Dephosphorylation of human fibrinogen, previously phosphorylated in vitro by protein kinase C, by whole blood or intestinal alkaline phosphatase. Effects on thrombin-induced gelation of in vitro dephosphorylated human fibrinogen

Human fibrinogen, a phosphoprotein, was either left untreated or phosphorylated by protein kinase C. Then both were dephosphorylated by calf intestinal alkaline phosphatase. The dephosphorylated fibrinogen gave an increased fibre thickness during thrombin-induced gelation. Whole blood anticoagulated by heparin, EDTA or sodium citrate, contained dephosphorylating activity against 32P-labeled fibrinogen, although there were significant differences in activity among the three anticoagulants.     

Effect of phosphorylation in vitro of human fibrinogen with protein kinase C on thrombin-induced gelation

Thrombin-induced gel formation of fibrinogen phosphorylated by protein kinase C yielded a transparent gel, whereas unphosphorylated fibrinogen yielded a coarse gel. The mass-length ratio was found to be one order of magnitude higher for the unphosphorylated than for the phosphorylated fibrinogen. Since the phosphorylated sites are located near the cross-linking sites in the A alpha-chain of fibrinogen, it is likely that the introduction of charged phosphate groups in this region prevent the lateral growth of the fibrin fibres.     

Dopamine transporters are dephosphorylated in striatal homogenates and in vitro by protein phosphatase 1

Dopamine transporters (DATs) undergo increased phosphorylation upon treatment of striatal tissue or cultured cells with protein kinase C activators and protein phosphatase inhibitors. Phosphorylation conditions also lead to reductions in dopamine transport activity, which may function to regulate synaptic dopamine levels and control the extent and duration of dopaminergic signaling. Treatment of rat striatal tissue with okadaic acid (OA), a broad-spectrum protein phosphatase inhibitor, produces apparent maximal increases in DAT phosphorylation, suggesting that dephosphorylation is a crucial regulator of the DAT phosphorylation state. We used a combination of endogenous and in vitro approaches to identify the phosphatase(s) responsible for this activity. In homogenates prepared from (32)PO(4)-labeled rat striatal slices, OA inhibited DAT dephosphorylation with an IC(50) of 40 nM, a dose most compatible with inhibition of protein phosphatase 1 (PP1). Dephosphorylation of DAT in striatal homogenates was also inhibited by PP1 inhibitor 2, while little effect was produced by protein phosphatase 2A inhibitor 1. In vitro dephosphorylation assays showed substantial removal of (32)PO(4) from DATs by PP1 but not by protein phosphatase 2A, protein phosphatase 2B, or protein tyrosine phosphatase, and this effect was blocked by OA, verifying that the (32)PO(4) loss from DAT was due to dephosphorylation. These results demonstrate that DAT is a direct substrate for PP1 in vitro and suggest that PP1 is a major DAT phosphatase in rat striatum.     

Myelin-associated glycoprotein is phosphorylated by protein kinase C

The myelin-associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin-forming cells and neurons. To characterize the molecular mechanisms underlying post-translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of ³²P-orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol-12-myristoyl-13-acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2–3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [γ-³²P] ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet-derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes. © 1993 Wiley-Liss, Inc.     

Identification of a protein phosphorylated by cAMP-dependent protein kinase in drosophila brain

In the present study, we examined the effects of reagents that elevate cAMP concentration on protein phosphorylation in Drosophila brain cells. Application of forskolin or dopamine to brain cells that were prelabeled with (32)Pi enhanced phosphorylation of a 25-kDa protein (p25a). To identify p25a, we isolated the protein, and partial amino acid sequences were analyzed. Database searches showed that p25a is a J-domain protein, dJDP that contains the J-domain found in the DnaJ (heat shock protein 40) family of molecular chaperones.     

Identification of a protein phosphorylated by cAMP-dependent protein kinase in drosophila brain

In the present study, we examined the effects of reagents that elevate cAMP concentration on protein phosphorylation in Drosophila brain cells. Application of forskolin or dopamine to brain cells that were prelabeled with ³²Pi enhanced phosphorylation of a 25-kDa protein (p25a). To identify p25a, we isolated the protein, and partial amino acid sequences were analyzed. Database searches showed that p25a is a J-domain protein, dJDP that contains the J-domain found in the DnaJ (heat shock protein 40) family of molecular chaperones.     

New protein kinase C activator regulates amyloid precursor protein processing in vitro by increasing alpha-secretase activity

The beta amyloid (Abeta) cascade has been at the forefront of the hypothesis used to describe the pathogenesis of Alzheimer's disease (AD). It is generally accepted that drugs that can regulate the processing of the amyloid precursor protein (APP) toward the non-amyloidogenic pathway may have a therapeutic potential. Previous studies have shown that protein kinase C (PKC) hypofunction has an important role in AD pathophysiology. Therefore, the effects of a new PKC activator, alpha-APP modulator [(2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam (TPPB)], on APP processing were investigated. Using PC12 cells and SH-SY5Y(APP695) cells, it was found that TPPB promoted the secretion of sAPPalpha without affecting full-length expression of APP. The increase in sAPPalpha by TPPB was blocked by inhibitors of PKC, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and tyrosine kinase, suggesting the involvement of these signal transduction pathways. TPPB increased alpha-secretase activity [a disintegrin and metalloproteinase (ADAM)10 and 17], as shown by direct fluorescence activity detection and Western blot analysis. TPPB-induced sAPPalpha release was blocked by the metalloproteinase inhibitor TAPI-2, furin inhibitor CMK and by the protein-trafficking inhibitor brefeldin. The results also showed that TPPB decreased beta-secretase activity, Abeta40 release and beta site APP-cleaving enzyme 1 (BACE1) expression, but did not significantly affect neprilysin (NEP) and insulin-degrading enzyme (IDE) expression. Our data indicate that TPPB could direct APP processing towards the non-amyloidogenic pathway by increasing alpha-secretase activity, and suggest its therapeutic potential in AD.     

Localization of protein kinase C in human skeletal muscle.

The immunolocalization of protein kinase C (PKC) isozymes alpha, beta I and beta II, was investigated in human skeletal muscle. All three isozymes were present on the muscle fiber surface membrane and within the muscle fibers. The alpha-isozyme was most clearly delineated on the surface membrane of the muscle fiber and on small blood vessels in the connective tissue. The axons of myelinated intramuscular nerves stained intensely for the beta I isozyme, whereas the endoneurial connective tissue reacted more strongly for the alpha- and beta II-isozymes. PKC isozymes may regulate intracellular signal transduction in human skeletal muscle, as in the other tissues, but their exact role in muscle remains unknown.     

Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP-dependent and Ca2+/calmodulin-dependent protein kinase in the rat hippocampal slice.

Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long-term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylated of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle-associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose-dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho-site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP-dependent protein kinase and the multifunctional Ca2+/calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expressions of protein kinase C isozymes during proliferation and differentiation of human skeletal muscle cells in vitro

The mechanism of skeletal muscle regeneration in vivo can be well modeled in vitro by culturing skeletal muscle cells. In these cultures mononuclear satellite cells fuse to form polynuclear myotubes by proliferation and differentiation. The aim of this study was to determine how the different protein kinase C (PKC) isozymes were expressed during differentiation of human skeletal muscle in vitro. The expressions of desmin, used as a muscle-specific intermediate filament protein marker of differentiation, and of different PKC isozymes were detected by single and double immunohistochemical labeling, and by Western blot analysis. In skeletal muscle cells we could identify five PKC isozymes (PKCα, -γ, -η, -θ and -ζ). The expressions of PKCα and -ζ did not change significantly during differentiation; their levels of expression were high in the early immature cells and remained unchanged in later phases. In contrast, the expression levels of PKCγ and -η increased with differentiation. Furthermore, the cellular localization of PKCγ markedly altered during differentiation, with a perinuclear-nuclear to cytoplasmic translocation. The change in the level of expression of PKCθ during differentiation showed different pattern; its expression was high during the early phases, but a decreased immunostaining was detected in the matured, well-differentiated myotubes. We conclude, therefore, that cultured human skeletal muscle cells possess a characteristic PKC isozyme pattern, and that the different phases of differentiation are accompanied by different expression patterns of the various isozymes. These data suggest the possible functional and differential roles of PKC isozymes in human skeletal muscle differentiation. Received: 28 February 1999 / Revised, accepted: 24 June 1999     

Opposing influences of protein kinase activities on neurite outgrowth in human neuroblastoma cells: initiation by kinase A and restriction by kinase C.

The respective roles of cAMP-dependent protein kinase (protein kinase A [PKA]) and protein kinase C (PKC) in the early stages of neurite outgrowth were examined in SH-SY-5Y human neuroblastoma cells. Forskolin or dbcAMP, agents that increase intracellular cAMP levels, and intracellular delivery of PKA catalytic subunit induced neurite outgrowth. The PKA inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), prevented the increases, and decreased further the percentage of cells possessing short, filopodia-like neurites in the absence of inducers. In contrast to effects on PKA activation, PKC activation by 12-0-tetradecanoylphorbol-13-acetate (TPA) reduced the percentage of filopodia-like neurites elaborated by otherwise untreated cells, and prevented neurite outgrowth induced by PKA activators. PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), staurosporine, and sphingosine induced neurite outgrowth. Neurites induced by PKA activation contained higher levels of tubulin immunoreactivity than those induced by PKC inhibition. Furthermore, PKA-induced neurites rapidly retracted in the presence of colchicine, while those elaborated following PKC inhibition were more resistant. These data suggest that neurites elaborated in response to PKA activation are dependent upon microtubule polymerization, and that neurite induction following PKC inhibition is mediated by a different mechanism. PKA activators and PKC inhibitors exerted additive effects on neurite outgrowth, suggesting that the distinct pathways regulated by these two kinases function cooperatively during neuritogenesis.     

Involvement of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse formalin pain model

In the present study, we investigated the role of phosphorylated calcium/calmodulin-dependent protein kinase II (pCaMK-II) and phosphorylated extracellular signal-regulated protein kinase (pERK) in nociceptive processing at the spinal and supraspinal levels in the formalin subcutaneous induced mouse pain model. In the immunoblot assay, subcutaneous (s.c.) injection with formalin increased the pERK and pCaMK-IIalpha level in the spinal cord, and an immunohistochemical study showed that the increase of pERK and pCaMK-IIalpha immunoreactivity mainly occurred in the laminae I and II areas of the spinal dorsal horn. At the supraspinal level, although pERK was not changed in the hippocampus induced by formalin s.c. injection, pCaMK-IIalpha was increased in the hippocampus and hypothalamus by s.c. formalin injection, and an increase of pCaMK-IIalpha immunoreactivity mainly occurred in the pyramidal cells and the stratum lucidum/radiatum layer of the CA3 region of hippocampus and paraventricular nucleus of the hypothalamus. Moreover, pERK immunoreactivity in the hypothalamic paraventricular nucleus was also increased. The second phase of nociceptive behavior induced by formalin administered either i.t. or intracerebroventricularly (i.c.v.) was attenuated by PD98059 (ERK inhibitor) as well as KN-93(a CaMK-II inhibitor). On the other hand, the first phase of nociceptive behavior induced by formalin s.c. injection was not affected by i.t. KN-93. Our results suggest that pERK and pCaMK-II located at both the spinal cord and supraspinal levels are an important regulator during the nociceptive processes induced by formalin administered s.c. respectively.     

Production of recombinant human protein C in vitro and in vivo by muscle cells.

Protein C plays a key role in a natural anticoagulation mechanism, and is also implicated in fibrinolytic and anti-inflammatory functions. Here we describe the production of biologically active human protein C by muscle-targeted gene transfer. Human protein C expression vectors were designed and constructed to produce human protein C in skeletal muscle cells. These vectors were tested in transient and stable transfections of SCID mice myoblasts. Stably transfected cells produced as high as 2.27 microg/10(6) cells/day. Human protein C produced had a relative activity of 92+/-8% compared to the plasma derived human protein C, and was composed of alpha and beta forms, 69% and 31%, respectively. After implantation of stably transfected myoblasts into the hind limb muscles of SCID mice, systemic stable production of human protein C in a range of 31-116 ng/ml serum was obtained up to at least 2.5 months.     

Translocation of protein kinase C by halothane in cholinergic cells

Protein kinase C (PKC) is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for volatile anaesthetic actions. However, the effects of these agents on PKC activity are not yet fully understood. Volatile anaesthetics increase intracellular calcium concentration ([Ca(2+)](i)) in a variety of cells, thus their effects on PKC activity may be indirect due to [Ca(2+)](i) increase. Alternatively, the anaesthetics could directly stimulate PKC activity. In order to distinguish these two possibilities in intact cells, we used a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII) and confocal microscopy to evaluate the dynamic redistribution of PKC in living SN56 cells, a cholinergic cell line, in response to halothane. Halothane induced PKC translocation in SN56 cells transfected with GFP-PKCbetaII. This effect was not suppressed by dantrolene, a drug that blocks halothane-induced Ca(2+) release from intracellular stores in these cells. These findings indicate that halothane induces PKC translocation in SN56 cells independently of its ability to release calcium from internal stores.     

Inhibitory effects of omega-3 fatty acids on protein kinase C activity in vitro

Preliminary clinical data indicate that omega-3 fatty acids may be effective mood stabilizers for patients with bipolar disorder. Both lithium and valproic acid are known to inhibit protein kinase C (PKC) activity after subchronic administration in cell culture and in vivo. The current study was undertaken to determine the effects of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on protein kinase C phosphotransferase activity in vitro. Various concentrations of DHA, EPA, and arachidonic acid (AA) were incubated with the catalytic domain of protein kinase C beta from rat brain. Protein kinase C activity was measured by quantifying incorporation of (32)P-PO(4) into a synthetic peptide substrate. Both DHA and EPA, as well as the combination of DHA and EPA, inhibited PKC activity at concentrations as low as 10 micromol l(-1). In contrast, arachidonic acid had no effect on PKC activity. Thus, PKC represents a potential site of action of omega-3 fatty acids in their effects on the treatment of bipolar disorder.     

Neuritogenesis in cerebellar granule cells in vitro: a role for protein kinase C

We have used short-term (8 h) cultures of week-old rat cerebellar granule cells to examine the effects on neuritogenesis of activation and down-regulation of protein kinase C by phorbol esters. We have previously demonstrated that endogenously released glutamate promoted neurite outgrowth in the same system acting via N-methyl-D-aspartate receptors. Low levels (0.1-1 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) evoked increases in the number of granule cells which extended neurites; higher levels (10-250 nM) which caused a down-regulation of total protein kinase C, inhibited outgrowth in a dose-dependent manner. N-Methyl-D-aspartate by itself also stimulated process outgrowth but could not reverse the inhibition evoked by either TPA or the protein kinase C inhibitor sphingosine. Stimulation of protein kinase C with 0.1 nM TPA resulted in a general increase in the incorporation of 32P-labelled inorganic orthophosphate into granule cell polypeptides. The results indicate that the activation of protein kinase C is involved in neuritogenesis in granule cells and are consistent with the idea that N-methyl-D-aspartate receptor activation may exert its effect on neuritogenesis through protein kinase C.     

Uridine induces differentiation in human neuroblastoma cells via protein kinase C epsilon

Uridine metabolism has an important role in the physiopathology of the nervous system. In this paper, we have explored the effects of exogenous uridine on LAN-5 human neuroblastoma cells. Cells were exposed to uridine for 4 days and cell proliferation, neurite outgrowth, and 160 kDa neurofilament (NF) expression were the parameters measured. Our results showed that 10 microg/ml uridine decreased cell proliferation, this effect being associated with an increase in cell differentiation, as evidenced by neurite outgrowth and NF expression. These effects can be prevented by dipyridamole (10 microM), an inhibitor of nucleotides and nucleosides uptake. In the literature, neuroblastoma cells differentiation has been demonstrated to involve Protein Kinase C epsilon (PKCepsilon). After treatment with uridine, we observed in LAN-5 cells an increase in PKCepsilon protein level. This increase was inhibited by dipyridamole. Moreover, the increase of neurite outgrowth induced by uridine was inhibited by treatment with bisindolylmaleimide I (GF109203X), an inhibitor of PKC. Our data suggest that PKCepsilon is involved in uridine-induced cell differentiation in human neuroblastoma cells.     

Augmentation of procoagulant activity in monokine stimulated human endothelial cells by calmodulin/protein kinase C inhibitors

The incubation of human umbilical cord endothelial cell cultures with inflammatory mediators results in the induction of procoagulant activity. As many of these mediators activate protein kinase C, the effect of calmodulin and protein kinase C inhibitors on IL-1, TNF, phorbol ester and LPS stimulated procoagulant activity was determined. Incubation of endothelial cell cultures with these inflammatory agents in the presence of phenothiazine derivatives or other classes of calmodulin and protein kinase C antagonists resulted in a 2-4 fold increase in procoagulant activity compared to parallel stimulated cultures in the absence of antagonists. The augmented response of IL-1 stimulated endothelial cells to these antagonists was actinomycin D sensitive.     

Granulocyte enzyme mediated degradation of human fibrinogen in plasma in vitro

Incubation of plasma with granulocyte enzymes in the presence of kallikrein inhibitor resulted in a prolonged thrombin time of the plasma and in an increased level of fibrinogen fragments, indicating that fibrinogen was digested. In accordance with this, fibrinogen digestion products could be purified by affinity chromatography from plasma after incubation with granulocyte enzymes. The isolated products resembled early X-like fibrinogen fragments, which are produced by limited digestion of purified fibrinogen with elastase. On SDS gel electrophoresis both had no intact Aα-chains, but apparently intact fibrinogen Bβ- and γ-chains. Also, both fragments isolated from plasma and the X-like fragments produced with purified elastase had a low anticoagulant activity. Although elastase, the main fibrinolytic enzyme of the granulocyte, was rapidly complexed with inhibitors, 10–20% of the elastase activity towards succinyl-trialanyl-paranitro-anilide was detectable in plasma no matter if the mixture of granulocyte enzymes or purified elastase had been added. A possible role for the α₂-macroglobulin-granulocyte elastase complex in the production of the digestion products in plasma is discussed.     

In vitro effects of metrifonate on neuronal amyloid precursor protein processing and protein kinase C level

Alteration in the processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease (AD). It has been suggested that acetylcholinesterase (AChE) inhibitors, which promote the cholinergic function and consequently improve the cognitive deficits, may also exert a neuroprotective effect by activating normal APP processing. We now report that an irreversible AChE inhibitor (metrifonate) increase the cell-associated APP level in a basal forebrain neuronal culture and also elevate the amount of APP secreted into the medium. The alterations in APP processing were accompanied by increased protein kinase C (PKC) levels. The results suggest that AChE inhibitors modulate the metabolism of APP, possibly via their stimulatory effects on PKC. Since changes in the activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the beneficial effect of metrifonate in AD therapy may be due not only to the stimulatory cholinergic function, but also to its activating effect on PKC.