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 tyrosinephosphorylated IR ␤-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 ␤-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 ␤-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 insulininduced 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.Insulin binding to its cell surface transmembrane receptor stimulates intrinsic protein tyrosine kinase activity, autophosphorylation, and subsequent tyrosyl phosphorylation of insulin receptor substrate (IRS) proteins and adapter proteins such as Shc and Cbl/CAP (reviewed in references 5, 40, 54, and 55). Tyrosyl phosphorylation of IRS and adapter proteins generates docking sites for src homology 2 (SH2) domain-containing signaling proteins such as the p85 subunit of phosphatidylinositol 3-kinase. Collectively, these signaling proteins mediate the biological effects of insulin, which include antilipolysis, glucose uptake, glycogen synthesis, and cell growth (reviewed in references 5, 40, 54, and 55).Protein tyrosine phosphatases (PTPs) have a prominent role in the control of insulin receptor (IR) signaling. PTPs dephosphorylate the IR and its substrates and thus serve to inactivate the IR and terminate signaling. The IR and IRS proteins are rapidly and transiently phosphorylated in response to insulin, with phosphotyrosine (pTyr) content in the IR returning to basal levels within minutes of stimulation (10, 16). It is probable that numerous PTPs participate in the dephosphorylation of the IR and the multiple downstream tyrosine-phosphorylated signaling proteins. Several PTPs have been implicated in the negative regulation of the IR, including the endoplasmic reticulum-associated PTP1B (6,8,49,50) and the transmembrane PTPs LAR (34) and PTP␣ (30,41). PTP␣ has been shown to inhibit insulin-induced prolactin gene expression without altering the phosphorylation status of the IR, IRS-1, or Shc (26). Similarly, LAR may not dephosphorylate the IR in vivo but may downregulate insulin signaling by dephos...

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.

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. INTRODUCTIONProgrammed cell death is a means whereby metazoans can remove unwanted cells, with failure of programmed cell death enabling cancer and autoimmune disease, and inappropriate cell death contributing to neurodegenerative disease. Several of the proteins that regulate cell death, including Bcl-2 family members, signal-transduction receptors, effector proteases and DIABLO/Smac, are specifically enclosed within subcellular structures allowing precise control over the commitment of a cell to die. Understanding where and how these key regulators are localized is crucial in understanding their normal biological function and their role in the pathogenesis of malignant diseases.A family of intracellular proteases called caspases implement programmed cell death (Ekert et al., 1999). The activity of caspases is regulated by a family of inhibitor of apoptosis proteins (IAPs) that bind and neutralize active caspases (Deveraux and Reed, 1999). For example, the inhibitor MIHA/XIAP/hILP/BIRC4 can bind and inhibit processed caspases 3, 7, and 9 (Duckett et al., 1996;Liston et al., 1996;Uren et al., 1996;Deveraux et al., 1997Deveraux et al., , 1998. This damping of caspase activity provides a layer of regulation over the cell death-promoting activities of this family of effector proteases.In mammalian cells, signals for cell death can lead to rupture of the mitochondrial outer membrane, and under these conditions the inhibition of caspases can be antagonized by the mitochondrial proteins DIABLO/Smac Verhagen et al., 2000) and HtrA2/Omi (Suzuki et al., 2001;Martins et al., 2002;Verhagen et al., 2002). Structural studies Liu et al., 2000;Wu et al., 2000) have shown that purified DIABLO is a homodimer , and each DIABLO dimer can bind to the BIR domains of inhibitor proteins via contacts made to the N-terminal residues of DIABLO (Liu et al., 2000;Wu et al., 2000). The avid interaction DIABLO makes with the inhibitor MIHA competes the inhibitor away from active caspase 9, freeing caspase 9 to proteolytically activate downstream caspases .Mouse DIABLO is translated as a 237-residue precursor protein (preDIABLO) with an N-terminal presequence that must be cleaved to generate the mature form with the amino-ter...

Context:The pathogenic origin of preeclampsia is defective placental development (placentation) and function. Preeclampsia is not diagnosed until later in pregnancy, and reliable early detection is highly desirable. HtrA3 is a recently cloned gene with high expression during placentation in the mouse, rhesus monkey, and human.Objective: The present study examined the placental production and the serum profile of HtrA3 across gestation in women, the potential molecular mechanisms regulating HtrA3 production, and the association between maternal HtrA3 serum levels and preeclampsia. Methods:Immunohistochemistry determined HtrA3 expression pattern and cellular localization in first-, second-, and third-trimester placenta. The maternal serum HtrA3 levels were analyzed by Western blotting. The regulation of placental HtrA3 production and the secretion by oxygen tension was investigated in first-trimester placental explants and trophoblast cells.Results: Placental HtrA3 protein was maximally produced in the first trimester and then dramatically down-regulated, especially in the syncytiotrophoblast. HtrA3 was secreted into the maternal circulation with a serum profile reflecting placental production. Oxygen tension regulated HtrA3; low oxygen enhanced, whereas the transition from low to high oxygen decreased, HtrA3 protein production in syncytiotrophoblast. Maternal serum HtrA3 levels at approximately 13-14 wk of gestation were significantly higher in women who subsequently developed preeclampsia. It appeared that HtrA3 down-regulation was delayed in preeclamptic pregnancies. Conclusions:HtrA3 protein production is closely associated with changing in oxygen tension in the placenta. The decline in HtrA3 at the end of first trimester may reflect the placental low to high oxygen switch. Abnormally high levels of serum HtrA3 at approximately 13-14 wk of gestation is associated with preeclampsia. (J Clin Endocrinol Metab 96: 403-411, 2011) P reeclampsia is a serious disorder of human pregnancy affecting 5-10% of pregnant women (1). It contributes significantly to both maternal and fetal morbidity and mortality, accounting for about 18% of maternal and perinatal deaths in industrialized countries (2, 3). Preeclampsia is not diagnosed until it becomes clinically apparent late in pregnancy, at which time delivery is the only effective cure, often inflicting prematurity. The burden of preeclampsia thus also falls on the neonate because fetal survival rates inversely correlate with gestational age; survival rate for a baby born at 23 wk is less than 10% (4).

Mammalian HtrA3 (high temperature requirement A3) is a serine protease of the HtrA family. It has two isoforms [long (HtrA3-L) and short (HtrA3-S)] and is important for placental development and cancer progression. Recently, HtrA3 was identified as a potential diagnostic marker for early detection of preeclampsia, a life-threatening pregnancy-specific disorder. Currently there are no high-throughput assays available to detect HtrA3 in human serum. In this study we generated and fully tested a panel of five HtrA3 mouse monoclonal antibodies (mAbs). Three mAbs recognised both HtrA3-L and HtrA3-S and the other two detected HtrA3-L only. All five mAbs were highly specific to HtrA3 and applicable in western blotting and immunohistochemical analysis of endogenous HtrA3 proteins in the mouse and human tissues. Amplified luminescent proximity homogeneous assays-linked immunosorbent assays (AlphaLISAs), were developed to detect HtrA3 isoforms in picomolar levels in serum. The HtrA3 AlphaLISA detected significantly higher serum levels of HtrA3 in women at 13–14 weeks of gestation who subsequently developed preeclampsia compared to gestational-age matched controls. These HtrA3 mAbs are valuable for the development of immunoassays and characterisation of HtrA3 isoform-specific biology. The newly developed HtrA3 AlphaLISA assays are suitable for large scale screening of human serum.

This study characterised the impact of active metazoan apoptotic proteases (caspases) on Saccharomyces cerevisiae viability. Expression of active caspase-3 or caspase-8 in yeast ruptured plasma and nuclear membranes and dramatically impaired clonogenic survival, but did not damage DNA. Deletion of the proposed yeast apoptosis regulators YCA1 or Aif1p did not affect the ability of human, insect or nematode caspases to kill yeast. These data indicate that expression of active metazoan caspases causes irreversible damage to yeast membranes and organelles, in a manner independent of YCA1 and Aif1p.

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.

A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases.