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At least 1,135 records · Page 63Linked to original sources
PubMed1997

Bik and Bak induce apoptosis downstream of CrmA but upstream of inhibitor of apoptosis.

Recent studies have identified a number of cell death pathway components. In this study, we describe the role that two such components, Bik and Bak, play in initiating the apoptotic program. These Bcl-2 family members engage the death pathway downstream of the block imposed by the serpin CrmA, but upstream of the block initiated by cellular inhibitors of apoptosis, which are a family of molecules characterized by a conserved baculovirus inhibitor of apoptosis repeat motif. Distal death pathway components activated by Bik and Bak are similar to those activated by the CD-95 (Fas/Apo1) and tumor necrosis factor death receptors.

Apoptosis↗
PubMed1997

Cis-elements required for the demethylation of the mouse M-lysozyme downstream enhancer.

The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.

Animals↗
PubMed1997

Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation.

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.

Base Sequence↗
PubMed1997

Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells.

Interaction of type I collagen (COL(I)) with alpha2beta1 integrin causes differentiation and transforming growth factor (TGF)-beta receptor down-regulation in osteoblastic cells (Takeuchi, Y., Nakayama, K., and Matsumoto, T. (1996) J. Biol. Chem. 271, 3938-3644). The TGF-beta receptor down-regulation enables cells to escape from the inhibition of differentiation by TGF-beta. To clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL(I) stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase by herbimycin A, destruction of focal adhesion by cytochalasin D, or overexpression of antisense FAK mRNA prevented the activation of ERK/MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-beta receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-alpha2beta1 integrin interaction facilitates differentiation and down-regulates TGF-beta receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.

3T3 Cells↗
PubMed1997

Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport.

Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-zeta (PKC-zeta) in rat adipocytes. Concomitantly, insulin provoked increases in 32P labeling of PKC-zeta both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-zeta; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-zeta pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-zeta was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-zeta enzyme activity. Also, PI-3,4-(PO4)2, PI-3,4,5-(PO4)3, and PI-4,5-(PO4)2 directly stimulated enzyme activity and autophosphoralytion in control PKC-zeta immunoprecipitates to levels observed in insulin-treated PKC-zeta immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-zeta enzyme activity in vitro by both the PKC-zeta pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigen-tagged GLUT4, co-transfection of wild-type or constitutive PKC-zeta stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-zeta partially inhibited it. Our findings suggest that insulin activates PKC-zeta through PI 3-kinase, and PKC-zeta may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.

Adipocytes↗
PubMed1997

The testis-specific histone H1t gene is strongly repressed by a G/C-rich region just downstream of the TATA Box.

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5' of the TATA box and 3' of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5'-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.

3T3 Cells↗
PubMed1997

Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway. Divergence from downstream CT-1 signals for myocardial cell hypertrophy.

Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development (Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase.

Animals↗
PubMed1997

Characterization of downstream Ras signals that induce alternative protease-dependent invasive phenotypes.

Invasive and metastatic cells require protease expression for migration through the extracellular matrix. Metastatic NIH 3T3 fibroblasts transformed by different activated ras genes showed two different protease phenotypes, rasuPA+/CL- and rasCL+/uPA- (Zhang, J-Y., and Schultz, R. M. (1992) Cancer Research 52, 6682-6689). Phenotype rasuPA+/CL- is dependent on expression of the serine-type protease urokinase plasminogen activator (uPA) and the phenotype rasCL+/uPA- on the cystine-type protease cathepsin L (CL) for lung colonization in experimental metastasis. The existence of multiple invasive phenotypes on ras-isoform transformation implied the activation of alternative pathways downstream from Ras. We now show that c-Raf-1, extracellular signal-regulated protein kinase (ERK)-1, and ERK-2 are hyperphosphorylated, and the ERK activity is high in both the uPA- and CL-dependent ras-transformed invasive phenotypes. Levels of c-Jun and c-Jun NH2-terminal kinase (JNK) activity are also high in the uPA-dependent phenotype, but they are almost undetectable in the CL-dependent phenotype. The uPA Ras-response element is a PEA3/URTF element, and mobility shift assays show a strong PEA3/URTF protein band in the uPA-dependent phenotype. This band is competed by a consensus AP-1 DNA sequence and by antibodies to PEA3 and c-Jun. Thus, the uPA-invasive phenotype appears to require the activation of Ets/PEA3 and c-Jun transcription factors activated by the ERK and JNK pathways, while the CL-invasive phenotype appears to require ERK activity with suppression of JNK and c-Jun activities. These postulates are supported by the introduction of a dominant negative c-Jun, TAM67, into cells of phenotype rasuPA+/CL-, which down-regulated the high uPA mRNA levels characteristic of this phenotype to basal levels and up-regulated basal levels of CL mRNA to levels similar to those observed in cells of phenotype rasCL+/uPA-. We conclude that the JNK pathway acts as a switch between two distinct protease phenotypes that are redundant in their abilities to grow tumors and metastasize.

3T3 Cells↗
PubMed1998

Prostaglandin F2alpha (PGF2alpha) and the isoprostane, 8, 12-iso-isoprostane F2alpha-III, induce cardiomyocyte hypertrophy. Differential activation of downstream signaling pathways.

Prostaglandin receptors may be activated by their cognate ligand or by free radical catalyzed isoprostanes, products of arachidonic acid peroxidation. For example, prostaglandin F2alpha (PGF2alpha) causes hypertrophy of neonatal rat ventricular myocytes, via the PGF2alpha receptor (FP). However, the FP may also be activated by the isoprostane, 8,12-iso-iPF2alpha-III (Kunapuli, P., Lawson, J. A., Rokach, J., and FitzGerald, G. A. (1997) J. Biol. Chem. 272, 27147-27154). Both ligands induce myocyte hypertrophy with overlapping potencies. Interestingly, the hypertrophic effects of these two agonists on cardiomyocytes are additive. Furthermore, the preference of these two agonists for activation of intracellular signal transduction pathways differs in several respects. Thus, PGF2alpha and 8,12-iso-iPF2alpha-III stimulate inositol phosphate formation with EC50 values of 50 +/- 12 nM and 3.5 +/- 0.6 microM, respectively. Moreover, PGF2alpha causes a robust activation ( approximately 50-fold) of Erk2, whereas 8,12-iso-iPF2alpha-III has no effect. Similarly, PGF2alpha causes translocation of cytosolic phospholipase A2 and also results in a 7-fold increment in the formation of 6-keto-PGF1alpha, whereas 8,12-iso-iPF2alpha-III exerts no effect on this pathway. On the other hand, both agonists are equally potent in activating JNK1 and c-Jun, whereas neither activates the p38 kinase. Both PGF2alpha and 8,12-iso-iPF2alpha-III activate the p70S6 kinase (p70(S6K)), but not Akt, downstream of phosphatidylinositol-3-kinase (PI3K). However, both wortmannin, a PI3K inhibitor, and rapamycin, an inhibitor of p70(S6K) activity, inhibit 8,12-iso-iPF2alpha-III -induced myocyte hypertrophy, with IC50 values of 60 +/- 12 and 3 +/- 1.7 nM, respectively, whereas neither compound abrogates the PGF2alpha-mediated response. Thus, both PGF2alpha and 8,12-iso-iPF2alpha-III induce myocyte hypertrophy via discrete signaling pathways. Although both agonists signal via the JNK pathway to initiate changes in c-Jun-dependent gene transcription, PGF2alpha preferentially activates the MEK-Erk2- cytosolic phospholipase A2 pathway. In contrast, the PI3K-p70(S6K) pathway appears to be essential for 8,12-iso-iPF2alpha-III-induced myocyte hypertrophy.

Animals↗
PubMed1998

Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex.

The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing signaling complex (DISC) and by increased expression of Bcl-xL (Peter, M. E., Kischkel, F. C., Scheuerpflug, C. G., Medema, J. P., Debatin, K.-M., and Krammer, P. H. (1997) Eur. J. Immunol. 27, 1207-1212). This raises the possibility that Bcl-xL directly prevents caspase-8 activation by the DISC. To test this hypothesis a cell line in which CD95 signaling was inhibited by overexpression of Bcl-xL was used. In these MCF7-Fas-bcl-xL cells Bcl-xL had no effect on the recruitment of caspase-8 to the DISC. It did not affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10. In contrast, cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, was inhibited in comparison with the control-transfected CD95-sensitive MCF7-Fas cells. To test whether Bcl-xL would inhibit active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed. Using monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-xL antibodies, or fusion proteins of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical interaction between caspase-8 and Bcl-xL was found. Moreover, overexpression of Bcl-xL did not inhibit the activity of the caspase-8 active subunits p18/p10. Therefore, in this cell line that has become resistant to CD95-induced apoptosis due to overexpression of Bcl-xL, Bcl-xL acts independently and downstream of caspase-8.

Apoptosis↗
PubMed1998

Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells.

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-alpha-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or double transfected with Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibited apoptosis induced by either anti-Fas or TNF-alpha. In addition, Bcl-xL prevented the change in cytochrome c immunolocalization induced by anti-Fas or TNF-alpha treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-alpha, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 was processed normally while processing of the downstream caspase-7 was markedly attenuated. Moreover, apoptosis induced by direct microinjection of recombinant, active caspase-8 was completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step.

Apoptosis↗
PubMed1999

Nuclear localization of mitogen-activated protein kinase kinase 1 (MKK1) is promoted by serum stimulation and G2-M progression. Requirement for phosphorylation at the activation lip and signaling downstream of MKK.

Stimulation of mammalian cells results in subcellular relocalization of Ras pathway enzymes, in which extracellular signal-regulated protein kinases rapidly translocate to nuclei. In this study, we define conditions for nuclear localization of mitogen-activated protein kinase kinase 1 (MKK1) by examining effects of perturbing the nuclear export signal (NES), the regulatory phosphorylation sites Ser218 and Ser222, and a regulatory domain at the N terminus. After disrupting the NES (Delta32-37), nuclear uptake of MKK was enhanced when quiescent cells were activated with serum-phorbol 12-myristate 13-acetate or BXB-Raf-1 cotransfection. Uptake was enhanced by mutation of Ser218 and Ser222 to Glu and Asp, respectively, and blocked by mutation of these residues to Ala, although mutation of Lys97 to Met, which renders MKK catalytically inactive, did not interfere with uptake. Therefore, nuclear uptake of MKK requires incorporation of phosphate or negatively charged residues at the activation lip but not enzyme activity. On the other hand, uptake of an active MKK mutant with disrupted NES (Delta32-51) was elevated in quiescent as well as stimulated cells, and pretreatment of cells with the MKK inhibitor 1,4-diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio]butadiene blocked nuclear uptake. Thus, signaling downstream of MKK is also necessary for translocation. Finally, wild type MKK containing an intact NES translocates to nuclei during mitosis before envelope breakdown. Comparison of mutants with Ser to Glu and Asp or Ala substitutions indicates that Ser phosphorylation is also required for mitotic nuclear uptake of MKK.

3T3 Cells↗
PubMed1999

Transmembrane helix 7 of the endothelin B receptor regulates downstream signaling.

Endothelin is a 21-amino acid peptide with a striking diversity of important biological responses, including, vasoconstriction, bronchoconstriction, and mitogenesis. Endothelin-1 binding to the endothelin B receptor (ETB), a member of the superfamily of G-protein-coupled receptors, was associated with catalytic activation of the extracellular-regulated kinase 2 (ERK2) and stimulation of AP-1 transcriptional reporter activity. A panel of single point mutations in transmembrane helix 6 (TM6), intracellular loop 3, and transmembrane helix 7 (TM7) were developed to study the structural requirements for ETB activation. Point mutations within highly conserved regions of TM6 and intracellular loop 3 were without effect on agonist-stimulated ERK activation. However, mutations within TM7 of the ETB significantly impacted ligand-stimulated downstream signaling. For example, nine point mutations within TM7 of the ETB were identified that prevented endothelin-stimulated ERK activation. Interestingly, the TM7 mutants fell into two classes; several exhibited greatly decreased AP-1 activity, relative to wild type ETB, whereas others displayed augmented endothelin-stimulated AP-1 transcriptional activity relative to wild type ETB. Our results suggest that TM7 of the ETB is involved in its activation mechanism and regulates agonist-stimulated ERK activation.

Amino Acid Sequence↗
PubMed1999

The ability of p53 to activate downstream genes p21(WAF1/cip1) and MDM2, and cell cycle arrest following DNA damage is delayed and attenuated in scid cells deficient in the DNA-dependent protein kinase.

scid mouse embryonic fibroblasts are deficient in DNA-dependent protein kinase activity due to a mutation in the C-terminal domain of the catalytic subunit (DNA-PKcs). When exposed to ionizing radiation, the increase in levels of p53 was the same as in normal mouse embryonic fibroblasts. However, the rise in p21(WAF1/cip1) and mdm2 was found to be delayed and attenuated, which correlated in time with delayed onset of G1/S arrest by flow cytometric analysis. The p53-dependent G1 checkpoint was not eliminated: inactivation of p53 by the E6 protein in scid cells resulted in the complete loss of detectable G1/S arrest after DNA damage. Immunofluorescence analysis of normal cells revealed p53 to be localized predominantly within the cytoplasm prior to irradiation and then translocate to the nucleus after irradiation. In contrast, scid cells show abnormal accumulation of p53 in the nucleus independent of irradiation, which was confirmed by immunoblot analysis of nuclear lysates. Taken together, these data suggest that loss of DNA-PK activity appears to attenuate the kinetics of p53 to activate downstream genes, implying that DNA-PK plays a role in post-translational modification of p53, without affecting the increase in levels of p53 in response to DNA damage.

Animals↗
PubMed1999

Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins.

Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway.

Animals↗
PubMed1999

CPG16, a novel protein serine/threonine kinase downstream of cAMP-dependent protein kinase.

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested, e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase.

3T3 Cells↗
PubMed1999

A small 2'-OH- and base-dependent recognition element downstream of the initiation site in the RNA encapsidation signal is essential for hepatitis B virus replication initiation.

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate. In contrast to retroviral reverse transcriptases, their replication enzyme, P protein, does not use a nucleic acid primer but initiates DNA synthesis de novo from within an RNA stem-loop structure called epsilon. A short DNA oligonucleotide is copied from epsilon and covalently attached to P protein, and then synthesis is arrested. The information for initiation site selection and synthesis arrest must be contained in the structure of the P protein/epsilon complex. Because P protein activity depends on cellular chaperones this complex can as yet only be generated by in vitro translation of duck hepatitis B virus P protein in rabbit reticulocyte lysate; functional interaction with its cognate RNA element Depsilon can be monitored by the covalent labeling of P protein during primer synthesis. Combining this in vitro priming reaction and a set of chimeric RNA-DNA Depsilon analogues, we found that only five ribose residues in the 57-nucleotide stem-loop were sufficient to provide a functional template; these are a single residue in the template region and the two base pairs at the tip of the lower stem. The base identities in the very same region are essential as well. The presence of this 2'-OH- and base-dependent determinant shortly downstream of the initiation site suggests a mechanism that can account for both initiation site selection and programmed primer synthesis arrest.

Animals↗
PubMed2000

The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance.

The intracellular concentration of K(+)-glutamate, chromatin-associated proteins, and a downstream regulatory element (DRE) overlapping with the coding sequence, have been implicated in the regulation of the proU operon of Salmonella typhimurium. The basal expression of the proU operon is low, but it is rapidly induced when the bacteria are grown in media of high osmolarity (e.g. 0.3 M NaCl). It has previously been suggested that increased intracellular concentrations of K(+)-glutamate activate the proU promoter in response to increased extracellular osmolarity. We show here that the activation of the proU promoter by K(+)-glutamate in vitro is nonspecific, and the in vivo regulation cannot simply be mimicked in vitro. In vivo specificity requires both the chromatin-associated protein H-NS and the DRE; they are both needed to maintain repression of proU expression at low osmolarity. How H-NS and the DRE repress the proU promoter in vivo has so far been unclear. We show that, in vivo, the DRE acts at a distance to inhibit open complex formation at the proU promoter.

Bacterial Proteins↗