Stage-specific regulation of caspase activity in drosophila oogenesis.

In Drosophila oogenesis, the programmed cell death of germline cells occurs predominantly at three distinct stages. These cell deaths are subject to distinct regulatory controls, as cell death during early and midoogenesis is stress-induced, whereas the cell death of nurse cells in late oogenesis is developmentally regulated. In this report, we show that the effector caspase Drice is activated during cell death in both mid- and late oogenesis, but that the level and localization of activity differ depending on the stage. Active Drice formed localized aggregates during nurse cell death in late oogenesis; however, active Drice was found more ubiquitously and at a higher level during germline cell death in midoogenesis. Because Drice activity was limited in late oogenesis, we examined whether another effector caspase, Dcp-1, could drive the unique morphological events that occur normally in late oogenesis. We found that premature activation of the effector caspase, Dcp-1, resulted in a disappearance of filamentous actin, rather than the formation of actin bundles, suggesting that Dcp-1 activity must also be restrained in late oogenesis. Overexpression of the caspase inhibitor DIAP1 suppressed cell death induced by Dcp-1 but had no effect on cell death during late oogenesis. This limited caspase activation in dying nurse cells may prevent destruction of the nurse cell cytoskeleton and the connected oocyte.

Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3.

The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kappa B family. v-Rel causes rapidly fatal lymphomas in young chickens, and transforms and immortalizes chicken lymphoid cells in vitro. Several mutations that have enhanced the oncogenicity of v-Rel have been selected during in vitro and in vivo passage of v-Rel-containing retroviruses. In this report, we show that the C-terminal deletion and two point mutations (Asp-->Gly at residue 91 and Asp-->Asn at residue 437) in v-Rel make it resistant to cleavage by the cell-death protease caspase-3. In contrast, c-Rel, which has Asp residues at these sites, can be cleaved by caspase-3 in vitro as well as in vivo in cells induced to undergo apoptosis. We have characterized activities of v-Rel mutants with recreated single caspase-3 cleavage sites, two cleavage sites, or an introduced artificial cleavage site. All of these mutant v-Rel proteins are sensitive to caspase-3 cleavage in vitro, and show wild-type activity in terms of nuclear localization in chicken fibroblasts and DNA binding in vitro. Moreover, all caspase-3-sensitive v-Rel mutants transform chicken spleen cells in vitro and induce fatal lymphoid tumors in vivo to approximately the same extent as wild-type v-Rel. As with v-Rel mutants, caspase-3-resistant c-Rel mutants behave similarly to caspase-3-sensitive wild-type c-Rel in terms of DNA binding, transcriptional activation, in vitro transformation, and tumorigenicity. Mammalian c-Rel proteins can also be cleaved by caspase-3 in vitro, and a c-Rel mutant from a human pre-T lymphoma cell line is less sensitive than wild-type human c-Rel to cleavage by caspase-3. Taken together, these results demonstrate that specific mutations render oncogenic forms of Rel proteins resistant to cleavage by a cell-death caspase; however, the biological relevance of this resistance remains unclear. Nevertheless, to our knowledge, this is the first demonstration of mutations in caspase-3 recognition sites occurring during the evolution of an oncogenic protein.

Control of apoptosis by Rel/NF-kappaB transcription factors.

Apoptosis is a physiological process critical for organ development, tissue homeostasis, and elimination of defective or potentially dangerous cells in complex organisms. Apoptosis can be initiated by a wide variety of stimuli, which activate a cell suicide program that is constitutively present in most vertebrate cells. In diverse cell types, Rel/NF-kappaB transcription factors have been shown to have a role in regulating the apoptotic program, either as essential for the induction of apoptosis or, perhaps more commonly, as blockers of apoptosis. Whether Rel/NF-kappaB promotes or inhibits apoptosis appears to depend on the specific cell type and the type of inducer. An understanding of the role of Rel/NF-kappaB transcription factors in controlling apoptosis may lead to the development of therapeutics for a wide variety of human diseases, including neurodegenerative and immune diseases, and cancer.

Phosphorylation of IkappaB-alpha inhibits its cleavage by caspase CPP32 in vitro.

IkappaB proteins function as direct regulators of Rel/NF-kappaB transcription complexes. We show that the cell-death protease CPP32 (caspase-3) in vitro specifically cleaved chicken and human IkappaB-alpha at a conserved Asp-Ser sequence. This cleavage site appears to be identical to the site at which chicken IkappaB-alpha is cleaved in vivo in temperature-sensitive v-Rel-transformed chicken spleen cells undergoing apoptosis. Other caspases, namely interleukin-1beta-converting enzyme (caspase-1) and Ich-1 (caspase-2), did not cleave IkappaB-alpha. CPP32 also cleaved mammalian IkappaB-beta in vitro at the analogous Asp-Ser sequence. Cleavage of IkappaB-alpha by CPP32 was blocked by serine phosphorylation of IkappaB-alpha. Cleavage of IkappaB-alpha by a CPP32- like protease could generate a constitutive inhibitor of Rel transcription complexes. This report provides evidence for a direct biochemical interaction between the NF-kappaB signaling pathway and a cell-death protease signaling pathway.

Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1.

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.

Hematopoietic growth factor receptor genes as markers of lineage commitment during in vitro development of hematopoietic cells.

We have used two in vitro models to identify genes whose expression may serve as markers of lineage commitment during the development of hematopoietic stem cells. One system involves the development in vitro of blastocyst-derived embryonic stem cells into embryoid bodies. The second involves culturing of day 3.5 blastocysts in vitro under conditions that support their development into yolk saclike cysts. In both cases, hematopoietic cells arise in a manner that closely mimics the normal process occurring in the yolk sac of the early mouse embryo. We have focused our analysis on the expression of mRNAs for 15 hematopoietic growth factor receptor genes and other genes expressed in a hematopoietic lineage-specific manner. Although some growth factor receptor genes are apparently expressed constitutively during in vitro development, there are several classes of genes that undergo a highly consistent pattern of induction in both model systems. Genes induced early include those encoding the shared beta subunits of the interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors; those induced at intermediate times include the c-fms, G-CSF receptor, and CD34 genes; and a gene induced late during in vitro development is the IL-7 receptor gene. The defined temporal order for the expression of these genes suggests that they may be useful as markers for multiple stages in the development of different hematopoietic cell lineages during embryogenesis.