Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.     

Lymphoma protein kinase C is associated with the transmembrane glycoprotein, GP85, and may function in GP85-ankyrin binding

In this study, several complementary techniques have been used to investigate the involvement of a protein kinase C (PKC) molecule in the plasma membrane-cytoskeleton interactions that occur in mouse T-lymphoma cells. Our data indicate that the lymphoma plasma membrane contains a 78-kDa polypeptide that exists in a complex with one of the major transmembrane glycoproteins, GP85 (a wheat germ agglutinin-binding protein). This membrane-associated 78-kDa protein appears to have PKC-like properties based on the following criteria: 1) it cross-reacts with a specific antibody raised against brain PKC; 2) it has a pI of 5.6-5.8, which is similar to that of the PKC described previously in other cell types; and 3) it displays characteristic PKC enzymatic activity by phosphorylating histone H1 in a Ca2+- and phospholipid-dependent manner. Double immunocytochemical staining experiments reveal that the lymphoma PKC-like molecules translocate from the cytoplasm to the cell membrane and accumulate directly underneath receptor capped structures following addition of various ligands. Studies we have done to identify the cellular substrate(s) of the lymphoma plasma membrane-associated PKC have shown that GP85 is preferentially phosphorylated in isolated membrane preparations following addition of the PKC activator, TPA (phorbol-12-O-tetradecanoyl-phorbol 13-acetate), but not the biologically inactive TPA analogue, 4 alpha-PDD (4 alpha-phorbol 12,13-didecanoate). In addition, we have found that GP85 can be phosphorylated by purified brain protein kinase C. Analysis of the resulting phosphoamino acids indicates that phosphorylation of GP85 occurs primarily at serine residues, occurs in minor amounts (approximately 5%) at threonine residues, and does not occur at tyrosine residues. These data indicate that the lymphoma GP85 is a substrate for PKC. Furthermore, we have established that phosphorylation of GP85 by PKC enhances its binding affinity with the membrane linker molecule, ankyrin. These findings suggest that PKC-mediated phosphorylation of GP85 may be an important part of the lymphoma plasma membrane-cytoskeleton interaction.     

The rat lacrimal gland expresses the alpha isoform of PKC. Further evidence for the PMA-activated and phospholipid-independent protein kinase activity.

The molecular heterogeneity of protein kinase C (PKC) is now widely documented. In our first report, we characterized the rat lacrimal gland PKC along with a phorbol 12-myristate 13-acetate (PMA)-activated and phospholipid-independent protein kinase activity [Mauduit P., Zoukhri D. and Rossignol B. (1989) Fedn Eur. biochem. Socs Lett. 252, 5-11. In this work, we show that when the rat lacrimal gland cytosolic fraction is chromatographed on hydroxyapatite, only one peak of PKC activity can be detected. Comparison with a rat brain cytosolic fraction indicated that it is PKC-alpha which is expressed in the rat lacrimal gland. This result was confirmed by the use of polyclonal antibodies raised against rat brain PKC-alpha, beta and gamma isoforms. We also provide evidence that free arachidonic acid activates PKC, as does PMA, in a calcium and phospholipid-free system.     

Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons

Abstract: The ability of ethanol to enhance GABA_A receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA_A inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA_A IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABA_A IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA_A receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA_A receptors in the mammalian brain.     

Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-alpha stimulation, and effects of a phosphodiesterase inhibitor

Diabetic nephropathy is a major complication of diabetes leading to end-stage renal disease, which requires hemodialysis. Although the mechanism by which it progresses is largely unknown, the role of hyperglycemia-derived oxidative stress has recently been the focus of attention as the cause of diabetic complications. Constituent cells of the renal glomeruli have the capacity to release reactive oxygen species (ROS) upon stimulation of NADPH oxidase activated by protein kinase C (PKC). Hyperglycemia and insulin resistance in the diabetic state are often associated with activation of PKC and tumor necrosis factor (TNF)-alpha, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and TNF-alpha in rat glomeruli. Isolated rat glomeruli were stimulated with phorbol 12-myristate 13-acetate (PMA) and TNF-alpha, and the amount of ROS was measured using a chemiluminescence method. Stimulation with PMA (10 ng/ml) generated ROS with a peak value of 136+/-1.2 cpm/mg protein (mean+/-SEM). The PKC inhibitor H-7, the NADPH oxidase inhibitor diphenylene iodonium and the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin inhibited PMA-induced ROS production by 100%, 100% and 80%, respectively. In addition, TNF-alpha stimulated ROS production (283+/-5.8/mg protein/20 min). The phosphodiesterase inhibitor cilostazol activates protein kinase A and is reported to improve albuminuria in diabetic rats. Cilostazol (100 microg/ml) inhibited PMA, and TNF-alpha-induced ROS production by 78+/-1.8, and 19+/-2.7%, respectively. The effects of cilostazol were not additive with wortmannin. Cilostazol arrests oxidative stress induced by PKC activation by inhibiting the PI-3 kinase-dependent pathway, and may thus prevent the development of diabetic nephropathy.     

Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Immunochemical characterization of rat brain protein kinase C

Polyclonal antibodies against rat brain protein kinase C (the Ca2+/phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca2+, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca2+/phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of [3H]phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. This degraded form of the phorbol ester-binding protein still requires phospholipid for activity but, unlike the native enzyme, becomes less dependent on Ca2+. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca2+-independent/phospholipid-dependent phorbol ester-binding protein.     

Protein Kinase C-Mediated Phosphorylation of Kvβ2 in Adult Rat Brain

The phosphorylation of Kvβ2 was investigated by different protein kinases. Protein kinase A catalytic subunit (PKA-CS) yielded the greatest phosphorylation of recombinant Kvβ2 (rKvβ2), with limited phosphorylation by protein kinase C catalytic subunit (PKC-CS) and no detectable phosphorylation by casein kinase II (CKII). Protein kinase(s) from adult rat brain lysate phosphorylated both rKvβ2 and endogenous Kvβ. The PKA inhibitor, PKI 6-22, fully inhibited PKA-mediated phophorylation of rKvβ2 yet showed minimal inhibition of kinase activity present in rat brain. The inhibitor Gö 6983, that blocks PKCα, PKCβ, PKCγ, PKCδ and PKCζ activities, inhibited rKvβ2 phosphorylation by rat brain kinases, with no inhibition by Gö 6976 which blocks PKCα and PKCβΙ activities. Dose-response analysis of Gö 6983 inhibitory activity indicates that at least two PKC isozymes account for the kinase activity present in rat brain. Τhus, while PKA was the most active protein kinase to phosphorylate rKvβ2 in vitro, Kvβ2 phosphorylation in the rat brain is mainly mediated by PKC isozymes.     

Expression of protein kinase C subspecies in human leukemia-lymphoma cell lines

Expression of protein kinase C (PKC) subspecies was studied in various human leukemia-lymphoma cell lines. The PKC in most cell lines examined was resolved into two major fractions corresponding to type II (beta-sequence) and type III (alpha-sequence) PKC of the rat brain. The amounts of these two subspecies greatly varied among the cell lines. Type I PKC (gamma-sequence) was expressed in none of the cell lines tested, but PKCs with undefined structures were frequently detected. The differential co-expression of several PKC subspecies is presumably related to the state of cell differentiation.     

Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia

Protein kinase C (PKC) activity was determined in different (membrane, nuclear and soluble) subcellular fractions prepared separately from the CA1 and CA3 subfields ofMongolian gerbils hippocampus at various time intervals following a single 5-min occlusion of the common carotid arteries.Soluble andnuclear PKC activities of the CA1 sector were found to be elevated at 24 hours following the ischemic injury, while PKC activities did not increase in the CA3 region until the 3rd day after ischemia. The ratio ofsoluble/membrane-associated PKC activities followed a similar pattern, predominantly because the activation/elevation and then down regulation of the cytosolic enzyme pool changing correspondingly to the ongoig pathological processes. PKC activity returned to the normal level in each subfraction of the CA3 subfield by the 7th day. However, PKC activity remained elevated in the soluble fraction of the CA1 sector even after the delayed death of pyramidal neurons, presumably because of the reactive response of astrocytes. Conceivably, the transient activation and rapid down regulation of PKC in the CA1 sector may contribute to the initiation of postischemic neuronal death in the CA1 subfield.Abbreviations BSA bovine serum albumin - GFAP glial fibrillary acidic protein - PKC Ca²⁺/phospholipid-dependent protein kinase - PMSE phenylmethylsulfonyl fluoride     

Atrial natriuretic factor inhibits autophosphorylation of protein kinase C and A 240-kDa protein in plasma membranes of bovine adrenal glomerulose cells: Involvement of cGMP-dependent and independent signal transduction mechanisms

To clarify the intracellular signalling mechanisms of atrial natriuretic factor (ANF), we studied its effect on protein phosphorylation in plasma membranes of bovine adrenal cortical cells. ANF (1×10^–7 M) inhibited phosphorylation of the 78-kDa protein kinase C (PKC) and a 240-kDa protein in specific manner. In parallel experiments, cGMP (0.5 mM) inhibited phosphorylation of only the 78-kDa PKC but it did not affect phosphorylation of the 240-kDa protein. Phosphorylation of the 78-kDa PKC was enhanced in a Ca²⁺-/phospholipid-dependent manner. However, after prolonged preincubation of plasma membranes with Ca²⁺ (0.5 mM), the incorporation of³²P-radioactivity rapidly decreased in the 78-kDa PKC and subsequently increased in the 45- and 48-kDa protein bands due to Ca²⁺-dependent proteolytic degradation of 78-kDa PKC. Polyclonal antibodies against brain PCK were used to immunoblot and immunoprecipitate the 78-kDa PKC. Preincubation of plasma membranes with Ca²⁺ for varying times, followed by immunoblotting revealed a gradual loss of the immunoreactive 78-kDa PKC band in a time-dependent manner. Immunoprecipitation of phosphorylated 78-kDa PKC in plasma membranes showed that its phosphorylation was significantly inhibited in the presence of ANF as compared to control membranes, phosphorylated in the absence of ANF. The results in this present study document a new signal transduction mechanism of ANF at molecular level which possibly involves dephosphorylation of the 78-kDa PKC and a 240-kDa protein in a cGMP-dependent and-independent manner in bovine adrenal glomerulosa cell membranes. (Mol Cell Biochem141: 103–111, 1994)     

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.     

Hyperexcitability and changes in activities of Ca2+/calmodulin-dependent kinase II and mitogen-activated protein kinase in the hippocampus of rats exposed to 1-bromopropane

Chronic inhalation of 1-bromopropane (1-BP), a substitute of ozone-depleting chlorofluorocarbons, has been suspected of having central neurotoxicity (Clinical Neurology and Neurosurgery 101 (1999) 199; Journal of Occupational Health 44 (2002) 1) for humans. In animal experiments, 1-BP inhalation (1500 ppm) caused hyperexcitability in the CA1 and the dentate gyrus (DG) [Journal of Occupational Health 42 (2000) 149, Journal of Occupational Health 44 (2002) 156]. We studied whether the hyperexcitability is associated with changes of Ca2+/calmodulin-dependent kinase II (CaMKII), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC). Male Wistar rats were exposed to 1-BP for 6 hours in a day in an exposure chamber with a concentration of 700 ppm for 8 weeks. After the inhalation, paired-pulse ratios of field excitatory postsynaptic potentials and population spikes (PSs) were analyzed in the CA1 and DG of hippocampal slices. Control rats were then given fresh air in the inhalation chamber. Semiquantitative immunoblotting analyses of protein kinases using antibodies against active and conventional protein kinases were done using the whole hippocampus. A paired-pulse ratio of PS was increased at the 5 ms interpulse interval in the CA1 and at the 10-20 ms interpulse intervals in the DG. The amount of active MAPK and total amount of CaMKIIalpha and beta were significantly increased by 28, 29, and 46% compared to control, respectively, without any change in PKC activity. In contrast, the amount of active CaMKIIbeta was decreased to 78%. These results suggest that modifications of intracellular signaling cascades are associated with hyperexcitability that occurred in the hippocampal formation of rats exposed to the chronic inhalation of 1-BP.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

Human recombinant chromogranin A-derived vasostatin-1 mimics preconditioning via an adenosine/nitric oxide signaling mechanism

The acidic protein chromogranin A (CgA) is the precursor of several regulatory peptides generated by specific proteolytic processes. Human recombinant CgA NH(2)-terminal fragment STA-CgA(1-78) (hrSTA-CgA(1-78)), containing vasostatin-1 (CgA(1-76)) domain, exerts a negative inotropic effect and counteracts the beta-adrenergic positive inotropic effect on the rat heart. We hypothesized an involvement of nitric oxide (NO)-dependent pathway in both cardiodepression and cardioprotection by hrSTA-CgA(1-78). We also hypothesized an involvement of adenosine A(1) receptor and protein kinase C (PKC) in cardioprotection by hrSTA-CgA(1-78). Therefore, we evaluated whether 1) the cardioinhibition mediated by hrSTA-CgA(1-78) involves the G(i/o) proteins/NO-dependent signal transduction cascade, 2) hrSTA-CgA(1-78) induces ischemic preconditioning-like protective effects on the myocardium, and 3) inhibition of NO synthase (NOS), adenosine A(1) receptor, or PKC affects hrSTA-CgA(1-78) protection. Using the isolated rat heart, we found that the reduction of left ventricular pressure (LVP), rate-pressure product, and maximal values of the first derivative of LVP elicited by hrSTA-CgA(1-78) at 33 nM is abolished by blocking G(i/o) proteins with pertussis toxin, scavenging NO with hemoglobin, and blocking NOS activity with N(G)-monomethyl-l-arginine or N(5)-(iminoethyl)-l-ornithine, soluble guanylate cyclase with 1H-[1,2,4]oxadiazole-[4,4-a]quinoxalin-1-one, and protein kinase (PKG) with KT5823. Data suggest the involvement of the G(i/o) proteins/NO-cGMP-PKG pathway in the hrSTA-CgA(1-78)-dependent cardioinhibition. When given before 30 min of ischemia, hrSTA-CgA(1-78) significantly reduced the size of the infarct from 64 +/- 4 to 32 +/- 3% of the left ventricular mass. This protective effect was abolished by either NOS inhibition or PKC blockade and was attenuated, but not suppressed, by the blockade of A(1) receptors. These results suggest that hrSTA-CgA(1-78) activity triggers two different pathways: one of these pathways is mediated by A(1) receptors, and the other is mediated by NO release. As with repeated brief preconditioning ischemia, hrSTA-CgA(1-78) may be considered a stimulus strong enough to trigger both pathways, which may converge on PKC.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS

To study the cell signaling events leading to 17beta-estradiol (E(2))-induced acute cardioprotection, we subjected isolated rat hearts to three 5-min cycles of 10 microM E(2) before 30 min of regional ischemia, followed by 2 h of reperfusion. Protection was judged by changes in infarct size in percentage of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC), or reactive oxygen species (ROS) in E(2)-induced protection, we combined wortmannin (1 microM), chelerythrine (2 microM), and 2-mercaptopropionylglycine (300 microM), respectively, with E(2) exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E(2) preconditioning were investigated. E(2) preconditioning led to significant reduction in infarct size from 31.8 +/- 5.3 to 20.2 +/- 2.6% in male hearts and from 42.7 +/- 4.7 to 17.1 +/- 3.4% in female hearts (P < 0.05). Protection was abolished by wortmannin (30.0 +/- 3.2%), chelerythrine (45.1 +/- 4.4%), and 2-mercaptopropionylglycine (36.8 +/- 4.7%). E(2) preconditioning induced phosphorylation of PKB, PKCalpha, and PKCepsilon and membrane translocation of PKCepsilon and PKCdelta. Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E(2) to induce preconditioning-like cardioprotection via cell signaling events shared by classic ischemic preconditioning.     

The activation of protein kinase C by the polyphosphoinositides phosphatidylinositol 4,5-diphosphate and phosphatidylinositol 4-monophosphate

Protein kinase C(PKC) is a Ca2+- and phospholipid-dependent protein kinase which can be activated by diacylglycerol, a product of polyphosphoinositide hydrolysis. In this report, we show that the polyphosphoinositides L-alpha-phosphatidylinositol 4-monophosphate (PI 4P) and L-alpha-phosphatidylinositol 4,5-diphosphate (PI 4.5DP) can serve as phospholipid cofactors of isolated rat brain PKC. The order of potency of the phosphoinositides in the activation of PKC, PI greater than PI 4P greater than PI 4,5DP, shows a negative correlation with the degree of acidity of the phospholipid head group, whether 1 mM Ca2+ or 200 nM TPA is present in the reaction assay mixture. Although the polyphosphoinositides are by themselves weaker activators of PKC than PI, small amounts of PI 4,5DP cause a two-fold enhancement of PKC in the presence of Ca2+ and PI. While the endogenous phospholipid cofactors of PKC remain to be identified, these results suggest that the small amounts of polyphosphoinositides which are present in cell membranes may play a direct role in the activation of PKC in vivo, by serving as phospholipid cofactors of the enzyme.