Postnuclear Extracts Research Articles

Poly r(C) Binding Protein 1 Regulates Posttranscriptional Expression of the Ubiquitin Ligase TRIM56 in Ovarian Cancer.

Our earlier work has shown that the E3 ligase TRIM56 messenger RNA (mRNA) level and vimentin protein expression followed an inverse correlation in ovarian carcinoma patients; however, the regulatory mechanisms underlying TRIM56 expression is unclear. Steady state expression of TRIM56 mRNA expression in the normal ovarian cell line Moody and ovarian cancer cell lines SKOV-3, A2780, and Caov-3 were not significantly different; however, TRIM56 protein expression was significantly lower in the ovarian cancer cell lines compared to the Moody cell line. Polysome profiling showed significant increase in translation of TRIM56 messenger RNA in the Moody cells compared to the SKOV-3 cells. We performed RNA-affinity pulldown using biotinylated TRIM56 5 'and 3'-UTR and postnuclear extracts from Moody and SKOV-3 cells. Whereas no notable difference was observed in affinity pull-down obtained with the 5'-UTR, there was obvious difference in protein binding patterns with the 3'-UTR. Mass spectrometry was used to determine the most differentially binding protein as poly r (c) binding protein 1 (PCBP1). PCBP1 expression and binding to the 3'-UTR was both higher in SKOV-3 cells compared to the Moody cells. Silencing of TRIM56 in Moody cells cause an increase in in vitro migration and invasion, and a similar effect was mimicked by overexpression of PCBP1. Conversely, silencing of PCBP1 or overexpression of TRIM56 in SKOV-3 cells significantly decreased in vitro migration and invasion. In xenograft assays, SKOV-3 cells stably overexpressing shRNA targeting PCBP1 decreased metastasis, whereas shRNA-targeting TRIM56 potentiated detection of metastatic lesions, compared to the parental SKOV-3 cells themselves. Taken together our results reveal a yet undefined posttranscriptional regulatory mechanism underlying low expression of TRIM56 in ovarian cancer. © 2018 IUBMB Life, 71(1):177-182, 2019.

Involvement of inhibitory factors in the inefficient entry of HIV-1 into the human CD4 positive HUT78 cell line

Little is known about whether human CD4 positive T cells, the principal natural target of HIV-1, have intrinsic factors, other than the receptor/coreceptor molecules, which modulate the entry efficiency of HIV-1. In the present study, we found that human T cell lines, HUT78 and PM1, were less permissive to VSV-G-mediated HIV-1 infection compared with the Jurkat cell line. Furthermore, HUT78 cells were also less sensitive to HIV-1 Env-mediated infection, while PM1 cells became susceptible to HIV-1. Real-time PCR analyses showed that less susceptibility of the cells to HIV-1 was due to block at, or prior to, reverse transcription of viral RNA. To clarify the entry efficiency of HIV-1 into these cell lines, we analyzed the internalization of p24 Ag into the cytosolic and vesicular fractions of post-nuclear extracts at 4 h post-infection. When the cells were infected with HIV-1 pseudotyped with VSV-G, the amount of p24 Ag in the cytosolic fractions in both HUT78 and PM1 cells was lower than that observed in Jurkat cells. In the case of HIV-1 Env-mediated infection, however, PM1 cells exhibited comparable amounts of p24 Ag in the cytosolic fraction compared with Jurkat cells, while the amount of p24 Ag in HUT78 cells remained low. Heterokaryon experiments between susceptible and less susceptible cell lines suggested that some inhibitory factors counteracted VSV-G-mediated viral entry in PM1 and HUT78 cells, and HIV-1 Env-mediated viral entry in HUT78 cells.

Origin of a metal-binding protein in serum of Mytilus edulis

The most abundant humoral protein of the haemolymph of Mytilus edulis is known to bind a variety of heavy metals (Renwrantz et al., 1998Comp. Biochem. Physiol. A, 121: 175–180). This serum protein band 1 (SPB1) was isolated from Mytilus serum by a two-step purification procedure. For the purified protein a molecular weight of 34 kDa was estimated under denaturing conditions and of 37 kDa in its reduced form. After blotting of Mytilus serum onto a nitrocellulose membrane, Cu-binding by SPB1 was visualized. Furthermore, indications were obtained of Ca-binding properties of SPB1 and of some bands postulated to be SPB1-oligomers. Formation of dimers was confirmed by their reaction with SPB1-specific antibodies. On immunoblots, anti-SPB1 detected the metal-binding protein in postnuclear haemocyte extracts. After further fractionation of the cell homogenate by sucrose gradient centrifugation, the indicator antibodies recognized SPB1 in the cytosol, as a component of the plasma membrane and in the fraction of granules which was assumed to include secretory vesicles. Investigation of the supernatant from a 24 h haemocyte culture indicated release of the metal-binding protein by blood-cells. Incubations of different haemocyte monolayers on slides with anti-SPB1 indicated expression of SPB1 in amounts of 55% to almost 100% of the cells, whereby the intensity of antibody-staining varied between individual haemocytes. Intensity was independent of cell type as density gradient separation of haemocytes into basophilic and eosinophilic granulocytes resulted in similar staining patterns in both groups. Thus, all granulocytes seemed to be able to produce SPB1 with a varying degree of activity indicating a hitherto unknown regulation system.

Glia Protects Neurons against Extracellular Human Neuromelanin

Background: Neuromelanin-containing neurons of the substantia nigra are highly vulnerable to degenerate in Parkinson’s disease. Inhibition of the respiratory chain or formation of reactive oxygen species (ROS) by intracellular neuromelanin and triggering of inflammatory processes by extracellular neuromelanin emanating from melanized neurons after their demise are thought to be causally implicated in the high vulnerability of melanized neurons. Objective: We addressed the direct effect of purified neuromelanin on mitochondrial complex I activity, and its influence on ROS production and survival of primary mesencephalic neurons in the presence or absence of glia. Methods: Neuromelanin was isolated from midbrain of postmortem human brains. The content in iron and other elements was measured by inductively coupled mass spectrometry. The effect of neuromelanin on mitochondrial complex I activity was analyzed in post-nuclear extracts. Primary neuronal enriched and neuron-glia mixed cultures from midbrain were treated with different concentrations of neuromelanin. The generation of ROS was determined by fluorochrome detection. MAP2-positive and TH-positive neuronal viability was analyzed. Results: Neuromelanin did not affect complex I activity, but concentration-dependently increased ROS production in neurons and reduced the number of MAP2-positive and TH-positive cultured neurons. Glia protected neurons against the neuromelanin toxicity. Conclusion: Extracellular neuromelanin is detrimental to neurons implicating a mechanism of intracellular ROS production, but not complex I inhibition. ROS formation may be catalyzed by iron, which was sensitively identified in purified neuromelanin (3.3 mg/g). Importantly, we demonstrate that glial cells have the potential to mitigate the neurotoxic effect of neuromelanin.

Mammalian Smaug Is a Translational Repressor That Forms Cytoplasmic Foci Similar to Stress Granules

Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile alpha motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons.

Molecular Cloning of a Sixth Member of the K+-dependent Na+/Ca2+ Exchanger Gene Family, NCKX6

Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K(+)-dependent Na+/Ca2+ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the alpha-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K(+)-dependent Na+/Ca2+ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na+/Ca2+ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K(+)-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis in diverse tissues and cell types.

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism.

The selective RNA-binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src-PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src-PTK-dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src-PTK-dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.

Purification and characterization of a novel intracellular 17 beta-estradiol binding protein in estrogen-resistant New World primate cells.

Compared to Old World primates, including man, New World primates display target-tissue resistance to gonadal steroid hormones. In female New World primates this resistant phenotype is characterized by elevated concentrations of plasma estradiol and progesterone. Here we describe the discovery of an intracellular estrogen binding protein (IEBP) that acts to concentrate 17 beta-estradiol (E2) in the cytoplasm of New World primate target cells. IEBP was purified by E2-affinity chromatography from postnuclear extracts of the B95-8 cells established from an E2-resistant New World primate. Compared with unpurified extract, affinity-purified IEBP demonstrated a 300-fold enrichment in specific E2 binding activity; half-maximal displacement of [3H]E2 from affinity-purified IEBP was observed with 0.1 nM E2. Affinity-purified extracts were subjected to SDS-PAGE with isolation of a dominant 27-28 kDa protein. N-terminal sequencing of tryptic peptides of the protein showed sequence homology with human heat shock protein-27 (hsp27). By immunoblot and E2 binding capacity, IEBP was 1] 2-3-fold greater in New World than in Old World primate tissues and cell lines, 2] heat-inducible and 3] up-regulated in vivo in the presence of the functioning female gonad. In conclusion, IEBP is a specific E2-interacting heat shock protein in the hsp-27 family that is relatively overexpressed in estrogen-resistant cells.

Poly(ADP-ribose) degradation by post-nuclear extracts from human cells

The nuclear metabolism of poly(ADP-ribose) is mainly regulated by poly(ADP-ribose) polymerase-1 (PARP-1) and by poly(ADP-ribose) glycohydrolase (PARG). A PARP-like enzyme, V-PARP, and a PARG isoform are present in the extra-nuclear compartment of mammalian cells, even if poly(ADP-ribose) has never been detected therein. In this work, we demonstrate the ability of post-nuclear extracts from HeLa and HL60 cells to degrade synthetic 32P-polymers of ADP-ribose to ADP-ribose and, further, to AMP. This reaction implies the combined action of PARG and of an ADP-ribose-degrading activity, possibly corresponding to a phosphodiesterase and/or to an ADP-ribose pyrophosphatase. The inhibition of PARG or ADP-ribose-degrading enzymes allowed the demonstration that in vitro synthesized 32P-poly(ADP-ribose) is first digested to ADP-ribose monomers by a typical PARG reaction, and that ADP-ribose is further rapidly converted into AMP by an Mg 2+-dependent activity. Collectively, our results demonstrate the ability of the human cell post-nuclear fraction to convert synthetic poly(ADP-ribose) into utilizable AMP units by the concerted action of PARG and ADP-ribose-degrading activities.

Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release.

BID, a pro-apoptotic Bcl-2 family member, promotes cytochrome c release during apoptosis initiated by CD95L or TNF. Activation of caspase-8 in the latter pathways results in cleavage of BID, translocation of activated BID to mitochondria, followed by redistribution of cytochrome c to the cytosol. However, it is unclear whether BID participates in cytochrome c release in other (non-death receptor) cell death pathways. Here, we show that BID is cleaved in response to multiple death-inducing stimuli (staurosporine, UV radiation, cycloheximide, etoposide). However BID cleavage in these contexts was blocked by Bcl-2, suggesting that proteolysis of BID occurred distal to cytochrome c release. Furthermore, addition of cytochrome c to Jurkat post-nuclear extracts triggered breakdown of BID at Asp-59 which was catalysed by caspase-3 rather than caspase-8. We provide evidence that caspase-3 catalysed cleavage of BID represents a feedback loop for the amplification of mitochondrial cytochrome c release during cytotoxic drug and UV radiation-induced apoptosis.

Preferential Perinuclear Localization of Poly(ADP-ribose) Glycohydrolase

The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.

Spermine triggers the activation of caspase-3 in a cell-free model of apoptosis

Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post-nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP-dependent processing of pro-caspase-3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro-apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.

Molecular Cloning of a Novel Potassium-dependent Sodium-Calcium Exchanger from Rat Brain

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.

Discovery of Human Antibodies to Cell Surface Antigens by Capture Lift Screening of Phage-Expressed Antibody Libraries

An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates. This screening method, termed capture lift, results in the immobilization of greater quantities of Fab and decreases the binding of unrelated host proteins, resulting in a more sensitive plaque lift assay. The capture lift permits the simulataneous analysis of thousands of antibody clones and, more importantly, can be used with crude detergent-solubilized cell extracts, permitting the discovery of Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Optimal conditions were identified utilizing phage-expressed BR96 Fab and a horseradish peroxidase conjugate of Lewis Y, a soluble cross-reactive antigen. Subsequently, it was demonstrated that the assay was functional with postnuclear detergent extracts isolated from surface-biotinylated tumor cells expressing the BR96 tumor antigen. Purification of the target antigen was not required. To demonstrate the application of the capture lift assay for the discovery of Fabs reactive with novel cell surface antigens a phage-expressed human antibody library constructed from tumor-infiltrating B lymphocytes was screened. Multiple antibody clones which reacted with detergent-solubilized biotinylated surface antigens were identified. Upon further characterization a portion of these displayed selectivity for tumor cells, as demonstrated by the binding of Fab to fixed and live tumor cells but not normal fibroblasts.

Syk is required for BCR-mediated activation of p90Rsk, but not p70S6k, via a mitogen-activated protein kinase-independent pathway in B cells.

The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk- and DT40Lyn- B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn- cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk- and DT40Lyn- cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines.

Functional Characterization and Purification of an Intracellular Vitamin D-binding Protein in Vitamin D-resistant New World Primate Cells: AMINO ACID SEQUENCE HOMOLOGY WITH PROTEINS IN THE HSP-70 FAMILY

Most genera of New World primates exhibit resistance to vitamin D. These monkeys harbor high circulating concentrations of the prohormone 25-hydroxyvitamin D and the active vitamin D hormone 1, 25-dihydroxyvitamin D. Previous work from this laboratory indicated that resistance is associated with the overexpression of a 60-65-kDa intracellular protein that binds vitamin D metabolites competitively. In the current studies 25-[3H]hydroxyvitamin D3 (25-OHD3) was used as a competitive ligand to investigate the ability of a number of small lipid molecules to interact with this intracellular vitamin D-binding protein (IDBP) in post-nuclear extracts of a prototypical lymphoblast cell line from the common marmoset, a vitamin D-resistant New World primate. Only those vitamin D metabolites with a hydroxyl moiety in the C-25 position were bound by IDBP. Disruption of the C-25 hydroxyl obviated binding, whereas more proximal alterations in the vitamin D side chain did not. Modifications in the A-ring of 25-hydroxylated vitamin D metabolites, most specifically hydroxylation of C-1, diminished but did not abolish ligand binding. Of more than two dozen other small lipid molecules examined, only the C-19 17-hydroxysteroids, 17beta-estradiol and testosterone, and the C-21 steroid progesterone were found to be capable of binding specifically to IDBP. Using a combination of physical and serial chromatographic techniques, we enriched IDBP 25-OHD3 binding activity 17,588-fold in extracts of B95-8 cells. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified fraction demonstrated a predominant 65-kDa molecular species with a pI approximately 4.5. Seven different peptide fragments were isolated from the 65-kDa protein, each possessing sequence similarity to the hsp-70 family of proteins. Ligand binding analyses confirmed that human inducibly expressed hsp-70-bound 25-OHD3 with approximately similar affinity ( approximately 10(-7) M) as did purified IDBP. In summary, these results suggest a novel action for the hsp-70 family of proteins as intracellular vitamin D- and gonadal steroid hormone-binding molecules.

Vitamin D and gonadal steroid-resistant New World primate cells express an intracellular protein which competes with the estrogen receptor for binding to the estrogen response element.

New World primates (NWP) exhibit a form of compensated resistance to vitamin D and other steroid hormones, including 17beta-estradiol. One postulated cause of resistance is that NWP cells overexpress one or more proteins which block hormone action by competing with hormone for its cognate hormone response element. Here we report that both nuclear and postnuclear extracts from NWP, but not Old World primate, cells contained a protein(s) capable of binding directly to the estrogen response element (ERE). This ERE binding protein(s) (ERE-BP) was dissociated from the ERE by excess of either unlabeled ERE or excess of the ERE half-site motif AGGTCAcag. DNA affinity chromatography using concatamers of the latter resulted in > 20,000-fold purification of the ERE-BP. The intensity of the ERE-BP-ERE complex in electromobility shift assay was indirectly related to the amount of wild-type Old World primate estrogen receptor (ER) but not affected when potential ligands, including 17beta-estradiol (up to 100 nM), or anti-ER antibody was added to the binding reaction. We conclude that vitamin D-resistant and gonadal steroid-resistant NWP cells contain a protein(s) that may "silence" ER action by interacting directly with the ERE and interfering with ER binding.

Inhibition of vitamin D receptor-retinoid X receptor-vitamin D response element complex formation by nuclear extracts of vitamin D-resistant New World primate cells.

Most New World primate (NWP) genera evolved to require high circulating levels of steroid hormones and vitamin D. We hypothesized that an intracellular vitamin D binding protein (IDBP), present in both nuclear and cytoplasmic fractions of NWP cells, or another protein(s) may cause or contribute to the steroid hormone-resistant state in NWP by disruption of the receptor dimerization process and/or by interference of receptor complex binding to the consensus response elements present in the enhancer regions of steroid-responsive genes. We employed electromobility shift assay (EMSA) to screen for the presence of proteins capable of binding to the vitamin D response element (VDRE). Nuclear and post-nuclear extracts were prepared from two B-lymphoblastoid cell lines known to be representative of the vitamin D-resistant and wild type phenotypes, respectively. The extracts were compared for their ability to retard the migration of radiolabeled double stranded oligomers representative of the VDREs of the human osteocalcin and the mouse osteopontin gene promoters. A specific, retarded band containing VDR-RXR was identified when wild type cell but not when vitamin D-resistant cell nuclear extract was used in the binding reaction with either probe. In addition, vitamin D-resistant cell nuclear extract contained a protein(s) which was bound specifically to the VDRE and was capable of completely inhibiting VDR-RXR-VDRE complex formation; these effects were not demonstrated with nuclear extract from the wild type cell line or with the post-nuclear extract of the vitamin D-resistant cell line. We conclude that a VDRE-binding protein(s), distinct from IDBP and present in nuclear extract of cells from a prototypical vitamin D-resistant NWP, is capable of inhibiting normal VDR-RXR heterodimer binding to the VDRE.

Lytic reaction of in vivo primed peritoneal exudate CTL. Induction of high-conductance single channels in the target cell membrane.

CTL, primary effectors in immune responses, deliver a "lethal hit" signal to target cells, causing their destruction. The precise membrane events associated with the lethal hit remain elusive. We investigated the signal(s) mediating destruction of tumor target cells (EL4) by perforin-deficient peritoneal exudate CTL (PEL). We utilized patch clamp techniques to record electrophysiological events associated with the cytolytic interaction of PEL and EL4 in isolated conjugates. PEL-EL4 interaction resulted in induction in EL4 cells, of single channels (followed by EL4 destruction), with a mean conductance of 437 pS and a reversal potential of -1.0 mV, suggestive of nonselective pathways. Similar channels were induced in EL4 cells conjugated with perforin-rich PEL blasts (PEB), by perforin, postnuclear extract from PEL (pnPEL) and from other cytotoxic lymphocytes, but not from noncytolytic lymphocytes. As similar channels were induced by pnPEL in EL4 membrane patches, we propose that these channels result from a direct effect of PEL-derived channel-forming substance(s) on the target cell's membrane. Importantly, postnuclear extracts from perforin-devoid cytotoxic PEL-hybridomas induced similar channels, suggesting the presence of a nonperforin, channel-forming activity in PEL and PEL-hybridomas. Based on the present study, we conclude that the delivery of the lethal hit by cytolytic PEL and PEL-hybridoma is associated with induction in the target cell of high-conductance channels, which most likely mediate its destruction. We propose that these channels are related to the Fas pathway of lymphocytotoxicity.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.