Oxidant-Induced Activation of Protein Kinase C in Uc11mg Cells

Free radical formation and subsequent lipid peroxidation may participate in the pathogenesis of tissue injury, including the brain injury induced by hypoxia or trauma and cardiac injury arising from ischemia and reperfusion. However, the exact cellular mechanisms by which the initial oxidative insult leads to the ultimate tissue damage are not known. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this work, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H₂O₂. Incubation with 0.5 mM H₂O₂ increased malondialdehyde levels by as early as 15 minutes. To assess the effects of H₂O₂ treatment on PKC activation, we measured phosphorylation of an endogenous PKC substrate, the MARCKS (myristoylated alanine-rich C kinase substrate) protein. Treatment of cells with 0.2-1.0 mM H₂O₂ resulted in a rapid increase in MARCKS phosphorylation. Phosphorylation was stimulated approximately 2.5-fold following treatment with 0.5 mM H₂O₂ for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, increased MARCKS phosphorylation approximately 4-fold. The H₂O₂-induced MARCKS phosphorylation was inhibited by the addition of the kinase inhibitors H-7 and staurosporine. Furthermore, specific down-regulation of PKC by phorbol ester also inhibited H₂O₂-induced MARCKS phosphorylation. These results indicate that PKC is rapidly activated in cells following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidative damage in the cell.     

Effects of Hyperglycemia and Protein Kinase C on Connexin43 Expression in Cultured Rat Retinal Pigment Epithelial Cells

Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions. Received: 21 July 2000/Revised: 19 January 2001     

Involvement of Desmoplakin Phosphorylation in the Regulation of Desmosomes by Protein Kinase C,in HeLa Cells

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, anti-phosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation of desmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.     

蛋白激酶C分子的成熟与活化

蛋白激酶C（Proteinkinase C,PKc）是细胞内一类重要的Ser／Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环（Acti．vation-loop,A—loop）、转角模体（Tummotif,T1V1）以及疏水模体（hydrophobic motif,HM）的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.     

蛋白激酶C分子的成熟与活化

蛋白激酶C(Protein kinase C,PKC)是细胞内一类重要的Ser/Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环(Activation-loop,A-loop)、转角模体(Turn motif,TM)以及疏水模体(hydrophobic motif,HM)的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

Abstract: Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin O-permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of ³²P incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serine was the only phosphorylated residue, (b) ³²P incorporation was inhibited by the protein kinase inhibitors H7, GF 109203X, and staurosporine, and (c) activators of this enzyme, 12- O -tetradecanoylphorbol 13-acetate and 1,2-dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases.     

Distinct Effects of Protein Kinase C on the Barrier Function at Different Developmental Stages

We show here, that activation of protein kinase C by the phorbol ester PMA improves barrier function in colon carcinoma (HT 29) cells. By contrast, in canine kidney (MDCK I) cells it caused increased permeability and opening of tight junctions; the latter has also been noticed in other studies. Thus, with PMA confluent HT 29 cells responded with a reduced passage of 330 kDa sodium fluorescein, increased transepithelial electrical resistance, and a change in the cell shape of the HT 29 cells from an irregular to a regular, hexagonal form. Confocal imaging revealed parallel distinct changes in the staining of occludin and caludin-1, viz. a translocation from cytoplasmic clusters to apical cell–cell contacts. Interestingly, in both cell lines protein kinase A activation caused a decreased in the threonine phosphorylation of occludin that correlated with tight junction assembly in HT 29 cells and tight junction disassembly in MDCK I cells. We conclude that protein kinase C regulation of the epithelial barrier involves specific molecular mechanisms and achieves distinct effects at different developmental stages.     

Long-Term Activation of Protein Kinase C by Nicotine in Bovine Adrenal Chromaffin Cells

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.     

Phosphorylation and Activation of Tryptophan Hydroxylase by Exogenous Protein Kinase A

Abstract: The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in V_max without alterations in the K_m for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.     

Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor-Mediated Phospholipase C Activation in PC12 Cells

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT-induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT-stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)-α-methyl-4-carboxyphenylglycine; (+)-MCPG]. Furthermore, the rise in IP₃ level was blocked by pretreating cells with a PLC inhibitor (U-73122) or by a combination of atropine and (+)-MCPG. This pretreatment also significantly decreased TMT-stimulated PKC translocation, indicating that TMT-mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)-MCPG did not provide protection against TMT-induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein-coupled receptors, ultimately leading to PKC translocation.     

Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells

Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.     

The Src Family Kinases and Protein Kinase C Synergize to Mediate Gq-dependent Platelet Activation

The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y₁₂ deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking G_q. We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5′-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and G_q deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from G_q/Ca²⁺ signaling. Moreover, A23187-induced thromboxane A₂ synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A₂ production in wild-type and P2Y₁₂ deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin α_IIbβ₃ activation, and aggregation of P2Y₁₂ deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data indicate that Ca²⁺/SFKs/PI3K and PKC represent two alternative signaling pathways mediating G_q-dependent platelet activation.     

Phosphorylation of the Conditioning-Associated GTP-Binding Protein cp20 by Protein Kinase C

Abstract: The phosphorylation state of cp20, a low molecular weight membrane-associated GTP-binding protein, was previously shown to increase two- to threefold 24 h after associative conditioning. Here, cp20 is shown to be phosphorylated by protein kinase C (PKC) in vitro. Pronounced differences in activity were observed with the three major isoforms of PKC, whereas casein kinase, calcium/calmodulin-dependent protein kinase II, and cyclic AMP-dependent protein kinase produced no detectable phosphorylation of cp20. Phosphorylation of cp20 had no effect on its GTPase or GTP-binding activity but caused a translocation of cp20 from cytosol to the nuclei/mitochondrial particulate fraction. These results suggest that the increase in phosphorylation of cp20 after conditioning may be due to PKC.     

Role of Cx43 Phosphorylation and MAP Kinase Activation in EGF Induced Enhancement of Cell Communication in Human Kidney Epithelial Cells

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.