ABSTRACT
Although the anti-apoptotic activity of Bcl-2 has been extensively studied, its mode of action is still incompletely understood. In the nematode Caenorhabditis elegans, 131 of 1090 somatic cells undergo programmed cell death during development. Transgenic expression of human Bcl-2 reduced cell death during nematode development, and partially complemented mutation of ced-9, indicating that Bcl-2 can functionally interact with the nematode cell death machinery. Identification of the nematode target(s) of Bcl-2 inhibition would help clarify the mechanism by which Bcl-2 suppresses apoptosis in mammalian cells. Exploiting yeast-based systems and biochemical assays, we analysed the ability of Bcl-2 to interact with and regulate the activity of nematode apoptosis proteins. Unlike CED-9, Bcl-2 could not directly associate with the caspase-activating adaptor protein CED-4, nor could it inhibit CED-4-dependent yeast death. By contrast, Bcl-2 could bind the C. elegans pro-apoptotic BH3-only Bcl-2 family member EGL-1. These data prompt us to hypothesise that Bcl-2 might suppress nematode cell death by preventing EGL-1 from antagonising CED-9, rather than by inhibiting CED-4.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Calcium-Binding Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/physiology , Repressor Proteins/metabolism , Animals , Animals, Genetically Modified , Apoptosis/physiology , Apoptosis Regulatory Proteins/metabolism , Caenorhabditis elegans , Humans , Protein Binding , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , Saccharomyces cerevisiae/metabolism , Two-Hybrid System Techniques , bcl-2-Associated X Protein/antagonists & inhibitorsABSTRACT
DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX(5)P in Imp1 and NX(5)S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space.
Subject(s)
Endopeptidases/metabolism , Intracellular Membranes/enzymology , Intracellular Membranes/metabolism , Mitochondria/enzymology , Mitochondrial Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/biosynthesis , Amino Acid Motifs , Amino Acid Sequence , Blotting, Western , Catalytic Domain , Conserved Sequence , Endopeptidases/chemistry , Green Fluorescent Proteins/metabolism , Microscopy, Confocal , Models, Biological , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
The proinflammatory cytokine tumor necrosis factor (TNF) modulates cellular responses through the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways, but the molecular mechanisms underlying MAPK activation are unknown. T cell protein tyrosine phosphatase (TCPTP) is essential for hematopoietic development and negatively regulates inflammatory responses. Using TCPTP-deficient fibroblasts, we show here that TCPTP regulates TNF-induced MAPK but not NF-kappaB signaling. TCPTP interacted with the adaptor protein TRAF2, and dephosphorylated and inactivated Src tyrosine kinases to suppress downstream signaling through extracellular signal-regulated kinases and production of interleukin 6. These results link TCPTP and Src tyrosine kinases to the selective regulation of TNF-induced MAPK signaling and identify a previously unknown mechanism for modulating inflammatory responses mediated by TNF.
Subject(s)
MAP Kinase Signaling System , Protein Tyrosine Phosphatases/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/pharmacology , src-Family Kinases/metabolism , Animals , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Interleukin-6/metabolism , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2 , TNF Receptor-Associated Factor 2/metabolism , Up-RegulationABSTRACT
The human protein tyrosine phosphatase TCPTP exists as two forms: an endoplasmic reticulum-targeted 48-kDa form (TC48) and a nuclear 45-kDa form (TC45). Although targeted to the nucleus, TC45 can exit in response to specific stimuli to dephosphorylate cytoplasmic substrates. In this study, we investigated the downregulation of insulin receptor (IR) signaling by TCPTP. In response to insulin stimulation, the TC48-D182A and TC45-D182A "substrate-trapping" mutants formed stable complexes with the endogenous tyrosine-phosphorylated IR beta-subunit in 293 cells. Moreover, in response to insulin stimulation, the TC45-D182A mutant accumulated in the cytoplasm of cells overexpressing the IR and in part colocalized with the IR beta-subunit at the cell periphery. These results indicate that the IR may serve as a cellular substrate for both TC48 and TC45. In immortalized TCPTP(-/-) murine embryo fibroblasts, insulin-induced IR beta-subunit tyrosine phosphorylation and protein kinase PKB/Akt activation were enhanced relative to the values in TCPTP(+/+) cells. Importantly, the expression of TC45 or TC48 to physiological levels suppressed the enhanced insulin-induced signaling in TCPTP(-/-) cells. These results indicate that the differentially localized variants of TCPTP may dephosphorylate the IR and downregulate insulin-induced signaling in vivo.
