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.

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.

scholarly journals Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C

2000 ◽  
Vol 350 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Philip A. HELLIWELL ◽  
Michael RICHARDSON ◽  
Julie AFFLECK ◽  
George L. KELLETT
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Sugar Absorption ◽  
Kinase C

Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC βII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3–4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.

scholarly journals Protein kinase C-independent stimulation of activator protein-1 and c-Jun N-terminal kinase activity in human endometrial cancer cells by the LHRH agonist triptorelin

2001 ◽  
pp. 651-658 ◽  
Author(s):  
C Grundker ◽  
L Schlotawa ◽  
V Viereck ◽  
G Emons
Keyword(s):  
Cell Proliferation ◽  
Endometrial Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Fold Increase ◽  
Lhrh Agonist ◽  
Activator Protein 1 ◽  
Kinase C ◽  
Lhrh Receptor

OBJECTIVE: The expression of luteinizing hormone-releasing hormone (LHRH) and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumours, including cancers of the endometrium. The signalling pathway through which LHRH acts in endometrial cancer is distinct from that in pituitary gonadotrophs. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors via activation of a phosphotyrosine phosphatase, resulting in down-regulation of cancer cell proliferation. In addition, LHRH activates nucleus factor kappaB (NFkappaB) and protects the cancer cells from apoptosis. This study was conducted to investigate additional signalling mechanisms of the LHRH receptor cooperating with NFkappaB in endometrial cancer cells. DESIGN: The LHRH agonist triptorelin-induced activator protein-1 (AP-1) activation was analysed using a pAP-1-SEAP reporter gene assay. Expression of c-jun mRNA was quantified using quantitative reverse transcription (RT)-PCR. c-Jun N-terminal kinase (JNK) activity was measured by quantification of phosphorylated c-Jun protein. RESULTS: Treatment of Ishikawa and Hec-1A human endometrial cancer cells with 100 nM triptorelin resulted in a 3.1-fold and 3.5-fold activation of AP-1 respectively (P<0.05). If the cells had been made quiescent, treatment with triptorelin (100 nM) resulted in a 41.7-fold and 48.6-fold increase of AP-1 activation respectively (P<0.001). This effect was completely blocked by simultaneous treatment with pertussis toxin (PTX). A 17.6-fold and 17.3-fold increase of c-jun mRNA expression respectively (P<0.001) was obtained after 20 min of stimulation with triptorelin (100 nM). Treatment with 1 nM triptorelin resulted in a 12.5-fold or an 11.9-fold increase, and treatment with 10 pM triptorelin resulted in a 6.5-fold or a 5.2-fold increase of maximal c-jun mRNA expression respectively (P<0.001). Maximal c-Jun phosphorylation (68.5-fold and 60.2-fold, respectively, P<0.001) was obtained after 90 min incubation with triptorelin (100 nM). CONCLUSIONS: These results suggest that the LHRH agonist triptorelin stimulates the activity of AP-1 in human endometrial cancer cells mediated through PTX-sensitive G-protein alphai. In addition, triptorelin activates JNK, known to activate AP-1. In earlier investigations we have shown that triptorelin does not activate phospholipase and protein kinase C (PKC) in endometrial cancer cells. In addition, it has been demonstrated that triptorelin inhibits growth factor-induced mitogen activated protein kinase (MAPK, ERK) activity. Thus triptorelin-induced activation of the JNK/AP-1 pathway in endometrial cancer cells is independent of the known AP-1 activators, PKC or MAPK (ERK).

scholarly journals N-Acetylcysteine and α-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-δ and PKC-ζ and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro

2007 ◽  
Vol 192 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Mattias Gäreskog ◽  
Parri Wentzel
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
High Glucose ◽  
Culture Medium ◽  
Hydroxycinnamic Acid ◽  
Oxygen Species ◽  
Rat Embryos ◽  
Protein Kinase C Inhibitors ◽  
Kinase C

Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of α-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-δ and protein kinase C-ζ in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-δ and protein kinase C-ζ. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-δ and protein kinase C-ζ activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-δ and protein kinase C-ζ were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-δ and protein kinase C-ζ.

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