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S Stabel

Publications and source records attributed to S Stabel.

At least 37 records · Page 2Linked to original sources
PubMed1992

Effect of tumour-promoting phorbol ester, thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium.

Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean +/- S.E.M.) of 24 +/- 3 and 46 +/- 4% respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278 +/- 27 to 198 +/- 24 (mean +/- S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mM concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.

Amino Acid Sequence↗
PubMed1992

Sequence of a rat cDNA encoding the ERK1-MAP kinase.

Mitogen-activated protein (MAP) kinases are cytoplasmic and/or nuclear protein kinases which are activated by one or several signal transduction pathways from the cell surface into the nucleus. Their activity is regulated by phosphorylation on Tyr as well as on Ser/Thr residues. A cDNA encoding the rat ERK1 member of the MAP kinase family was isolated and sequenced. The longest cDNA consisted of 1875 nucleotides and coded for a polypeptide of 380 amino acids with a predicted M(r) of 42987.

Amino Acid Sequence↗
PubMed1992

Protein kinase C group B members PKC-delta, -epsilon, -zeta and PKC-L(eta). Comparison of properties of recombinant proteins in vitro and in vivo.

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.

Animals↗
PubMed1992

Studies on the phosphorylation of protein kinase C-alpha.

A kinase-defective protein kinase C-alpha mutant is shown to be a phosphoprotein when expressed in COS-1 cells, indicating that intramolecular phosphorylation does not fully account for the phosphate content of protein kinase C-alpha. Furthermore, evidence is presented that the intermolecular phosphorylation of protein kinase C-alpha is due to an activity other than protein kinase C-alpha itself, and this phosphorylation appears to be necessary for protein kinase C-alpha activity. By contrast, the characteristic shift in apparent molecular mass consequent on phosphorylation in vivo can be accounted for by autophosphorylation, as demonstrated in vitro. The relationship between these phosphorylated protein kinase C-alpha species is discussed.

Acid Phosphatase↗
PubMed1992

Immunocharacterization of delta- and zeta-isoenzymes of protein kinase C in rat renal mesangial cells.

The isoforms of protein kinase C (PKC) present in rat mesangial cells were identified by immunoblot analysis with antibody raised against isotype-specific peptides. In addition to the previously observed alpha- and epsilon-subspecies, mesangial cells also express the delta- and zeta-isoenzymes of PKC. On exposure to phorbol 12,13-dibutyrate (PDB) a complete depletion of PKC-delta is observed within 8 h. Removal of PDB results in a recovery of PKC-delta. In contrast, PKC-zeta is unaffected by addition or removal of PDB.

Amino Acid Sequence↗
PubMed1992

Expression of a pheromone-binding protein in insect cells using a baculovirus vector.

A cDNA encoding a pheromone-binding protein from the male silkmoth Antheraea pernyi has been integrated into the genome of the Autographa californica multiple nuclear polyhydrosis virus such that the transcription was under the control of the strong polyhedrin promoter. Recombinant pheromone-binding protein was expressed in a baculovirus-infected insect cell line (Sf9) and secreted from the cells into the culture medium. Using a two-step protocol, recombinant pheromone-binding protein has been isolated and purified to homogeneity. Pheromone binding of recombinant protein has been demonstrated using a tritiated analog of (E,Z)-6,11-hexadecadienyl acetate.

Affinity Labels↗
PubMed1992

Activation of the c-Raf protein kinase by protein kinase C phosphorylation.

The product of the c-raf-1 proto-oncogene is a cytoplasmic serine/threonine protein kinase that appears to be activated in signal transduction from a variety of cell-surface receptors. The mechanism of c-Raf activation upon stimulation of cell-surface receptors is not clear, but there seem to exist multiple pathways of activation which involve tyrosine and/or serine phosphorylation of the c-Raf protein in vivo. The activated state of Raf is reflected in an increased apparent molecular weight of the Raf protein in sodium dodecyl sulfate-polyacrylamide gels owing to hyperphosphorylation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) is one of the agents able to induce this hyperphosphorylation of Raf in vivo, suggesting that protein kinase C (PKC) may be involved in the activation of c-Raf in particular situations. Using recombinant baculoviruses expressing PKC and Raf polypeptides, we show here that conventional PKC types (alpha, beta, gamma) but not novel types (delta, zeta, eta) or the unrelated Mos kinase are able to activate c-Raf in a TPA-dependent manner upon coexpression in insect cells. Direct phosphorylation of the Raf protein with PKC in vitro also enhanced the kinase activity of c-Raf, suggesting that c-Raf acts immediately downstream of PKC in a protein kinase cascade which is triggered by TPA and may lead to transcriptional activation of TPA-inducible genes and tumor promotion.

Amino Acid Sequence↗
PubMed1991

Preferential release of catecholamine from permeabilized PC12 cells by alpha- and beta-type protein kinase C subspecies.

Protein kinase C (PKC) is now recognized as comprising two groups of closely related subspecies. The PKC gamma enzyme is apparently present only in central nervous tissues, and hence was expected to participate in neurotransmitter release. We have utilized a 'depletion-insertion' method to identify the PKCs participating in the exocytotic response. PC12 cells were 'down-regulated' by prior treatment (24 h) with phorbol 12-myristate 13-acetate (PMA; 1 microM), which nearly abolished endogenous PKC activity. Down-regulated PC12 cells were loaded with [3H]dopamine, permeabilized with digitonin, and recombinant or purified PKCs were inserted and activated with a low dose of PMA (20 nM). Among group A PKCs, PKC alpha was the most effective activator of [3H]dopamine release (215%), followed by beta II (185%) and beta I (150%). PKC gamma had no consistent effect on neurotransmitter release. PC12 cells express PKC alpha and PKC beta, but not PKC gamma, as revealed by Northern-blot analysis. We therefore postulate that PKC alpha and PKC beta participate in neurotransmitter release, whereas PKC gamma might be involved in other neuronal functions.

Animals↗
PubMed1991

Downregulation of protein kinase C-gamma is independent of a functional kinase domain.

Prolonged activation of protein kinase C (PKC) types alpha and beta by tumor-promoting phorbol esters leads to desensitization of the phorbol ester response, downregulation of protein kinase C activity and depletion of the protein kinase C polypeptide. When the gamma isoenzyme of PKC is transiently expressed in COS-1 cells and exposed to phorbol esters, PKC-Gamma is downregulated in COS cells although these cells do not normally express this subtype. A point mutation in the putative ATP-binding site (Lys-380----Met-380) of the protein kinase C gamma isoenzyme which results in a kinase-deficient enzyme does not interfere with this downregulation. Our results suggest that autophosphorylation or constitutive signalling through the protein kinase C-gamma kinase domain is not a prerequisite for downregulation of PKC activity.

Adenosine Triphosphate↗
PubMed1991

Protein kinase C.

Based on the molecular structure of the individual members of the protein kinase C family, general properties and the mode of activation of this enzyme family are discussed. Examples are presented of how the investigation of protein kinase C function in vivo has been approached at the molecular level.

Amino Acid Sequence↗
PubMed1989

Tumor promotion and depletion of protein kinase C in epidermal JB6 cells.

Promotion of JB6 epidermal cells to anchorage-independent growth requires exposure to TPA for greater than 4 days. Over a similar time span, a practically complete loss of enzymic and immunoreactive proteinkinase C (PKC) equivalents was observed at greater than 10 nM TPA. Promotion did not appear to require (transient) activation of PKC since PKC inhibitors H7 and HA1004 did not prevent but enhanced colony formation in soft agar at concentrations greater than IC50-values. The efficacy of the inhibitors in vivo was shown by their ability to suppress PKC-induced transcription of c-fos gen. PKC inhibitors that interfered with cell proliferation at lower concentrations than those required for PKC inhibition (sphingosine, staurosporin, sangivamycin, trifluoperazine) did not stimulate anchorage-independent growth. As H7 as well as HA1004 were able to promote JB6 cells in the complete absence of TPA, and induced neither depletion nor processing of PKC we postulate that depletion/inactivation rather than activation of PKC correlates with the promotion of epidermal JB6 cells to anchorage-independent growth.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Arrangement and expression of integrated adenovirus type 12 DNA in the transformed hamster cell line HA12/7: amplification of Ad12 and c-myc DNAs and evidence for hybrid viral-cellular transcripts.

In the genome of the adenovirus type 12 (Ad12)-transformed hamster cell line HA12/7 about three copies of the viral DNA are fixed by integration. The results of blot-hybridization, molecular cloning, and nucleotide sequencing experiments suggest a model for the arrangement of Ad12 DNA molecules in which the left hand terminus of one of the Ad12 DNA copies is linked to unique hamster DNA. The right hand end of this DNA molecule is fused to an inverted copy of a left terminal approximately 4.3 kb fragment of Ad12 DNA. This ensemble is followed by the second Ad12 DNA copy whose right terminus is again joined to an inverted, supernumerary left terminal approximately 4.3 kb Ad12 DNA fragment. There is a third Ad12 DNA copy whose right terminus is linked to cellular DNA. In this sequence arrangement, the left terminus of Ad12 DNA is overrepresented, as had been shown earlier (S. Stabel, W. Doerfler and R.R. Friis (1980) J. Virol. 36, 22-40). In the presented model, cellular DNA sequences are interspersed in between the three copies of Ad12 DNA. In the left terminus of the integrated Ad12 DNA, transcription of RNA is initiated which extends out into cellular DNA. The interviral DNA junctions are also transcribed. The c-myc gene in cell line HA12/7 is amplified about 10-fold and considerably more c-myc RNA has been identified in the Ad12-transformed cells than in BHK21 or in LSH hamster cells. It has been shown previously that the E1 region of Ad12 DNA is transcribed into mRNA in HA12/7 cells (Ortin et al. (1976) J. Virol. 20, 355-372). It remains to be investigated whether c-myc amplification and expression are related to the transformed phenotype of HA12/7 cells.

Adenoviridae↗
PubMed1989

Protein kinase C--a family affair.

The structural analysis of protein kinase C has led to the identification of a family of related gene products. This family of kinases consists of six unique genes that give rise to at least seven polypeptides. The high degree of conservation and the differential distribution of these mRNAs/proteins suggest that they perform distinct functions in vivo. Characterization of the activities of some of these proteins in vitro shows that there are functional differences with respect to both their regulation and substrate specificity. This indicates that each member of this family may play a unique role in signal transduction.

Amino Acid Sequence↗
PubMed1989

Expression of a functional protein kinase C-gamma using a baculovirus vector: purification and characterisation of a single protein kinase C iso-enzyme.

The cloning of complementary DNAs for protein kinase C (PKC) has revealed a multi-gene family of closely related protein kinases [Parker et al. (1986) Science 233, 853-859; Coussens et al. (1986) Science 233, 859-866]. In vivo, the distribution of the PKC isoenzymes follows a fairly tissue-specific pattern suggesting that functional differences exist between the members of this kinase family. To initiate a detailed characterisation of the individual isoenzymes, and as an alternative approach to purifying and separating the individual PKC types and their splice variants from mammalian tissues, we have expressed the bovine PKC type gamma in insect cells using a baculovirus expression vector. The bovine protein constitutes one of the major proteins in infected cells and can be purified to near homogeneity by a 2-step procedure. Analysis of the purified protein confirms that it has authentic mammalian PKC characteristics with respect to phospholipid dependence and phorbol ester binding. The bovine PKC gamma purified from infected cells is post-translationally modified and resolves into a doublet of molecular weights 82,000 and 84,000 upon SDS-polyacrylamide gel electrophoresis. These two size classes of polypeptides appear to result from differential phosphorylation as demonstrated by sensitivity to protein phosphatase treatment. The applicability and the potential of this system for the analysis of the various mammalian PKC isoenzymes is discussed.

Animals↗
PubMed1987

Down-regulation of protein kinase C is due to an increased rate of degradation.

The phorbol-ester-induced loss of protein kinase C that has been documented in many cell types appears to be a critical event in the generation of a cellular refractory state. We have investigated here the synthesis and degradation of the protein kinase C polypeptide in order to determine why its steady-state amounts are depleted in response to phorbol esters. These results indicate that depletion is due to an increased rate of degradation, with no change either in mRNA amounts or in rates of polypeptide synthesis.

Animals↗