Biomedical subjects

D Mochly-Rosen

Publications and source records attributed to D Mochly-Rosen.

At least 73 records · Page 4Linked to original sources

PubMed1994

Distinct responses of protein kinase C isozymes to c-erbB-2 activation in SKBR-3 human breast carcinoma cells.

We have studied the effect of activation of the c-erbB-2 receptor tyrosine kinase on protein kinase C (PKC) in cultured SKBR-3 human breast cancer cells. Treatment with the agonistic anti-receptor monoclonal antibody TAb 250 induces receptor autophosphorylation and stimulates phospholipase C-gamma 1 (L. K. Shawver et al. Cancer Res., 54: 1367-1373, 1994). TAb 250 induced a rapid and marked translocation of PKC histone phosphorylation activity to the particulate fraction of SKBR-3 cells. By immunoblot, however, this translocation was limited to specific PKC isozymes. beta PKC and zeta PKC translocated to the particulate fraction, whereas epsilon PKC underwent "partial reversed translocation" to the cell soluble fraction after receptor stimulation. Furthermore, beta PKC was rapidly degraded following TAb 250 treatment. By immunocytochemistry, beta IPKC translocated from the perinuclear area to the cytosol and into the nucleus, whereas zeta PKC translocated to the perinuclear region and into the nucleus. Consistent with the Western blot results, epsilon PKC translocated from the nucleus to the perinuclear area and the cytosol. These changes in the localization of PKC isozymes were not observed after addition of normal IgG1 or a nonagonistic anti-c-erbB-2 monoclonal antibody to SKBR-3 cells. alpha, beta II, or delta PKC present in these cells did not translocate following receptor stimulation. These data indicate that c-erbB-2 signal transduction may involve the activation of specific PKC isozymes. The biological role of these enzymes in the phenotype and cellular responses of c-erbB-2-overexpressing carcinoma cells remains to be studied.

Biological Transport↗

PubMed1992

Inhibition of protein kinase C function by injection of intracellular receptors for the enzyme.

We tested the hypothesis that the translocation and function of protein kinase C (PKC) requires the binding of PKC to its intracellular receptors (RACKs), using insulin-induced maturation of Xenopus oocytes. We show that after exposure of oocytes to insulin, PKC translocated from the cytosol to the particulate fraction. PKC is also required for insulin-induced oocyte maturation: microinjection of a PKC inhibitory peptide delayed maturation. To determine whether translocation of PKC was a result of the binding of PKC to the RACKs in the particulate fraction, we microinjected purified rat brain RACKs into oocytes before insulin exposure. Microinjection of RACKs, but not inactive phosphorylated RACKS, inhibited PKC translocation and delayed oocyte maturation. These results suggest an in vivo role for RACKs in a function mediated by PKC.

Animals↗

PubMed1992

p65 fragments, homologous to the C2 region of protein kinase C, bind to the intracellular receptors for protein kinase C.

Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.

Amino Acid Sequence↗

PubMed1992

Nicotinic acetylcholine receptor desensitization is regulated by activation-induced extracellular adenosine accumulation.

Adenosine modulation of nicotinic ACh receptor (nAChR) function was studied in primary cultures of rat skeletal muscle. Activation of the nAChR by carbachol increased extracellular adenosine concentration in a dose-dependent manner. Furthermore, carbachol activation of the nicotinic receptor resulted in a twofold increase in cAMP levels in the muscle cells. The carbachol-dependent increase in cAMP levels was inhibited by adenosine receptor antagonists as well as by nicotinic receptor antagonists. These results suggest that the increased cAMP levels were due to adenosine receptor activation by the extracellular adenosine accumulated on nAChR activation. Others have shown that desensitization of the nAChR by agonist is mediated, in part, by phosphorylation. Since we found that nicotinic cholinergic agonists also cause adenosine accumulation with concomitant cAMP increases, we determined whether the accumulated adenosine has a role in desensitization. We found that the adenosine receptor antagonist, BW1434U, significantly inhibited carbachol-induced nAChR desensitization, indicating that extracellular adenosine is involved in nAChR desensitization. Our data suggest that nAChR function is regulated via a feedback mechanism mediated by adenosine released from muscle on activation of the nAChR.

Adenosine↗

PubMed1991

Intracellular receptors for activated protein kinase C. Identification of a binding site for the enzyme.

Protein kinase C (PKC) isozymes comprise a family of cytosolic enzymes that translocate to different intracellular sites on activation. We have recently characterized at least two intracellular receptor proteins for PKC (termed RACKs for receptors for activated C-kinase) in the Triton-insoluble material of the particulate fraction from neonatal rat heart. Here, we identify a sequence that appears to resemble the PKC binding site on these RACKs. A peptide (peptide I) with the sequence KGDYEKILVALCGGN bound PKC, and binding was markedly increased in the presence of PKC activators. Furthermore, peptide I inhibited PKC binding to RACKs in a dose-dependent manner. These data suggest that these RACKs have a common PKC binding sequence. Since peptide I inhibited PKC binding to RACKs in vitro, it may be a useful tool to inhibit PKC translocation and subsequent function in vivo.

Amino Acid Sequence↗

PubMed1991

Identification of intracellular receptor proteins for activated protein kinase C.

Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC.

Animals↗

PubMed1991

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Animals↗

PubMed1991

The role of adenosine and adenosine transport in ethanol-induced cellular tolerance and dependence. Possible biologic and genetic markers of alcoholism.

Acute exposure to ethanol in culture inhibits adenosine uptake into cells, thereby increasing the concentration of extracellular adenosine. Extracellular adenosine then reacts with adenosine A2 receptors to stimulate intracellular cAMP production. During prolonged exposure to ethanol, the increase in cAMP is followed by the development of heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory GTP-binding protein. Ethanol-induced heterologous desensitization appears to be due to a reduction in mRNA and protein for G alpha s, a subunit of Gs. This is an example of cellular dependence on ethanol. The important implication of these findings is that a selective inhibitory effect of ethanol on adenosine uptake can lead to desensitization of diverse receptors coupled to cAMP production. Such changes could contribute to the pleiotropic effects of ethanol in the brain and other organs. Prolonged exposure to ethanol also alters the nucleoside transport system. While ethanol inhibits adenosine uptake into naive cells, ethanol no longer inhibits adenosine uptake into cells that have adapted to ethanol. This resistance to ethanol inhibition appears to be a form of cellular tolerance to ethanol. Thus, there appears to be a synergism between ethanol-induced heterologous desensitization of receptor-stimulated cAMP production (cellular dependence) and resistance to ethanol inhibition of adenosine uptake (cellular tolerance), because both lead to reduced intracellular levels of cAMP. Our studies on cAMP signal transduction in cell culture are directly relevant to the pathophysiology of human alcoholism. Heterologous desensitization of cAMP production is demonstrable in lymphocytes taken from actively drinking alcoholics; this measurement appears to be a biologic marker of active alcohol consumption. In addition, regulation of adenosine receptor-dependent cAMP production may be altered in patients at risk to develop alcoholism because of genetic factors. Thus, lymphocytes from alcoholics cultured many generations in the absence of ethanol show increased adenosine receptor-dependent cAMP production and increased sensitivity to ethanol-induced heterologous desensitization. These persistent phenotypic abnormalities in cell culture could be used as genetic markers for alcoholism. Studies are under way to test this possibility.

Adenosine↗

PubMed1990

GTP-binding proteins are restricted to signal transduction sites.

We have used the Torpedo electric organ to study GTP-binding protein localization since functionally distinct membrane fractions can be isolated from this tissue. Postsynaptic membranes from the innervated face and membranes from the non-innervated face of the electrocyte, as well as presynaptic membranes from the innervating nerve, can be isolated. alpha s was restricted to the innervated face of the postsynaptic cell; alpha i, alpha o, and ras were found only in the presynaptic membrane fraction of the innervating nerve. 21 and 25 kDa GTP-binding proteins were present in all the membrane fractions. These results suggest that specific GTP-binding proteins are differentially restricted to membrane areas specialized in signal transduction.

Animals↗

PubMed1990

A protein kinase C isozyme is translocated to cytoskeletal elements on activation.

Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.

Actin Cytoskeleton↗

PubMed1990

Chronic ethanol-induced heterologous desensitization is mediated by changes in adenosine transport.

Chronic exposure of cultured cell lines to ethanol results in a heterologous desensitization of receptors coupled to adenylate cyclase via GS, the stimulatory guanine nucleotide regulatory protein. This heterologous desensitization is accompanied by a decrease in alpha S, the GTP-binding subunit of GS. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization. To determine the mechanism underlying the ethanol-dependent increase in extracellular adenosine, we investigated the effects of ethanol on the nucleoside transporter responsible for the bidirectional transport of adenosine into and out of the cell. We found that ethanol specifically and non-competitively inhibited nucleoside uptake. Inhibition of adenosine uptake was primarily due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake and, consequently, no longer increased extracellular adenosine concentration. Taken together with our previous studies, these results suggest that acute ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which in turn activates adenosine A2 receptors to increase cyclic AMP levels, leading to desensitization of receptor-dependent cyclic AMP signal transduction after chronic exposure to ethanol. We next determined whether the same effects of ethanol also occur in alcoholics. We isolated lymphocytes from alcoholics and non-alcoholics and found that alcoholics had a 75% decrease in basal and adenosine receptor-stimulated cyclic AMP production compared with non-alcoholics. To determine whether these differences were due to exposure to ethanol in vivo or to a possible genetic difference between alcoholics and non-alcoholics, we grew lymphocytes in culture in the absence of ethanol. Adenosine receptor-stimulated cyclic AMP levels were higher in alcoholics than non-alcoholics. Moreover, cultured cells from alcoholics were more sensitive to the effects of chronic alcohol on cyclic AMP signal transduction than cells from non-alcoholics. Our results suggest that the cyclic AMP signal transduction system may reflect a genetic alteration in alcoholics and that studies in cultured lymphocytes may allow us to identify individuals at risk of developing alcoholism.

Adenosine↗

PubMed1988

Chronic ethanol causes heterologous desensitization of receptors by reducing alpha s messenger RNA.

One of the biochemical results of ethanol exposure is a change in the amount of the intracellular second messenger cyclic AMP (cAMP) produced in response to receptor stimulation. In general, acute ethanol exposure increases the amount of cAMP produced on stimulation of receptors coupled to the enzyme adenylyl cyclase via the GTP-binding protein Gs, whereas chronic ethanol exposure has the opposite effect (results for receptors coupled via Gi have been more variable). We previously reported that adaptation to continuous ethanol exposure reduces receptor-stimulated cAMP production by 25-35% in a neuroblastoma cell line (NG108-15), and an even greater reduction of 75% was observed in lymphocytes taken from actively-drinking alcoholics. This reduction in receptor-stimulated cAMP levels was recently confirmed in platelets from alcoholics. None of these studies, however, determined whether more than one receptor coupled to adenylyl cyclase activity was affected in the same cell. Here we report that chronic ethanol exposure causes desensitization of heterologous receptors coupled to Gs as cAMP production mediated by prostaglandin E1 as well as by adenosine is reduced by approximately 30% in NG108-15 cells. We show that, after chronic ethanol exposure, the activity of the alpha subunit of Gs is decreased by 29%, the amount of alpha s protein is decreased by 38.5%, and alpha s messenger RNA is decreased by 30%. Thus, cellular adaptation to ethanol involves a reduction in alpha s mRNA and, as a consequence, reduced cAMP production by heterologous receptors coupled to Gs. Such changes in cAMP production may account for the tolerance and physical dependence on ethanol in alcoholism.

Cyclic AMP↗

PubMed1988

A general procedure for screening inhibitory antibodies: application for identifying anti-protein kinase C antibodies.

In this communication we describe a microfiltration assay to identify monoclonal antibodies that interfere with the activity of enzymes. This method is quick and sensitive to small changes in the activity of the enzyme and does not require highly purified enzyme or large quantities of antibodies. It has been applied to identify anti-protein kinase C antibodies which would have been impossible to identify by classical assays such as enzyme-linked immunosorbent assay.

Antibodies, Monoclonal↗

PubMed1987

Comparison of S100b protein with calmodulin: interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein kinase C.

To gauge similarities between S100b protein and calmodulin, interactions were observed between S100b and melittin and between S100b and tau, the microtubule-associated proteins. The interaction of melittin with S100b protein in the presence and absence of calcium was studied by fluorescence polarization, UV difference spectroscopy, and sulfhydryl derivatization. Whether calcium was present or not in the solution, melittin and S100b form a complex of molar ratios up to 2:1. Further binding of melittin occurred, but it resulted in precipitation of S100b, as is true of the corresponding case of melittin binding to calmodulin. In the absence of calcium, the interaction of melittin and S100b shielded the tryptophan (Trp) of the former protein and exposed cysteine-84 beta (Cys-84 beta) of the latter protein, leaving the tyrosine-16 beta (Tyr-16 beta) of S100b unaffected. Calcium addition to the complex partially restored the exposure of Trp of melittin and caused changes in the environment of Tyr-16 beta (unlike the environmental changes induced for Tyr-16 beta by calcium in the absence of melittin). The conformational changes induced in S100b by interaction with melittin increased its affinity for calcium and offset the inhibition of calcium binding otherwise observed in the presence of potassium ions. This corroborated the previous finding that S100b affinity for calcium greatly depends on the protein conformation. The phenomena described above are similar to the interactions of melittin with calmodulin and thus suggest that S100b and calmodulin have a common structural domain not only that binds melittin but also that may interact with common target proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties.

Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.

Animals↗

PubMed1987

Domain structure and phosphorylation of protein kinase C.

The phospholipid- and calcium-dependent protein kinase C has been shown to autophosphorylate on both the catalytic and the regulatory domains. The autophosphorylation displays zero-order kinetics, indicating that it is an intramolecular event. Autophosphorylation increases the activity of protein kinase C by decreasing the Km for the substrate H1 histone. The catalytic fragment obtained by limited proteolysis can no longer autophosphorylate and has a reduced affinity for H1 histone, exhibiting a Km 5-fold higher than that of the intact enzyme. Monoclonal antibodies produced against the enzyme can distinguish between the catalytic fragment and the intact enzyme by inhibiting their activities in a specific manner. Evidence suggesting that dimerization of protein kinase C occurs upon activation is presented.

Animals↗

PubMed1987

Distinct cellular and regional localization of immunoreactive protein kinase C in rat brain.

Monoclonal antibodies raised against highly purified protein kinase C were used to localize protein kinase C in the rat brain. Using various monoclonal antibodies, at least three distinct antibody-staining patterns were found. One monoclonal antibody exclusively labeled astroglial elements, including astrocytes, tanycytes, and cerebellar radial glia. Another monoclonal antibody exclusively labeled neural cells, including cortical and hippocampal pyramidal dendrites and Purkinje cells of the cerebellum. A third monoclonal antibody (which inhibited protein kinase C activity) intensely stained more limited brain regions, particularly thalamic neurons, and also stained astroglial structures in brain, spinal cord, and cerebellum. The possibility that the three staining patterns reflect the differential regional and cellular localization of related, but distinct, enzymes of protein kinase C is discussed.

Animals↗

PubMed1986

Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity.

The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.

Animals↗