scholarly journals Protein kinase C isoenzymes in airway smooth muscle

1997 ◽  
Vol 324 (1) ◽  
pp. 167-175 ◽  
Author(s):  
Benjamin L. J. WEBB ◽  
Mark A. LINDSAY ◽  
Peter J. BARNES ◽  
Mark A. GIEMBYCZ
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Oligonucleotide Primer ◽  
Mrna Levels ◽  
Kinase C ◽  
Gf 109203X ◽  
Bona Fide

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCα, PKCβI and PKCβII. In some experiments immunoreactive bands corresponding to PKCδ, PKCϵ and PKCθ were also labelled, whereas the γ, η and ζ isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCδ, PKCϵ and PKCζ, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using ϵ-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, βI and βII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCα′, PKCβI′ and PKCβII′. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs α′, βI′ and βII′ represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when ϵ-peptide is used as the substrate.

Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1

1998 ◽  
Vol 274 (5) ◽  
pp. L842-L853 ◽  
Author(s):  
Larissa A. Shimoda ◽  
J. T. Sylvester ◽  
James S. K. Sham
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Vascular Reactivity ◽  
Signal Transduction Pathway ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Kinase C ◽  
Gf 109203X

Although endothelin (ET)-1 is an important regulator of pulmonary vascular tone, little is known about the mechanisms by which ET-1 causes contraction in this tissue. Using the whole cell patch-clamp technique in rat intrapulmonary arterial smooth muscle cells, we found that ET-1 and the voltage-dependent K+(KV)-channel antagonist 4-aminopyridine, but not the Ca2+-activated K+-channel antagonist charybdotoxin (ChTX), caused membrane depolarization. In the presence of 100 nM ChTX, ET-1 (10−10to 10−7 M) caused a concentration-dependent inhibition of K+ current (56.2 ± 3.8% at 10−7 M) and increased the rate of current inactivation. These effects of ET-1 on K+ current were markedly reduced by inhibitors of protein kinase C (staurosporine and GF 109203X) and phospholipase C (U-73122) or under Ca2+-free conditions and were mimicked by activators of protein kinase C (phorbol 12-myristate 13-actetate and 1,2-dioctanoyl- sn-glycerol). These data suggest that ET-1 modulated pulmonary vascular reactivity by depolarizing pulmonary arterial smooth muscle, due in part to the inhibition of KV current that occurred via activation of the phospholipase C-protein kinase C signal transduction pathway.

scholarly journals Characterization and properties of protein kinase C from the filamentous fungus Trichoderma reesei

1998 ◽  
Vol 330 (2) ◽  
pp. 689-694 ◽  
Author(s):  
Thomas LENDENFELD ◽  
P. Christian KUBICEK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Trichoderma Reesei ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ligand Affinity ◽  
Kinase C ◽  
Endogenous Substrates

The Trichoderma reesei pkc1 gene encodes a fungal homologue of the protein kinase C (PKC) family. Using antibodies directed against the nt-sequence-deduced pseudosubstrate domain for identification, Pkc1p was purified by dye-ligand affinity chromatography and Mono Q anion-exchange chromatography. Both the denatured as well as the native enzyme showed an Mr of 116-118 kDa, indicating that Pkc1p is a monomer. The enzyme phosphorylates the mutated (A → S) pseudosubstrate peptide and myelin basic protein, but not histone. Replacing three of the five basic amino acids around the serine acceptor residue resulted in a 25-fold increase in the Km. Pkc1p activity was stimulated by phospholipids, but this stimulation was counteracted by micromolar concentrations of Ca2+. Three proteins (85, 48 and 45 kDa) were identified as preferred endogenous substrates of Pkc1p in vitro. The enzyme was capable of autophosphorylation, and neither phosphorylation nor dephosphorylation in vitro affected the activity of the enzyme. A 116 kDa protein of T. reesei was demonstrated to bind to the N-terminal C2-region of Pkc1p in vitro. These data define Pkc1p as a unique member of the PKC family.

Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

1989 ◽  
Vol 67 (6) ◽  
pp. 260-270 ◽  
Author(s):  
Gwyneth DeVries ◽  
Elaine D. Fraser ◽  
Michael P. Walsh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chicken Gizzard ◽  
Substrate Protein ◽  
Kinase C ◽  
Endogenous Substrates

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+-dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61 500 by gel filtration and 80 000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at ~5 × 10−7 M Ca2+ in the presence of L-α-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2− substrate, for activity. The Km for ATP was determined to be 20 μM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.Key words: protein kinase C, gizzard, inhibitor, endogenous substrates.

Growth factor activity of endothelin on vascular smooth muscle

1990 ◽  
Vol 258 (3) ◽  
pp. C408-C415 ◽  
Author(s):  
A. Bobik ◽  
A. Grooms ◽  
J. A. Millar ◽  
A. Mitchell ◽  
S. Grinpukel
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Phospholipase C ◽  
Pertussis Toxin ◽  
Calf Serum ◽  
Mrna Levels ◽  
Kinase C

Endothelin is a novel peptide secreted by endothelial cells, the vasoconstrictor effects of which appear dependent on the activation of phospholipase C. We examined in tissue culture its potential as a growth factor for vascular smooth muscle. In quiescent cultures of rat aortic smooth muscle cells, endothelin rapidly elevated levels of c-fos and c-myc mRNA. Peak effects on c-fos mRNA occurred between 15 and 30 min and were completely gone after 2 h. The elevation in c-fos mRNA was, in part, dependent on protein kinase C, since phorbol myristate acetate (PMA) also elevated c-fos mRNA and further increased c-fos mRNA expression by endothelin, but the effects were not additive. Furthermore, the endothelin-induced elevation in c-fos mRNA was attenuated but not abolished in protein kinase C-depleted cells. Maximum levels of c-myc mRNA occurred between 15 and 30 min after exposing the cells to endothelin and persisted for at least 6 h. The effects of simultaneous addition of endothelin and PMA on c-myc mRNA levels were essentially similar to those observed with c-fos mRNA. [3H]thymidine incorporation into DNA occurred 8 h after exposing the cells to endothelin. The mitogenic effect of endothelin was smaller than that observed with either fetal calf serum or epidermal growth factor and was dependent on both pertussis toxin-insensitive and -sensitive pathways. Sensitivity to the latter pathway did not appear dependent on attenuation of phospholipase C activity, since neither peak intracellular calcium concentrations nor c-fos mRNA levels were reduced in pertussis toxin-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

CCK stimulatesmob-1 expression and NF-κB activation via protein kinase C and intracellular Ca2+

2000 ◽  
Vol 278 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
Bing Han ◽  
Craig D. Logsdon
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dna Binding ◽  
Mrna Levels ◽  
Similar Extent ◽  
Pancreatic Acini ◽  
Chemokine Expression ◽  
Kinase C ◽  
Intracellular Ca ◽  
Gf 109203X

Supraphysiological concentrations of cholecystokinin (CCK) induce chemokine expression in rat pancreatic acini through the activation of the transcription factor NF-κB. In the current study, the intracellular signals involved in these pathophysiological effects of CCK were investigated. CCK induction of mob-1 expression in isolated rat pancreatic acini was blocked by the protein kinase C (PKC) inhibitors GF-109203X and Ro-32–0432 and by the intracellular Ca2+chelator BAPTA. CCK induced NF-κB nuclear translocation, and DNA binding was also blocked by GF-109203X and BAPTA. Direct activation of PKC with TPA induced mob-1 chemokine expression and activated NF-κB DNA binding to a similar extent as did CCK. Increasing intracellular Ca2+using ionomycin had no effect on mob-1 mRNA levels or NF-κB activity. Both CCK and TPA treatments decreased inhibitory κB-α (IκB-α) levels, whereas ionomycin had no effect. However, the effects of TPA on IκB-α degradation were less complete than for CCK. In combination, TPA and ionomycin degraded IκB-α to a similar extent as CCK. Therefore, activation of NF-κB and mob-1 expression by supraphysiological CCK is likely mediated by both PKC activation and elevated intracellular Ca2+.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Regulation of Scavenger Receptor Expression in Smooth Muscle Cells by Protein Kinase C

1997 ◽  
Vol 17 (5) ◽  
pp. 969-978 ◽  
Author(s):  
Michele Mietus-Snyder ◽  
Annabelle Friera ◽  
Christopher K. Glass ◽  
Robert E. Pitas
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Scavenger Receptor ◽  
Muscle Cells ◽  
Receptor Expression ◽  
Kinase C
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