiation of T cells. The TCR-CD3 complex is composed of two signaling modules represented by CD3ζ and CD3ε. Signaling through CD3ζ has been extensively analyzed, but that via CD3ε, which is also crucial in immature thymocyte development, is still not clearly understood. We isolated cDNA encoding a novel CD3ε-binding protein CAST. CAST specifically interacts in vivo and in vitro with CD3ε but not with CD3ζ or FcRγ via a unique membrane-proximal region of CD3ε. CAST is composed of 512 amino acids including a single tyrosine and undergoes tyrosine phosphorylation upon TCR stimulation. Overexpression of two dominant-negative types of CAST, a minimum CD3ε-binding domain and a tyrosine-mutant, strongly suppressed NFAT activation and interleukin-2 production. These results demonstrate that CAST serves as a component of preformed TCR complex and transduces activation signals upon TCR stimulation and represents a new signaling pathway via the CD3ε-containing TCR signaling module.

T cells recognize antigens (Ag) 1 presented by major histocompatibility complex on antigen presenting cells. The Ag recognition signal is then transmitted to the cytoplasm via the T cell receptor (TCR) complex, resulting in T cell activation leading to various cellular events such as proliferation, apoptosis, anergy, and differentiation as well as a variety of effector functions. The TCR complex is composed of eight polypeptides including clonally variant ␣␤ (or ␥␦) chains and invariant CD3 chains that contain two noncovalently associated dimers ␥⑀ and ␦⑀, and a disulfide-like chain dimer (either -, -, or -FcR␥) (1). Although ␣␤ chains (␥␦ chains) are responsible for Ag recognition, CD3␥, ␦, ⑀, and chains play crucial roles in delivering the Ag recognition signal to the cytoplasm (2). Each CD3 chain possesses activation motifs in the cytoplasmic domain, termed ITAM (immune receptor tyrosine-based activation motif). ITAM contains two YXX(L/I) sequences 7 or 8 amino acids apart. Whereas CD3␥, ␦, ⑀, and FcR␥ chains have one ITAM, and chains have three and two, respectively (3). Upon TCR stimulation, Src family protein-tyrosine kinases such as Lck and Fyn are activated to phosphorylate tyrosine residues within ITAMs, followed by recruitment of the second type of tyrosine kinase ZAP-70 (Syk) to phosphorylated ITAM (4,5). Subsequently, such initial events lead to ZAP-70 activation and tyrosine phosphorylation of various intracellular proteins including phospholipase C␥ (6 -8), Vav (9, 10), SLP-76 (11,12), HS-1 (13), LAT (14 -16), phosphatidylinositol 3-kinase (17), and Cbl (18), as well as Ca 2ϩ mobilization, phosphatidylinositol hydrolysis, and the activation of mitogen-activated protein kinase cascade (2).
It has been shown that ITAM in not only CD3 but also CD3⑀ mediates activation signals, as demonstrated by experiments in which chimeric molecules composed of the extracellular domain of CD8 or CD25 with the cytoplasmic tail of CD3⑀ or CD3 such as CD8-or CD25-⑀ can mimic the TCR signals by inducing various tyrosine phosphorylations of cytoplasmic proteins, Ca 2ϩ mobilization, and phosphatidylinositol hydrolysis (19,20). However, from experiments analyzing the function of T cells expressing ITAM-deficient CD3 chain, it has been shown that T cell activation through Thy-1 or Ly-6 depends on the presence of ITAM of the chain and could not be replaced by ITAMs from other CD3 chains, indicating that the TCR-CD3 complex is composed of two functionally distinct signaling modules represented by CD3 and CD3⑀ (21,22). Furthermore, physiological significance of such distinct signaling modules has also been demonstrated in immature thymocytes (23,24). In addition to the fact that CD3 appears to associate with pre-TCR complex and clonotype-independent CD3 complex very weakly (25,26), cross-linking of CD3⑀ induces differentiation of CD4 Ϫ 8 Ϫ thymocytes into CD4 ϩ 8 ϩ cells in RAG-2 knockout mice (27,28). This implies that the signaling pathway through the module containing CD3⑀ plays an important role in controlling immature thymocyte development.
Phosphorylation-dependent downstream signals upon TCR stimulation, particularly molecules triggered by phosphorylation of ITAM, have been extensively investigated (29). However, signaling molecules, which constitutively associate with the TCR-CD3 complex, still remain to be elucidated. Such molecules should play a crucial role in T cell activation as a preformed signaling component of the TCR-CD3 complex. We therefore attempted to identify molecules, which constitutively associate with the CD3⑀ chain to clarify the preformed TCR signaling machinery as well as the downstream signaling path-way of the activation module containing CD3⑀ as compared with CD3. Utilizing the West-Western method (30), we identified a novel CD3⑀-binding protein, CAST, as a CD3⑀-associated signal transducer. In this report, we have analyzed its structure and function in T cell activation.

EXPERIMENTAL PROCEDURES
Cells and Antibodies-The ovalbumin-specific murine T cell hybridoma DO11.10 and TAg-Jurkat cells, a Jurkat T cell line stably transfected with simian virus 40 large T antigen cells (provided by G. Crabtree, Stanford University, Stanford, CA), were maintained in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 5 mM glutamine, and 50 M 2-mercaptoethanol (complete RPMI). Anti-CD3⑀ mAb, 145-2C11, was kindly provided by J. Bluestone (University of Chicago, Chicago, IL). Anti-HA mAb, 12CA5, and anti-phosphotyrosine mAb, 4G10, were purchased from Roche Molecular Biochemicals and Upstate Biotechnologies, Inc. (Lake Placid, NY), respectively. Anti-CAST antisera were prepared by immunizing New Zealand White rabbits (Japan SLC, Inc., Hamamatsu, Japan) subcutaneously with GST fusion protein that included a part of CAST corresponding to amino acids 1-489 used as the immunogen.
Preparation of 32 P-Labeled GST Fusion Proteins-Constructs of GST fusion protein containing the cytoplasmic domain of CD3⑀ (GST-⑀) and preparation of 32 P-labeled GST-⑀ as a screening probe were described previously (31). Briefly, purified GST-⑀ was adsorbed onto glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), which were then subjected to kinase reaction by the catalytic subunit of cAMP-dependent protein kinase (Sigma), and the labeled GST-⑀ was eluted by in 10 -50 beads volumes of 20 mM reduced glutathione. The probes were labeled to high specific activity (approximately 5-10 ϫ 10 5 cpm/g of protein).
Screening of cDNA Library-CD3⑀-binding proteins were screened from a gt11 library, which was constructed from mRNA from an human T-cell lymphotrophic virus, type I transformed human T cell line, HAT109 (provided by Dr. M. Yoshida, University of Tokyo) as described previously (31). The total of 1.6 ϫ 10 6 plaques was screened with 32 P-labeled GST-⑀. Positive phages were subsequently isolated, and the cDNA inserts were sequenced after subcloning into pBluescript II.
Expressible Constructs and Transfection-An expressible construct of CAST was constructed by inserting a full-length CAST cDNA into an expression vector, pBCMGSNeo (a gift of Dr. H. Karasuyama, Tokyo, Japan) or pEF-BOS (a gift of Dr. S. Nagata, Osaka, Japan). For HAtagged CAST, oligonucleotides corresponding to 5Ј and 3Ј sequences of HA were annealed and subcloned into the XhoI-NotI fragment of pBluescript SK II (pBSK/HA). A full-length cDNA of CAST was inserted into the EcoRI site of this vector (pBSK/HA-CAST). Then a XhoI-NotI fragment from pBSK/HA-CAST was subcloned into the XhoI-NotI fragment of pBCMGSNeo (pBCMGS/HA-CAST).
For transient transfection, EcoRV-SmaI fragment containing a fulllength CAST cDNA was subcloned into a blunt-ended pEF-BOS (pEF-BOS/CAST). The minimal binding region (BR) of CAST to CD3⑀, which corresponds to nucleotides 442-594, and a mutant CAST containing a mutation (Tyr 3 Phe) were prepared by polymerase chain reaction and subcloned into a blunt-ended pEF-BOS vector (pEF-BOS/BR, pEF-BOS/YF).
Stable transfectants of CAST into DO11.10 T cell hybridoma cells were obtained by electroporation with 30 g of pBCMGSNeo/HA-CAST using Gene Pulser II (Bio-Rad) with 310 V and 975 microfarads. For transient transfections, TAg-Jurkat T cells were co-transfected with 20 g of NFAT-luciferase (a gift of Dr. G. Crabtree) and 40 g of pEF-BOS, pEF-BOS/CAST, pEF-BOS/BR, and pEF-BOS/YF in serum-free RPMI 1640.
In Vitro Binding Assays-Specific interaction between CAST and CD3⑀ chains was analyzed using in vitro translated 35 S-labeled CAST and its mutants and GST fusion protein of CD3⑀ and its truncated products as described previously (31). In vitro translation of a fulllength or partial cDNAs of CAST subcloned into pCITE-4a(ϩ)vector was performed with the use of TNT coupled reticulocyte lysate systems (Promega, Madison, WI) in the presence of [ 35 S]methionine. Preparation and purification of various GST fusion proteins were described previously (31). In vitro binding assay were performed in a binding buffer (1% Brij 97, 50 mM Tris, pH 7.6, 150 mM NaCl, 10 g/ml aprotinin, 12.5 g/ml antipain, 12.5 g/ml chymostatin, 50 g/ml leupeptin, 25 g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide) as described (31).
Analysis of IL-2 Production-T cell hybridoma cells were stimulated by immobilized anti-CD3⑀ mAb 2C11, the culture supernatants after 20 h were collected, and the amount of IL-2 was measured by using enzyme-linked immunosorbent assay.
Accession Number-The human CAST cDNA sequence has been submitted to the DDBJ/EBI/GenBank TM data bases under accession number AF017633.

RESULTS
Identification of cDNAs Encoding CD3⑀-associated Proteins-To isolate cDNAs encoding CD3⑀-associated proteins, a gt11 expression library derived from a human T cell line was screened by a 32 P-labeled GST fusion protein containing the cytoplasmic domain of CD3⑀. Eight positive clones were obtained as fusion proteins with ␤-galactosidase (31). Subsequent DNA sequencing of these clones revealed that they encoded five different proteins, and one of these clones encoded a novel protein, CAST (CD3⑀-associated signal transducer). The entire coding sequence of cDNA and the deduced amino acid sequence of CAST are shown in Fig. 1. CAST is comprised of 512 amino acids including a single tyrosine residue (Tyr-82). Because Tyr-82 was followed by a leucine residue located two amino acids downstream, the sequence around Tyr resembles a half of ITAM or tyrosine signal motif for the interaction with the 2 FIG. 1. Protein sequence of CAST. The human CAST sequence was deduced from the nucleotide sequence of the CAST cDNA, starting at the first in-frame methionine residue. CAST consists of 512 amino acids. The minimal region required for binding with CD3⑀ as shown in Fig. 4 is boxed. A unique tyrosine residue located at position 82 is underlined. The human CAST nucleotide sequence will appear in the EBI and GenBank TM nucleotide sequence data bases under the accession number AF017633. chain of adapter complex AP-2 (32) (Fig. 1).
Association between CAST and CD3⑀ in T Cells-CAST was identified as a CD3⑀-binding protein by West-Western method. To examine whether CAST interacts with CD3⑀ under physiological conditions, we prepared the transfectants of a widely used ovalbumin-specific T cell hybridoma, DO11.10, which expressed CAST attached with HA tag at the N terminus (HA-CAST). HA-CAST was abundantly expressed in the transfectant ( Fig. 2A, lane 1). When the cell lysate was immunoprecipitated with anti-CD3⑀ mAb and immunoblotted with anti-HA mAb, a band corresponding to HA-CAST was clearly detected (Fig. 2A, lane 2). In contrast, no band was observed in the immunoprecipitates with a control hamster Ab. (Fig. 2A, lane 3). The specific interaction of CD3⑀ and CAST was further confirmed for the endogenous CD3⑀ and CAST in T cells. Human Jurkat cells were lysed in the 1% Brij97-containing buffer, and the lysate was immunoprecipitated with anti-CD3⑀ mAb and blotted with anti-CAST Ab. As shown in Fig.  2B, CAST was co-precipitated with anti-CD3⑀ mAb OKT3 (Fig.  2B, lane 2) but not with anti-Tac mAb 7G7 as a control (Fig. 2B,  lane 3). The association was hardly detected in the cell lysates prepared in stronger detergent condition such as 1% Nonidet P-40 (data not shown), suggesting that the association between CD3⑀ and CAST is weak. CAST was also detected by immunoprecipitation and blotting with anti-CAST Ab (Fig. 2B, lane 1). Because rabbit anti-CAST Ab does not precipitate CAST very well, the amount of CAST by immunoprecipitation with anti-CAST Ab was considerably underestimated. The specificity of the association of CAST with the TCR complex was further analyzed by immunoprecipitation with various Abs against other cell surface molecules on T cells. As shown in Fig. 2C, anti-CD3⑀ mAb but not mAbs against CD2, CD45, or CD4 (not shown) co-precipitate CAST. These results demonstrate that CAST associates specifically and constitutively with the TCR complex under physiological condition.
Specific Association of CAST with CD3⑀ but Not CD3-We next analyzed the specificity of the binding of CAST with CD3⑀ within the TCR complex. We examined particularly whether CAST interacted with CD3 family molecules (, , and FcR␥), because these molecules contain ITAMs and constitute a different signaling module from CD3⑀ in the TCR complex (21,22). In addition, we investigated the CAST-binding region within the CD3⑀ by in vitro binding assays. [ 35 S]methioninelabeled CAST was translated in vitro and precipitated with a variety of GST fusion proteins containing the entire or partial deletion mutants of the cytoplasmic domain of CD3⑀ as well as the cytoplasmic tails of CD3 and FcR␥ chains (Fig. 3). CAST was co-precipitated with GST-CD3⑀ (Fig. 3B, lane 7) but not with GST-CD3 or GST-FcR␥ (Fig. 3B, lanes 8 and 9). The specificity of the binding between CAST and CD3⑀ was demonstrated by the fact that luciferase as a control was not precipitated with CD3⑀ (Fig. 3B, lane 11). This result indicates that the association of CAST with the TCR complex is mediated through specific interaction with CD3⑀ and implies that CAST functions in the downstream of the signaling module containing CD3⑀.
We determined the CAST-binding region within CD3⑀ in the in vitro binding assay by co-precipitation with GST fusion protein containing various deletion mutants of CD3⑀. As shown in Fig. 3B, labeled CAST interacted with all GST fusion proteins (⑀D1, ⑀D3, ⑀D5, and CD3⑀) except for GST-⑀D4 (Fig. 3B,  lanes 3-7). Because GST-⑀D1 precipitated CAST, the proximal region to the transmembrane portion of CD3⑀ composed of only 12 amino acids was found to be necessary and sufficient for the CAST/CD3⑀ binding. Taken together, the results demonstrated that CAST interacts specifically with CD3⑀ through its unique region proximal to the transmembrane domain.
Mapping of CD3⑀-binding Region in CAST-In addition to the CAST-binding region within CD3⑀, the CD3⑀-binding re- gion within CAST was also determined by in vitro binding assays. Serially truncated cDNAs of CAST (d1-d4) were subcloned into pCITE4a vector (Fig. 4A). [ 35 S]Methionine-labeled in vitro translation products of these mutant CASTs were prepared. Then CAST and its truncated mutants were precipitated with GST-CD3⑀. As shown in Fig. 4B, d1, d2, and d4 bound strongly to CD3⑀, whereas d3 appeared to bind very weakly (Fig. 4B, lanes 5-8). Collectively, these results demonstrate that the region of amino acid residues 149 -198 in the Nterminal half of CAST is responsible for the specific binding to CD3⑀. This region is boxed in Fig. 1.
Tyrosine Phosphorylation of CAST upon TCR Stimulation-Because CAST binds directly and constitutively to CD3⑀, it may have a function for signal transduction upon T cell activation. CAST contains a single tyrosine residue at amino acid position 82. If this unique tyrosine undergoes phosphorylation upon TCR stimulation, it is possible that phosphorylated CAST may function to transduce downstream signals. Therefore, we examined whether CAST undergoes tyrosine phosphorylation upon TCR stimulation. DO11.10 T cell hybridomas overexpressing HA-CAST were prepared and stimulated for 2 min by cross-linking with anti-CD3⑀ mAb. The cell lysates were immunoprecipitated with anti-HA mAb, followed by immunoblotting with anti-phosphotyrosine mAb, 4G10. As shown in Fig. 5, anti-HA mAb but not control anti-Tac mAb precipitated a tyrosine phosphorylated band of approximately 80 kDa in TCRstimulated cells, whereas such a band was only weakly detected in unstimulated cells. This 80-kDa phosphorylated band was confirmed to be CAST by blotting the same filter with anti-CAST Ab (Fig. 5). These results demonstrate that CAST undergoes rapid, although weak, tyrosine phosphorylation upon TCR stimulation.

CAST Mediates Signals for NFAT Activation and IL-2 Production-
To investigate the function of CAST in T cell activation, we first analyzed the T cell hybridoma transfectants overexpressing CAST for IL-2 production. However, we failed to observe significant effect on IL-2 secretion (data not shown). Therefore, we decided to analyze the function of dominantnegative forms of CAST. For this purpose, two mutant forms of  CAST were prepared to serve as dominant-negative CASTs (Fig. 6A). One mutant was termed BR (binding region to CD3⑀, amino acid residues 148 -198), which was exclusively comprised of the minimal CD3⑀-binding region of CAST as shown in Fig. 4. The other was a tyrosine mutant YF, in which a unique tyrosine (Tyr-82) was mutated to phenylalanine. Although BR and YF can bind to CD3⑀ in in vitro binding assay (Fig. 4), it is particularly important to demonstrate the interaction in T cells to connect to the functional analysis.
Therefore, we confirmed the specific association of YF and BR with CD3⑀ and function as dominant-negative forms in T cells. The association of YF with CD3⑀ was analyzed in the transient transfection system. TAg-Jurkat cells (33) were transfected with YF and BR, and the cell lysates were immunoprecipitated with anti-CD3⑀ mAb and blotted with anti-CAST Ab. As shown in Fig. 6B, YF mutant was clearly coprecipitated with CD3⑀ (lane 6). Endogenous CAST was also precipitated with anti-CD3⑀ mAb (Fig. 6B, lane 2) as shown in Fig. 2B. Furthermore, the overexpression of BR induced the disappearance of the band corresponding to the endogenous CAST (Fig. 6B, lane 4), supporting the idea that BR competes with the CD3⑀ binding with endogenous CAST and thereby demonstrating that it functions as a dominant-negative form of CAST. Regarding the analysis of BR, because anti-CAST Ab unfortunately did not immunoprecipitate BR, T cell hybridoma cells transfected with BR were immunoprecipitated with anti-CD3⑀ Ab and blotted with anti-CAST Ab to investigate the association of BR and CD3⑀. As shown in Fig. 6C, the specific precipitation of the BR protein with anti-CD3⑀ mAb (Fig. 6C,  lane 9) but not with the control hamster Ig (Fig. 6C, lane 10) was detected as a 20-kDa band, although it was a very weak band because of the weak reactivity of anti-CAST Ab with BR even for blotting. Further evidence for competitive capability of BR to CD3⑀ binding by CAST was demonstrated with an in vitro binding assay. CAST and BR were in vitro translated with [ 35 S]methionine, and the graded amount of BR was added to the mixture of CAST and GST-CD3⑀. As shown in Fig. 6D, BR inhibited the CAST-CD3⑀ binding in a dose-dependent manner. Collectively, these results of biochemical analysis demonstrate that BR and YF indeed associate with CD3⑀ in T cells and function as dominant-negative forms of CAST by overexpression.
To analyze the function of these mutant CASTs in T cell activation, we first utilized a transient assay for NFAT activation by using NFAT-luciferase (33), a reporter construct containing multiple copies of the NFAT binding site of the IL-2 promoter fused to luciferase cDNA. We transiently transfected NFAT-luciferase in TAg-Jurkat T cells in combination with either empty vector, BR, YF, or the wild-type CAST. As endogenous control, sea pansy luciferase was also transfected into all cells and the NFAT-luciferase activity was normalized by sea pansy activity. No significant difference was observed in relative luciferase activity when TAg-Jurkat T cells was trans-FIG. 6. Dominant-negative forms of CAST and their association with CD3⑀. A, schematic structures of the wild-type and two dominant-negative forms of CAST, BR and YF. BR consists exclusively of the binding region of CD3⑀ in CAST and is shown as a shaded box. YF has a tyrosine to phenylalanine mutation at amino acid residue 82. B, the YF mutant binds to CD3⑀, and the BR can compete with the CD3⑀ binding with CAST. TAg-Jurkat cells were transiently transfected with the expressible constructs of BR (lanes 4 and 5) or YF (lanes 6 and 7). The cell lysates (equivalent to 5 ϫ 10 7 cells) were immunoprecipitated with either anti-CD3⑀ mAb (OKT3) or a control mAb (9E10) and blotted with anti-CAST Ab. As a control, whole cell lysate was also blotted (lane 1). The arrow indicates the position of CAST. C, the association between BR and CD3⑀. Stable transfectants of DO-11-10 hybridoma expressing ha-BR-BR were lysed in 1% Brij97, and the cell lysate was immunoprecipitated with either anti-CD3⑀ mAb (2C11) (lane 9) or a control Ab fected with the empty vector or wild-type CAST (Fig. 7A). However, transfection with both BR or YF strongly reduced luciferase activity compared with those transfected with the empty vector (Fig. 7B), whereas the activities upon stimulation with phorbol myristate acetate plus ionophore are almost the same (see the legend of Fig. 7). NFAT activation was inhibited by BR in a dose-dependent manner (Fig. 7C). These functional data demonstrate that CAST exhibits signaling function for NFAT activation.
To analyze the effect on IL-2 production after NFAT activation, stable transfectants of DO11.10 hybridoma cells expressing BR were prepared. As shown in Fig. 7D, BR transfectants exhibited reduced levels of IL-2 production upon TCR stimulation. These results indicate that CAST mediates activation signals important for IL-2 production. DISCUSSION We described the identification and function of the novel CD3⑀-associated molecule CAST in this study. Because CAST was cloned on the basis of specific binding to the cytoplasmic domain of CD3⑀ independently of its phosphorylation, it is expected to be a constitutive component of the preformed TCR-CD3 complex (34). As expected, CAST constitutively associates with CD3⑀ in T cells. CAST does not merely function as a component of the preformed TCR complex but also plays an important role in transducing activation signals in T cells despite the fact that CAST does not contain any particularly conserved functional domain structure.
The first evidence that CAST is important for T cell activation was demonstrated by its tyrosine phosphorylation upon T cell stimulation. Only a minor portion of CAST appeared to be phosphorylated upon stimulation. Preliminary results demonstrated that most of the phosphorylated CAST disappeared when cell lysates from anti-CD3⑀-stimulated T cells were precleared extensively, suggesting that the phosphorylated CAST was mostly associated with CD3⑀ on the cell surface. 2 The  (10,20, and 40 g) (C). The total amount of plasmid was adjusted to 40 g by adding the vector plasmid. Transfected cells were either unstimulated (white bars) or stimulated (black bars) with anti-CD3⑀ mAb (OKT3) and assayed for luciferase activity. Luciferase activity was determined in triplicate in each experimental condition. The data are expressed as the means Ϯ S.D. of triplicates from a representative of six independent experiments. As the maximum responses, the luciferase activities in the cells upon stimulation with phorbol myristate acetate plus ionophore were 402 Ϯ 89 and 372 Ϯ 24 for the vector alone and 40 g of YF (YF 40), respectively. Even when these data were presented as fold increase, the results were not greatly changed (not shown). D, expression of dominant-negative CAST (BR) inhibits IL-2 production upon TCR stimulation. DO11.10 cells (f) and the stable transfectants expressing BR (E and q as two representative clones) were stimulated with immobilized anti-CD3⑀ mAb for 20 h. The IL-2 produced in the culture supernatants was determined by enzyme-linked immunosorbent assay. The amounts of IL-2 upon stimulation with phorbol myristate acetate plus A23187 on DO11.10 and the two BR transfectants were 3.7, 3.2, and 3.0 ng/ml, respectively. The data are presented as the means Ϯ S.D. of triplicates. second evidence was shown by demonstrating that dominantnegative forms of CAST inhibited NFAT activation. NFAT activation and IL-2 production was significantly reduced by overexpression of two dominant-negative mutants BR or YF. The structure of these two mutants provides insights for the mechanism of CAST function. Because BR is the minimal region of CAST for CD3⑀ binding, BR inhibits NFAT activation by blocking the CD3⑀ binding of endogenous CAST. Indeed, we demonstrated that BR competed with the binding with endogenous CAST in T cells. In addition, this result suggests that there is an important region(s) in CAST other than the CD3⑀-binding region, which is required for signaling function of CAST probably through association with other signaling molecules. Because the YF mutant also suppressed NFAT activation, it is likely that one of the CAST-binding molecules recognizes the unique phosphorylated Tyr-82 and then transduces downstream signals leading to NFAT activation. Because the YF mutant could not associate with such a downstream protein, its recruitment by CAST to the TCR complex on the cell surface was inhibited in YF-transfected cells. In contrast to the inhibitory effect of these two dominant-negative types of CAST mutants, overexpression of wild-type CAST had little effect on NFAT activation. This might be explained on the basis of preliminary results showing that CAST associated with the TCR complex on the cell surface, which is only a minor fraction of overexpressed CAST, is mainly phosphorylated to mediate signaling function. Alternatively, unidentified CAST-binding protein(s) may be limited.
It is noted that BR or YF did not suppress NFAT activation completely, and increasing the dose of BR cDNA for transfection did not result in further reduction. This is probably due to the presence of other pathways to induce NFAT activation upon TCR stimulation, which can not be blocked by dominantnegative CAST mutants. The TCR-CD3 complex is composed of two distinct signaling modules: one is composed of CD3⑀, and the other is composed of CD3 (21,22). It is possible that distinct signaling molecules may be associated with the downstream signals of each module. CAST is the first signaling molecule that dissects functions of these two signaling modules by specific association with CD3⑀. Therefore, even if the pathway immediately downstream of the CD3⑀-containing module was blocked by the expression of dominant-negative CAST mutants, NFAT activation signals may be transduced via CD3. This may explain why NFAT activation was not completely blocked by the overexpression of BR or YF and also is consistent with the observation that CAST binds specifically to CD3⑀ but not to CD3. Furthermore, this also suggests that CAST-mediated activation signal is not dispensable by the signal through the CD3 module. To confirm this hypothesis, we are currently analyzing NFAT activation in T cells expressing the CD3 chain lacking all ITAMs.
We determined that CAST-binding region within CD3⑀ was located in the N-terminal 12 amino acids of the cytoplasmic domain of CD3⑀. This membrane-proximal region of CD3⑀ is very unique because other signaling molecules such as Fyn and ZAP-70 are known to associate with CD3 via its ITAM (35,36). CAST is the first molecule possessing the binding specificity to CD3⑀ because other molecules associate with both CD3 and CD3⑀. Therefore, the membrane-proximal region of CD3⑀ with which CAST associates may serve as a signaling motif and mediate ITAM-independent signaling upon TCR stimulation. This idea is supported by the fact that the first 13 amino acids of the cytoplasmic region of CD3⑀ are completely conserved between mouse and human. Previous study on signaling function of the cytoplasmic tail of CD3⑀ by Letourneur and Klausner (19) is also consistent with our results. This study demon-strated the crucial role of ITAM of CD3⑀ for T cell activation. In addition, it revealed that CD3⑀ in the presence of the membrane-proximal region induced severalfold higher IL-2 response than that of CD3⑀ lacking this region, indicating the important contribution of the membrane-proximal region of CD3⑀ for T cell activation.
Signaling pathways through CD3⑀ are important not only in mature T cells but also in immature thymocytes (27,28,37). The maturation of thymocytes in RAG-2-deficient mice is blocked at the transitional stage from CD4 Ϫ 8 Ϫ thymocytes to CD4 ϩ 8 ϩ cells (38). However, it has been demonstrated that this blockade in maturation becomes reversed upon stimulation of thymocytes by cross-linking of CD3⑀ (27,28). Considering that pre-TCR complex has a very weak association with CD3 as compared with the TCR complex on mature T cells (25,26,39), these experiments suggest that signaling through the CD3⑀containing module plays an important role in differentiation and selection of thymocytes and that CAST may contribute partly to this pathway by exhibiting function for downstream signaling of the TCR or pre-TCR complex. The in vivo function of CAST in thymocytes development is now being addressed by studies on transgenic mice expressing a dominant-negative CAST under a T cell-specific promoter.
When the expression of CAST in various tissues was investigated, it was found that CAST is expressed in a wide range of tissues with relatively high expression in heart, skeletal muscle, ovary, and small intestine (data not shown). This finding indicates diverse functions of CAST in various types of cells. Furthermore, it is noted that the C terminus of CAST is rich in Lys and Arg, which may represent a characteristic for nuclear localization. Indeed, preliminary results of the analysis of cellular localization of CAST demonstrated that CAST is located both in the cytoplasm and the nucleus (data not shown). Because it has been shown that CD3⑀ also exist in the nucleus (31), CAST in the nucleus may be co-localized with the nuclear CD3⑀. Alternatively, these results suggest that CAST may possess a distinct function other than mediating activation signals in the nucleus. Further analysis is required to elucidate such diverse functions of CAST.