Fusion of the human gene for the polyubiquitination coeffector UEV1 with Kua, a newly identified gene.

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon-intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua-UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua-UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua-UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua-UEV protein confers new biological properties to this regulator of variant polyubiquitination.

The mouse SHIP2 (Inppl1) gene: complementary DNA, genomic structure, promoter analysis, and gene expression in the embryo and adult mouse.

SHIP2 is a new member of the inositol polyphosphate 5-phosphatase family showing homology to SHIP1. The structure of both enzymes is characterized by the presence of a 5' SH2 domain, a central catalytic domain, and a 3' proline-rich region. Recent results suggest that SHIP2 and SHIP1 act downstream of various receptors by removing a phosphate from the 5' position of the phosphatidylinositol 3'-kinase phosphatidylinositol 3,4, 5-triphosphate product and of inositol 1,3,4,5-tetrakisphosphate. Human SHIP2 is highly expressed in adult heart, skeletal muscle, and placenta, whereas SHIP1 expression is limited to the hematopoietic system. We report here the molecular analysis of the mouse SHIP2 cDNA and the corresponding protein, the structure of the gene, and the identification of its promoter. SHIP2 mRNA expression was analyzed in embryonic and adult mouse tissues by reverse transcription-polymerase chain reaction and in situ hybridization. In embryonic day 15.5 mice, SHIP2 was strongly expressed in the liver, specific regions of the central nervous system, the thymus, the lung, and the cartilage perichondrium. In adult mice, SHIP2 mRNA was markedly present in the brain and the thymus and at different stages of spermatozoa maturation in the seminiferous tubules. The subtle differences in the protein structure of SHIP2 and SHIP1 as well as their different patterns of expression are discussed.

Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB.

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-kappaB) and bound RICK, a CARD-containing kinase that also induces NF-kappaB activation. Nod1 mutants inhibited NF-kappaB activity induced by RICK, but not that resulting from tumor necrosis factor-alpha stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-kappaB activation pathways.

CIPER, a novel NF kappaB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10.

We have identified and characterized CIPER, a novel protein containing a caspase recruitment domain (CARD) in its N terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or Apaf-1. Expression of CIPER induced NF-kappaB activation, which was inhibited by dominant-negative NIK and a nonphosphorylable IkappaB-alpha mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was sufficient and necessary for NF-kappaB-inducing activity. Point mutations in highly conserved residues in the CARD of CIPER disrupted the ability of CIPER to activate NF-kappaB and to form homodimers, indicating that the CARD is essential for NF-kappaB activation and dimerization. We propose that CIPER acts in a NIK-dependent pathway of NF-kappaB activation.

Regulation of B cell apoptosis by Bcl-2 and Bcl-XL and its role in the development of autoimmune diseases (Review).

Cell death is a common event during B cell development. The demise of developing B cells is a regulated process that serves to select cell populations bearing functional receptors and to remove cells that are no longer needed or potentially autoreactive. Bcl-2 and Bcl-XL, two members of the bcl-2 gene family of programmed cell death regulators with anti-apoptotic activity, are expressed in a highly regulated pattern during B cell maturation. Overexpression of Bcl-2 in developing B cells of transgenic mice, in the presence of T cell dependent costimulatory signals, results in the generation of a modified B cell repertoire and in the production of pathogenic autoantibodies. While disregulation of programmed cell death in B cells may cause autoimmune manifestations in mice, the involvement of such alterations in the pathogenesis of autoimmune diseases in humans merits further investigation.

Diva, a Bcl-2 homologue that binds directly to Apaf-1 and induces BH3-independent cell death.

We have identified and characterized Diva, which is a novel regulator of apoptosis. Sequence analysis revealed that Diva is a member of the Bcl-2 family of proteins containing Bcl-2 homology domain 1, 2, 3, and 4 (BH1, BH2, BH3, and BH4) regions and a carboxyl-terminal hydrophobic domain. The expression of Diva mRNA was detected in multiple embryonic tissues but was restricted to the ovary and testis in adult mice. The expression of Diva promoted the death of 293T, Ramsey, and T47D cells as well as that of primary sensory neurons, indicating that Diva is a proapoptotic protein. Significantly, Diva lacks critical residues in the conserved BH3 region that mediate the interaction between BH3-containing proapoptotic Bcl-2 homologues and their prosurvival binding partners. Consistent with this, Diva did not bind to cellular Bcl-2 family members including Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1/Bfl-1. Furthermore, mutants of Diva lacking the BH3 region fully retained their proapoptotic activity, confirming that Diva promotes apoptosis in a BH3-independent manner. Significantly, Diva interacted with a viral Bcl-2 homologue (vBcl-2) encoded by the Kaposi's sarcoma-associated herpesvirus. Consistent with these associations, apoptosis induced by Diva was inhibited by vBcl-2 but not by Bcl-XL. Importantly, Diva interacted with Apaf-1, an adapter molecule that activates caspase-9, a central death protease of the apoptotic pathway. The expression of Diva inhibited the binding of Bcl-XL to Apaf-1, as determined by immunoprecipitation assays. Thus, Diva represents a novel type of proapoptotic Bcl-2 homologue that promotes apoptosis independently of the BH3 region through direct binding to Apaf-1, thus preventing Bcl-XL from binding to the caspase-9 regulator Apaf-1.

Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL.

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.

A1 demonstrates restricted tissue distribution during embryonic development and functions to protect against cell death.

Members of the bcl-2 gene family are essential regulators of cell survival in a wide range of biological processes. A1, a member of the family, is known to be expressed in certain adult tissues. However, the precise tissue distribution and function of A1 remains poorly understood. We show here that A1 is expressed in multiple tissues during murine embryonic development. In the embryo, A1 was detected first at embryonic day 11.5 in liver, brain, and limbs. At day 13.5 of gestation, A1 expression was observed in the central nervous system, liver, perichondrium, and digital zones of developing limbs in a pattern different from that of bcl-X. In the central nervous system of 15.5-day embryos, A1 was expressed at high levels in the ventricular zone and cortical plate of brain cortex. Significantly, the interdigital zones of limbs and the intermediate region of the developing brain cortex, two sites associated with extensive cell death, were devoid of A1 and bcl-X. The expression of A1 was retained in many adult tissues. To assess the ability of A1 to modulate cell death, stable transfectants expressing different amounts of A1 protein were generated in K562 cells. Expression of A1 was associated with retardation of apoptotic cell death induced by actinomycin D and cycloheximide as well as by okadaic acid. Confocal microscopy showed that the A1 protein was localized to the cytoplasm in a pattern similar to that of Bcl-2. These results demonstrate that the expression of A1 is wider than previously reported in adult tissues. Furthermore, its distribution in multiple tissues of the embryo suggests that A1 plays a role in the regulation of physiological cell death during embryonic development.

Completely allogeneic spleen cells induced cytolytic neonatal tolerance to alloantigens, but failed to establish allo-helper interactions with host T cells.

The injection of spleen cells from F1 mice into-newborns from a parental strain results in the establishment of cytolytic tolerance to donor alloantigens and the development of a lupus-like disease. This syndrome is the consequence of the recognition by alloreactive host CD4+ T cells of discordant major histocompatibility complex (MHC) class II antigens on semi-allogeneic donor B cells. We have analysed whether completely allogeneic spleen cells are as able as semi-allogeneic spleen cells to induce cytolytic tolerance to donor alloantigens and to co-operate with alloreactive T cells for autoantibody production. BALB/c mice were injected at birth with Thy-1-depleted spleen cells from (C57BL/6 x BALB/c)F1 or C57BL/6 mice, either alone or in combination. Cytolytic tolerance was always induced, as manifested by persistence of chimerism and acceptance of skin allografts. However, only F1 semi-allogeneic B cells were activated by alloreactive host T cells to produce anti-DNA IgG antibody. The deficient co-operation between BALB/c CD4+ T cells and completely allogeneic C57BL/6 B cells was confirmed after neonatal injection of (C57BL/ 6 x BALB/c)F1(Igha) spleen cells together with C57BL/6(Ighb) spleen cells. These mice developed anti-DNA antibodies bearing only the Igha allotype. Similar results were observed in experiments of allogeneic interaction in vitro, in which BALB/c CD4+ T cells were cocultured with either (C57BL/6 x BALB/c)F1 or C57BL/6 B cells. The present results demonstrate that completely allogeneic spleen cells efficiently induced cytolytic unresponsiveness to donor alloantigens, but B cells contained in this spleen cell population were unable to establish allo-helper interactions with alloreactive CD4+ T cells, suggesting that cytolytic and helper T-cell interactions involved in alloreactivity may be different.

Constitutive expression of bcl-2 in B cells causes a lethal form of lupuslike autoimmune disease after induction of neonatal tolerance to H-2b alloantigens.

The bcl-2 protooncogene has been shown to provide a survival signal to self-reactive B cells, but it fails to override their developmental arrest after encounter with antigen. Furthermore, constitutive expression of bcl-2 in B cells does not promote the development of autoimmune disease in most strains of mice, indicating that signals other than those conferred by bcl-2 are required for long-term survival and differentiation of self-reactive B cells in vivo. To further examine the factors that are required for the pathogenesis of autoimmune disease, we have assessed the effect of bcl-2 overexpression on the development of host-versus-graft disease, a self-limited model of systemic autoimmune disease. In this model, injection of spleen cells from (C57BL/6 x BALB/c)F1 hybrid mice into BALB/c newborn parental mice induces immunological tolerance to donor tissues and activation of autoreactive F1 donor B cells through interactions provided by allogeneic host CD4+ T cells. BALB/c newborns injected with spleen cells from (C57BL/6 x BALB/c)F1 mice expressing a bcl-2 transgene in B cells developed high levels of anti-single-stranded DNA and a wide range of pathogenic autoantibodies that were not or barely detectable in mice injected with nontransgenic spleen cells. In mice injected with transgenic B cells, the levels of pathogenic autoantibodies remained high during the course of the study and were associated with long-term persistence of donor B cells, development of a severe autoimmune disease, and accelerated mortality. These results demonstrate that bcl-2 can provide survival signals for the maintenance and differentiation of autoreactive B cells, and suggest that both increased B cell survival and T cell help play critical roles in the development of certain forms of systemic autoimmune disease.