Subcellular distribution of protein kinase C of GH3 cells: quantitation and characterization by polyacrylamide gel electrophoresis

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).     

Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7.

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques. The inhibition of these two PKC isoforms by the protein kinase inhibitor H7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H1 and endogenous proteins as substrates. Phosphorylations catalyzed by the calcium-dependent PKC isoform alpha were almost 90% inhibited when histone H1 was used, and only 55% when endogenous proteins were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phospholipid-dependent PKC delta was not significantly affected by the inhibitor.     

A Role for Amplified Protein Kinase C Activity in the Pathogenesis of Amyotrophic Lateral Sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a human neurodegenerative disorder of unknown origin that is characterized by progressive degeneration of corticospinal tracts and anterior horn cells in the brainstem and spinal cord. Previous studies have indicated that motoneuron degeneration associated with ALS may be triggered by mechanisms leading to increased intracellular Ca²⁺. In the present report, Ca²⁺-activated phospholipid-dependent protein kinase C (PKC) was evaluated in cervical spinal cords from ALS patients and control subjects. In patients who died with ALS, PKC histone H1 phosphotransferase activity was significantly increased by 330% in cytosolic- and 118% in particulate-derived extracts compared with controls. This increase in PKC phosphotransferase activity appeared to be partially due to an increase in the amount of PKC protein present in ALS spinal cord tissue. PKC histone H1 phosphotransferase activities of cytosolic- and particulate-derived extracts from motor and visual cortex of ALS patients and controls were not statistically different, nor were there differences in PKC histone H1 phosphotransferase activity in platelets and leukocytes. The specific nature of PKC alterations in affected regions of the CNS supports a role for PKC in the events leading to motoneuron death in sporadic ALS.     

Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm,Manduca sexta: The cytosolic enzyme

A calcium and diacylglycerol-activated and phospholipid-dependent protein kinase (protein kinase C; PKC) in the cytosol of the pupal brain of the tobacco hornworm Manduca sexta has been characterized. Two peaks of PKC activity were separated by DEAE-cellulose chromatography, both of which were dependent upon Ca²⁺, phosphatidylserine and 1,2 diolein. Maximal enzyme activity was obtained in the presence of 0.7 mM Ca²⁺ and 200 μg/ml of phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) enhanced PKC activity and calcium sensitivity markedly in the presence of phospholipids. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate substituted for diacylglycerol in the activation of PKC. By utilizing the differential inhibition of PKC and cyclic AMP-dependent protein kinase by trifluoperazine and protein kinase inhibitor, both enzymes were measured accurately in dilute, crude cytosol preparations using the common substrate histone H1.     

Assay of protein kinase C with an N-bromosuccinimide-cleavage fragment of histone H1.

N-Bromosuccinimide cleavage of a lysine-rich histone fraction (histone III-S) yields a peptide substrate, purified by reverse-phase h.p.l.c., for the Ca2+ + phospholipid-dependent protein kinase (protein kinase C). This substrate displays no reactivity with the cyclic AMP-dependent protein kinase, and may prove useful for the detection of protein kinase C activity in crude tissue extracts.     

Characterization of a Ca2(+)- and phospholipid-dependent ATPase reaction catalyzed by rat brain protein kinase C

Protein kinase C (PKC) consists of a family of Ca2(+)- and phospholipid-dependent protein kinases that catalyze the transfer of the gamma-phosphate of ATP to phosphoacceptor serine or threonine residues of protein and peptide substrates. In this report, we demonstrate that purified, autophosphorylated rat brain PKC catalyzes a Ca2(+)- and phospholipid-dependent ATPase reaction, that appears to represent the bond-breaking step of its phosphotransferase reaction. The histone kinase and ATPase activities of PKC each had a Kmapp of 6 microM for ATP, and their metal ion cofactor requirements were similar. The rate of the Ca2(+)- and phospholipid-dependent PKC-catalyzed ATPase reaction was approximately 5 times slower than the rate of histone phosphorylation, but the basal rates of the PKC-catalyzed ATPase and histone kinase activities differed by less than a factor of 2. The mechanism of the ATPase reaction could entail either direct hydrolysis of ATP by water or formation of a stable phosphoenzyme (PKC-P) followed by its hydrolysis (PKC + Pi). The latter mechanism appears unlikely since [gamma-32P]ATP failed to label autophosphorylated PKC. Furthermore, the PKC preparation did not contain contaminating protein phosphatases, excluding the possibility that the ATPase activity represented dephosphorylation of contaminating PKC substrates. Therefore, our results suggest that water may effectively compete with protein substrates of PKC for the gamma-phosphate of ATP. Using PKC inhibitors and activators, we found that the ATPase and protein kinase activities of PKC were regulated analogously, providing evidence that allosteric activation of PKC involves facilitation of the bond-breaking step of the phosphotransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis

Expression of the cloned levansucrase gene (sacB) was demonstrated in E. coli minicells by assay of the enzyme in crude extracts, SDS-polyacrylamide gel electrophoresis and immunoblotting. The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Protein kinase C in hydrozoans: involvement in metamorphosis of Hydractinia and in pattern formation of Hydra

A wealth of information has suggested the involvement of protein kinase C (PKC) in metamorphosis of Hydractinia echinata and in pattern formation of Hydra magnipapillata. We have identified a Ca²⁺- and phospholipid-dependent kinase activity in extracts of both species. The enzyme was characterized as being similar to mammalian PKC by ion exchange chromatography. Gel filtration experiments revealed a molecular weight of about 70 kD. In phosphorylation assays of endogenous Hydractinia proteins, a protein with a molecular weight of 22.5 kD was found to be phoshorylated upon addition of phosphatidylserine. Bacterial induction of metamorphosis of Hydractinia echinata caused an increase in endogenous diacylglycerol, the physiological activator of PKC, suggesting that the bacterial inducer acts by activating receptor-regulated phospholipid metabolism. Exogenous diacylglycerol leads to membrane translocation of PKC, indicative of an activation. On the basis of our results and those of Freeman and Ridgway (1990) a model for the biochemical events during metamorphosis is presented.     

Role of the fixGHI region of Azorhizobium caulinodans in free-living and symbiotic nitrogen fixation

Abstract Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated. The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 ± 1.69 vs 93.56 ± 10.40 nmol/mg protein/min) and was dose dependent. Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa. The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells. Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa. However, STa had no direct role on the enzyme activity. Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes.     

Effect of pentosan polysulphate, standard heparin and related compounds on protein kinase C activity.

Ca2+/phospholipid-dependent protein kinase (PKC) was inhibited by sulphated polysaccharides. Pentosan polysulphate (PPS) and heparin were 8-10-times more potent than dextran sulphate or heparan sulphate. Steady-state studies revealed that PPS was a competitive inhibitor with respect to ATP with an apparent Ki value of 0.32 micrograms/ml and a non-competitive inhibitor with respect to histones. In contrast, the inhibition of PKC by heparin was competitive with substrate and non-competitive with respect to ATP. The interaction of sulphated polysaccharides with the catalytic domain of PKC was further demonstrated by the absence of effect on [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC. Furthermore, PPS and heparin inhibited equally cAMP-dependent protein kinase and tyrosine protein kinase. Structure-function relationships indicated that the Inhibition of protein kinases by PPS and heparin fractions was highly dependent on molecular weight. Additionally, PKC-affinity chromatography revealed that a high-molecular-weight heparin fraction with strong anti-PKC activity was eluted. We set out to demonstrate that heparin and PPS, which are potent antiproliferative agents on vascular smooth muscle cells (SMC), alter intracellular PKC activity (both membrane and cytosolic). Therefore, it is suggested that the mechanism by which sulphated polysaccharides inhibit SMC growth may be by direct inhibition of PKC in SMC.     

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.     

Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy.

A simple and rapid method based on 15N labeling and 1H-15N heteronuclear single quantum coherence spectroscopy is presented to directly assess the structural integrity of overexpressed proteins in crude Escherichia coli extracts without the need for any purification. The method is demonstrated using two different expression systems and two different proteins, the B1 immunoglobulin-binding domain of streptococcal protein G (56 residues) and human interleukin-1 beta (153 residues). It is shown that high quality 1H-15N correlation spectra, recorded in as little as 15 min and displaying only cross-peaks arising from the overexpressed protein of interest, can be obtained from crude E. coli extracts.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M_r), KP-2 (pI 4.7/20,700 M_r) and KP-10 (pI 4.7/25,000 M_r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca²⁺-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca²⁺-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.     

Protein kinase C interaction with calcium: a phospholipid-dependent process

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.