scholarly journals Purification of two distinct proteins of approximate Mr 80,000 from human epithelial cells and identification as proper substrates for protein kinase C

1990 ◽  
Vol 270 (3) ◽  
pp. 583-589 ◽  
Author(s):  
M Hirai ◽  
N Shimizu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Specific Substrate ◽  
Type I ◽  
Type Iii ◽  
Human Squamous Cell Carcinoma ◽  
Kinase C

A Mr-80,000 acidic phosphoprotein (‘80K protein’) is a specific substrate for protein kinase C. We attempted to purify the 80K protein from a human squamous-cell carcinoma cell line, Ca9-22, by the sequential use of heat treatment, (NH4)2SO4 precipitation, Mono Q column chromatography, proRPC column chromatography and gel filtration. The 80K protein was assayed by phosphorylation in vitro by using partially purified human type III protein kinase C, and was fractionated into two distinct molecular species with slightly different Mr values, designated 80K-L and 80K-H proteins. Phosphorylation occurred mainly at serine residues of these proteins. Two-dimensional phosphopeptide maps after trypsin digestion and kinetic profiles of phosphorylation were different from each other. Ca2(+)- and phospholipid-dependency of the phosphorylation in vitro confirmed that both 80K-L and 80K-H proteins are true substrates for three subtypes of protein kinase C. The 80K-L protein was a preferential substrate for type III protein kinase C, and the 80K-H protein was phosphorylated more effectively by type I and type II protein kinase C. The possible roles of these two distinct 80K proteins in signal transduction are discussed.

scholarly journals Differential localization of protein kinase C isozymes in retinal neurons.

1991 ◽  
Vol 112 (6) ◽  
pp. 1241-1247 ◽  
Author(s):  
N Usuda ◽  
Y Kong ◽  
M Hagiwara ◽  
C Uchida ◽  
M Terasawa ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bipolar Cells ◽  
Type I ◽  
Rabbit Retina ◽  
Type Ii ◽  
Retinal Neurons ◽  
Type Iii ◽  
Kinase C ◽  
Rod Bipolar Cells

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, and III) in the rabbit retina using the monospecific monoclonal antibodies MC-1a, MC-2a, and MC-3a. Using immunoblot analysis of partially purified protein kinase C preparations of rabbit retina, types II and III isozymes alone were detected. The activity of type III was the stronger. By light microscopic immunohistochemical analysis, retinal neurons were negative for type I and positive for type II and type III isozymes. Type II was more diffusely distributed through the retinal layers, but was distinctive in ganglion cells, bipolar cells, and outer segments. The immunoreactivity was stronger for type III isozyme, and it was observed in mop (rod) bipolar cells and amacrine cells. By using immunoelectron microscopy, the cytoplasm of the cell body, the axon, and dendrites of the mop bipolar cells were strongly immunoreactive for type III. The so-called rod bipolar cells were for the first time seen to form synapses with rod photoreceptor cells. These differential localizations of respective isozymes in retinal neurons suggest that each isozyme has a different site of function in each neuron.

scholarly journals Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils

1992 ◽  
Vol 284 (2) ◽  
pp. 399-405 ◽  
Author(s):  
K J Balazovich ◽  
E L McEwen ◽  
M L Lutzke ◽  
L A Boxer ◽  
T White
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Neutrophils ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Western Blots ◽  
Type Iii ◽  
Kinase C ◽  
Endogenous Protein

Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.

scholarly journals Synergistic activation of type III protein kinase C by cis-fatty acid and diacylglycerol

1992 ◽  
Vol 282 (1) ◽  
pp. 33-39 ◽  
Author(s):  
S G Chen ◽  
K Murakami
Keyword(s):  
Protein Kinase C ◽  
Fatty Acid ◽  
Protein Kinase ◽  
Second Messengers ◽  
Type I ◽  
Physiological Concentration ◽  
Type Iii ◽  
Kinase C ◽  
Synergistic Activation ◽  
Protein Kinase C Activation

Micromolar concentrations of cis-fatty acid synergistically activate type III protein kinase C with diacylglycerol. This synergistic effect occurs at low concentrations of cis-fatty acid and diacylglycerol, and it is capable of inducing almost full activation of this protein kinase C subtype at a physiologically relevant Ca2+ concentration (2 microM). The synergistic activation mode can be observed even in the absence of Ca2+, but micromolar Ca2+ significantly enhances the type III protein kinase C activation. cis-Fatty acid also augments the diacylglycerol-induced activation of other subtypes (type I and II), although the effect is smaller than that observed in type III. Neither the diacylglycerol- nor the cis-fatty acid-dependent mode of activation can fully activate any of these subtypes at a physiological concentration of Ca2+ (2 microM). Our results suggest that the generation of three second messengers, i.e. the increase in intracellular Ca2+ concentration and the generation of both cis-fatty acid and diacylglycerol in the cell, may be necessary signals for protein kinase C activation, particularly for type III protein kinase C.

scholarly journals Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils

1999 ◽  
Vol 344 (3) ◽  
pp. 859-866 ◽  
Author(s):  
Emer P. REEVES ◽  
Lodewijk V. DEKKER ◽  
Louisa V. FORBES ◽  
Frans B. WIENTJES ◽  
Ann GROGAN ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Gel Filtration ◽  
Direct Interaction ◽  
Subcellular Fractionation ◽  
Intact Cells ◽  
Kinase C ◽  
C Terminus ◽  
Time Courses

p47phox is an essential component of the NADPH oxidase, and phosphorylation of p47phox is associated with activation of the enzyme. Here we have used p47phox affinity chromatography to extract a p47phox kinase from neutrophil cytosol. The kinase activity was purified by gel filtration and Mini Q chromatography and shown to be indistinguishable from the catalytic fragments of protein kinase C (PKC)-βI, -βII and -∆. The C-terminus of p47phox represented the site of interaction with PKC. Co-immunoprecipitation experiments revealed that the interaction between PKC isotypes and p47phox takes place in intact cells. However PKC-β and -∆ showed different time courses of co-immunoprecipitation, suggesting that the interactions may serve different functions for the various PKC isotypes. Using cells lacking p47phox, we investigated the functional relevance of the interaction between PKC and p47phox. Subcellular fractionation revealed an abnormal recruitment of PKC-βI and -βII, but not PKC-∆, to particulate fractions in p47phox-deficient cells. Phosphorylation of cytosolic proteins was generally increased in stimulated p47phox-deficient neutrophils as compared with normal neutrophils. Furthermore, the cytoskeletal protein coronin was not phosphorylated upon stimulation of p47phox-deficient neutrophils. These findings were confirmed in an in vitro-reconstituted system using rat brain cytosol in which addition of p47phox affected phosphorylation by PKC/PKM (PKM is the catalytic fragment of PKC). These results indicate that p47phox can act as a regulator of PKC in neutrophils.

scholarly journals Two components of type III protein kinase C with different substrate specificities and a phospholipid-dependent but Ca2+ -inhibited protein kinase in rat brain

FEBS Letters ◽  
1989 ◽  
Vol 249 (2) ◽  
pp. 324-328 ◽  
Author(s):  
Lázló Buday ◽  
György Mészáros ◽  
Gyöngyi Farkas ◽  
János Seprődi ◽  
Ferenc Antoni ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Rat Brain ◽  
Substrate Specificities ◽  
Type Iii ◽  
Kinase C
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