Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells.
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The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS-, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-gamma, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-gamma were combined. (+info)
ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.
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Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event. (+info)
A FERM domain governs apical confinement of PTP-BL in epithelial cells.
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PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins. Here we show, by transient expression of modular protein domains of PTP-BL in epithelial MDCK cells, that the presence of a FERM domain in the protein is both necessary and sufficient for its targeting to the apical side of epithelial cells. Furthermore, immuno-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revealed that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10-15 nm from the plasma membrane. Immunofluorescence microscopy suggested colocalization of the FERM domain moiety with the membrane-cytoskeleton linker ezrin. However, at the electron microscopy level this colocalization cannot be confirmed nor can we detect a direct interaction by immunoprecipitation assays. Fluorescence recovery after photobleaching (FRAP) experiments show that PTP-BL confinement is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes. Our observations suggest that relocation is mediated via a cytosolic pool, rather than by lateral movement. Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays. Both the highly restricted subcellular compartmentalization and its specific associative properties may provide the appropriate conditions for regulating substrate specificity and catalytic activity of this member of the PTP family. (+info)
Identification and molecular characterization of BP75, a novel bromodomain-containing protein.
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We here describe the identification and characterization of a novel bromodomain-containing protein, the bromodomain protein of 75 kDa (BP75). Initially, we identified BP75 in a two-hybrid screening for proteins that interact with the first PDZ (acronym for post-synaptic density protein PSD-95, Drosophila discs large tumor suppressor DlgA and the tight junction protein ZO-1) domain in protein tyrosine phosphatase-BAS-like (PTP-BL). We found that BP75 is expressed ubiquitously and show that both BP75 and a PTP-BL deletion mutant consisting of the first PDZ domain are located mainly in the nucleus, although cytoplasmic localization is also evident. Full-length PTP-BL, on the contrary, is predominantly localized in the cytoplasm, although some basal nuclear staining is observed. The described molecular interaction may reflect a mechanism of coupling submembraneous signalling events and nuclear events. (+info)
Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.
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The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain. (+info)
Semiquantitative reverse transcription-polymerase chain reaction to evaluate the expression patterns of genes involved in the oestrogen pathway.
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The increasing number of factors to be taken into account in the oestrogen transcriptional process has created a need to develop a rapid screening method to evaluate their role in physiology and pathology. Molecular biology techniques enable gene expression studies at the mRNA level with small amounts of tissues. We therefore developed a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique using fluorescent oligonucleotides to analyse simultaneously a large panel of interrelated genes involved in the oestrogen transcriptional pathway using a moderately expressed housekeeping gene, the hypoxanthine phosphoribosyltransferase gene (HPRT), as the reference gene. Expression levels of oestrogen receptors (ERalpha, ERbeta), cofactors AIB1, RIP140, SMRT and the Fas-associated protein-tyrosine phophatase-1 (FAP-1) genes were evaluated in breast, endometrial and ovarian cancer cell lines and in three ERalpha-positive and three ERalpha-negative breast cancer tumours. This technique provides a rapid and reliable way to quantitate simultaneously numerous mRNAs of genes involved in the oestrogen pathway from small amounts of tissues. (+info)
Mechanism of chronic obstructive uropathy: increased expression of apoptosis-promoting molecules.
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BACKGROUND: We have demonstrated that renal tubular and interstitial cells undergo pronounced apoptosis during the course of chronic obstructive uropathy (COU). Apoptosis is a complex cellular process consisting of multiple steps, each of which is mediated by families of related molecules. These families may include receptor/ligand molecules such as Fas, Fas ligand, tumor necrosis factor receptor-1 (TNFR-1), and TNF-related apoptosis inducing ligand (TRAIL); signal transduction adapter molecules such as Fas-associated death domain (FADD), TNFR-1 associated death domain (TRADD), receptor-interacting protein (RIP), Fas-associated factor (FAF), and Fas-associated phosphatase (FAP); or effector molecules such as caspases. However, the mechanism of tubular cell apoptosis, as well as the pathogenetic relevance of these apoptosis-related molecules in COU, remains poorly understood. METHODS: Kidneys were harvested from sham-operated control mice and mice with COU created by left ureter ligation sacrificed in groups of three at days 4, 15, 30, and 45. To detect apoptotic tubular and interstitial cells, in situ end labeling of fragmented DNA was performed. To detect the expression of apoptosis-related molecules, ribonuclease protection assay was used with specific antisense RNA probes for Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAF, FAP, and caspase-8. Immunostaining for Fas, Fas ligand, TRAIL, TRADD, RIP, and caspase-8 was also performed. To assess the role of these molecules in COU-associated renal cell apoptosis, the frequencies of apoptotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated with those of apoptosis-related molecules. RESULTS: The obstructed kidneys displayed increased apoptosis of both tubular and interstitial cells. Tubular cell apoptosis appeared at day 4 after ureter ligation, peaked (fivefold of control) at day 15, and decreased gradually until the end of the experiment. In contrast, interstitial cell apoptosis sustained a progressive increase throughout the experiment. Apoptosis was minimal at all experimental time points for control and contralateral kidneys. Compared with control and contralateral kidneys, the ligated kidneys displayed a dynamic expression of mRNAs for many apoptosis-related molecules, which included an up to threefold increase for Fas, Fas ligand, TNF-R1, TRAIL, TRADD, RIP, and caspase-8, and an up to twofold increase for FADD and FAP, but there was little change for FAF. These mRNAs increased between days 4 and 15, decreased until day 30, but then increased again until day 45. The rise and fall of mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis in that period. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis. We demonstrated a positive immunostaining for Fas and Fas ligand in the tubular cells at early time points as well as in interstitial inflammatory cells at later time points. Although increased expression of TRAIL, TRADD, RIP, and caspase-8 was noted in tubular cells, there was no staining for these molecules in interstitial cells. CONCLUSION: The current study documents a dynamic expression of several molecules that are known to mediate the most crucial steps of apoptosis. It implicates these molecules in COU-associated renal cell apoptosis and in the pathogenesis of this condition. It also lays the foundation for interventional studies, including genetic engineering, to evaluate the molecular control of apoptosis associated with COU. (+info)
Identification of IkappaBalpha as a substrate of Fas-associated phosphatase-1.
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Fas (APO-1/CD95), a member of the tumor necrosis factor receptor (TNFR)/nerve growth factor receptor (NGFR) superfamily, is a cell-surface molecule that induces apoptosis upon activation. Fas-associated phosphatase-1 (FAP-1) is a 250-kDa protein tyrosine phosphatase (PTP) that is associated with the negative regulatory domain of Fas (C-terminal 15 amino acids). Human tumor cell lines become resistant to Fas-mediated apoptosis when transfected with FAP-1, indicating that FAP-1 functions as a negative regulator in Fas-mediated death signaling. However, the mechanisms by which FAP-1 inhibits apoptosis are still unclear. In order to determine how FAP-1 affects the signaling mediated by Fas, we set out to identify substrates of FAP-1. Toward this end, we prepared synthetic proteins with either the catalytic domain of FAP-1 (C-terminal 399 amino acids) or its inactive form (Cys2408-->Ser) fused to glutathione-S-transferase (GST). Using an in vitro dephosphorylation reaction, we found that FAP-1 dephosphorylates IkappaBalpha. Furthermore, a substrate trapping mutant was found to bind tyrosine-phosphorylated IkappaBalpha. Taken together, our data confirm that IkappaBalpha is a substrate of FAP-1. (+info)