Transcription factors of the nuclear factor–κB/rel (NF-κB) family may be important in cell survival by regulating unidentified, anti-apoptotic genes. One such gene that protects cells from apoptosis induced by Fas or tumor necrosis factor type α (TNF), IEX- 1L, is described here. Its transcription induced by TNF was decreased in cells with defective NF-κB activation, rendering them sensitive to TNF-induced apoptosis, which was abolished by transfection with IEX- 1L. In support, overexpression of antisense IEX- 1L partially blocked TNF-induced expression of IEX -1L and sensitized normal cells to killing. This study demonstrates a key role of IEX- 1L in cellular resistance to TNF-induced apoptosis.

Members of the tumor necrosis factor receptor (TNFR) superfamily are important for cell growth and survival. In addition to providing costimulatory signals for cell proliferation, ligation of both TNFR1 and Fas can result in programmed cell death or apoptosis. The underlying mechanism requires an intact 80-aa stretch present in the cytoplasmic tails of both TNFR1 and Fas, termed the death domain (DD). Here we show that CD27, a member of the TNFR family, expressed on discrete subpopulations of T and B cells and known to provide costimulatory signals for T and B cell proliferation and B cell Ig production, can also induce apoptosis. Co-crosslinking of surface Ig receptors along with ligation of CD27 augments CD27-mediated apoptosis. Unlike TNFR1 and Fas, the cytoplasmic tail of CD27 is relatively short and lacks the DD. Using the yeast two-hybrid system, we have cloned a novel protein (Siva) that binds to the CD27 cytoplasmic tail. It has a DD homology region, a box-B-like ring finger, and a zinc finger-like domain. Overexpression of Siva in various cell lines induces apoptosis, suggesting an important role for Siva in the CD27-transduced apoptotic pathway.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cefl proliferation. (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss ofbinding, while mutation ofM194 caused partial loss of binding. Bding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C-and N-terminal SH2 domans with an afnity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of snaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-klipase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.Activation of the resting T lymphocyte involves at least two steps: an antigen-specific signal generated by the T-cell receptor (TcR)2/CD3 and CD4/CD8-p56lck complexes, followed by a second signal delivered by an accessory cell (1). The CD28 antigen predominates in providing the costimulatory signals for T-cell stimulation (ref. 2; for reviews see refs. 1 and 3). It contains a single immunoglobulin-like domain and a 51-amino acid cytoplasmic tail (4). B7-1 and related B7-2/ B70 are also members of the immunoglobulin supergene family and serve as ligands for CD28 (5-7). Suboptimal proliferation induced by antigen, anti-CD3 or anti-CD2 antibody is augmented by CD28 ligation (for review see ref. 8). Likewise, CD28 ligation by B7 serves as an obligatory second step in T-cell activation (for review see ref.3). CD28-transgenic mice exhibit profound defects in mitogenic responses (9). CD28 signaling stabilizes mRNA for various lymphokines, in particular interleukin 2 (reviewed in refs. 1 and 8). T-cell nonresponsiveness or anergy can be reversed by CD28 engagement (1-3, 10), an event of potential importance in the generation of cytolytic responses against tumors (11,12).An important question concerns the mechanism by which CD28 regulates T-cell growth and the manner in which the CD28 signaling pathway relates to signals generated by the TcRC/CD3-CD4 complex. CD4 and CD8 associate with the protein-tyrosine kinase p56lck, while TcRC/CD3 is cou- By contrast, intracellular events generated by CD28 differ from those induced by TcRC/CD3 ligation. CD28 signaling appears to occur independent of conventional phosphatidylinositol hydrolysis and the Ca2 -dependent cyclophilincalcineurin pathway and is resistant to cyclosporin and FK506 (3,8). CD28 signaling, however, is sensitive to inhibitors of tyrosine kinases and to coligation with the protein-tyrosine phosphatase CD45 (3,8). CD28 ligation in the presence of phorbol ester induced the tyrosine phosphorylation of proteins at 75 and 100 kDa (3,8). This potentially unique pathway leads to DNA binding activity at an element located in the 5' interleukin 2 enhancer region (for review see refs. 1 and 3). PI 3-kinase is a heterodimer consisting of an adaptor subunit (p85) with two SH2 domains that is coupled to a p110 catalytic subunit and is important to signali...

The T cell molecule CD28 provides a co-stimulatory signal that is required for T cell proliferation, and has been implicated in the control of T cell anergy. An important clue to the signaling mechanism of CD28 is the finding that CD28 can bind to phosphatidylinositol 3-kinase (PI 3-kinase) by means of a cytoplasmic phospho-YMNM (pYMNM) motif. A remaining issue concerns whether CD28 can recruit other intracellular signaling molecules. In this study, we show that CD28 uses the same pYMNM motif to recruit a second intracellular protein, GRB-2. CD28-associated GRB-2, as detected by anti-GRB-2 immunoblotting, was found in human peripheral T cells, HPB-ALL and Jurkat cells. As in the case of PI3-kinase, antibody-induced cross-linking of CD28 induces a time-dependent recruitment of GRB-2. Likewise, mutation of the pY-191 residue within the pYMNM motif reduces GRB-2 binding. Peptide binding studies show that the SH2 domain of GRB-2 binds to the pYMNM motif with an affinity comparable to GRB-2/SHC, but some 10- to 100-fold lower than the CD28/PI 3-kinase. Despite this, CD28/GRB-2 and CD28/PI 3-kinase complexes are found to co-exist in peripheral T cells. Finally, immunoblotting shows that CD28 also associates with the gene product of the human homolog of the Drosophila Son of sevenless gene (SOS), a GRB-2-complexed guanine nucleotide exchange factor responsible for converting p21ras to a GTP-bound active state. CD28-associated GRB2/SOS is likely to serve an important link in the regulation of p21ras and lymphokine expression mediated by CD28.

We previously cloned Siva-1 by using the cytoplasmic tail of CD27, a member of the tumor necrosis factor receptor family, as the bait in the yeast two-hybrid system. The Siva gene is organized into four exons that code for the predominant full-length Siva-1 transcript, whereas its alternate splice form, Siva-2, lacks exon 2 coding sequence. Various groups have demonstrated a role for Siva-1 in several apoptotic pathways. Interestingly, the proapoptotic properties of Siva-1 are lacking in Siva-2. The fact that Siva-1 is partly localized to mitochondria despite the absence of any mitochondrial targeting signal, it harbors a 20-aa-long putative amphipathic helical structure that is absent in Siva-2, and that its expression is restricted to double-positive (CD3 ؉ , CD4 ؉ , CD8 ؉ ) thymocytes like BCL-XL, prompted us to test for a potential interaction between Siva-1 and BCL-XL. Here, we show that Siva-1 binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis. Indeed, the unique amphipathic helical region (SAH) present in Siva-1 is required for its binding to BCL-XL and sensitizing cells to UV radiation. Natural complexes of Siva-1͞BCL-XL are detected in HUT78 and murine thymocyte, suggesting a potential role for Siva-1 in regulating T cell homeostasis. A poptosis, or programmed cell death, and cell survival are intimately connected, and any shift in the equilibrium between these two important cell functions can cause disease. The principal mediators belong to the BCL-2 and tumor necrosis factor receptor (TNFR) families (1-3). The discovery of BCL-2, a powerful promoter of cell survival, was soon followed by the identification of several proteins with structural similarity to BCL-2. There are four regions of homology termed BH domains; BCL-2 and BCL-X L have all four domains, whereas BAX and BAK, the proapoptotic members of the family, lack the BH4 domain. Also included in this family are several potent apoptotic molecules that have only a minimal BH3 domain (BAD and BID) (1-3).BCL-2 and BCL-X L have a conserved transmembrane region (TM) toward the carboxy terminus that localizes these proteins to the outer mitochondrial membrane, the outer leaflet of the nuclear membrane, and the endoplasmic reticulum. The bulk of the protein projects into the cytoplasm. Although BAX has such a TM region, in normal cells, it appears to be cytosolic and localizes to the mitochondria upon induction of apoptosis. The principal site of action for the BCL-2 family members seems to be the mitochondria (4-6). The proapoptotic members bind via their BH3 domain to the cleft formed by the BH1, BH2, and BH3 domains of the antiapoptotic members BCL-2 and BCL-X L (1-3, 7). Interestingly, the BH3 domain is a part of the amphipathic helix, underscoring the importance of the amphipathic helical structures in protein-protein interactions.Various members of the TNFR family regulate cell proliferation and death by means of their interaction with specific intracellular signaling molecules that can be divided broadly int...

The Src-related tyrosine kinase p59fyI(T) signaling (18, 19). PI 3-kinase phosphorylates the D-3 position ofthe inositol ring ofPI, PI-4-P, and 20). It is a heterodimer consisting of a regulatory subunit (p85) and a catalytic subunit (p110) (21-25). The yeast homologue of the catalytic subunit of PI 3-kinase (p110), Vps34, plays an essential role in protein sorting and transport (26).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Src-related kinases such as p59fyn(r) contain Src-homology 2 and 3 (SH2 and SH3) domains with the potential to bind to intracellular substrates (27). SH2 domains are formed of a (3-pleated sheet surrounded by two a-helices and bind to phosphotyrosine residues (28). Various intracellular proteins with enzymatic activities such as phospholipase C-y, the p85 subunit ofPI 3-kinase, and the Ras GTPase-activating protein carry related SH2 domains (27). Similarly, smaller, noncatalytic adaptor proteins such as NcK and SHC possess SH2 domains which bind to phosphotyrosine residues on surface tyrosine kinase receptors such as the PDGF and fibroblast growth factor receptors (29-31). The SH2 domain of p85 binds to specific tyrosine-phosphorylated residues on the polyoma middle-sized tumor antigen and the receptors for PDGF and colony-stimulating factor 1 (15,17,18,32,33). By contrast, crystal and NMR analysis of the SH3 domain reveals a hydrophobic pocket formed by two antiparallel 3-sheets oriented at right angles (34, 35). These domains have an affinity for proline-rich residues, as first defined by the 3BP1 protein (36,37). Related motifs have been found in a variety of intracellular proteins such as formin and the rat m4 muscarinic receptor (37). To date, SH3 domains have been implicated in binding to cytoskeletal proteins, the control of membrane ruffling, and the regulation of GTP-binding proteins (27, 37-39).Although PI 3-kinase has been found associated with Src-related kinases (40, 41) the nature and site of interaction have not been established. Here we demonstrate an association between PI 3-kinase and p59fyn(T) and the TCR;/CD3-p59fyn(T) complex. Further, we establish that the SH3 domain of p59fyn is the principal site of PI 3-kinase binding. p59fynm therefore uses a fundamentally distinct mechanism ofbinding to PI 3-kinase, a mechanism that may allow the recruitment of PI 3-kinase independent of tyrosine kinase activity or phosphorylation. PI 3-kinase is an SH3 domain-binding protein that is likely to play a key role in signaling by the TCRC/CD3-p59fYn(T) complex. T-lymphoblastoid cell line HPB-ALL was cultured in RPMI 1640 containing 10% (vol/vol) fetal bovine serum, MATERIALS AND METHODS

PI3K was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85 PI3K in vitro. However, although a small amount of CRKL was preassociated with p85 PI3K , the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120 CBL , both of which bind to c-Kit through p85 PI3K. These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.

SummaryCTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2-and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (IDs0:0.6 and 0.04/~M), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intraceUular signaling molecule with the capacity to regulate cell growth.