The rat lacrimal gland expresses the alpha isoform of PKC. Further evidence for the PMA-activated and phospholipid-independent protein kinase activity.

The molecular heterogeneity of protein kinase C (PKC) is now widely documented. In our first report, we characterized the rat lacrimal gland PKC along with a phorbol 12-myristate 13-acetate (PMA)-activated and phospholipid-independent protein kinase activity [Mauduit P., Zoukhri D. and Rossignol B. (1989) Fedn Eur. biochem. Socs Lett. 252, 5-11. In this work, we show that when the rat lacrimal gland cytosolic fraction is chromatographed on hydroxyapatite, only one peak of PKC activity can be detected. Comparison with a rat brain cytosolic fraction indicated that it is PKC-alpha which is expressed in the rat lacrimal gland. This result was confirmed by the use of polyclonal antibodies raised against rat brain PKC-alpha, beta and gamma isoforms. We also provide evidence that free arachidonic acid activates PKC, as does PMA, in a calcium and phospholipid-free system.

Constitutive expression of FosB and its short form, FosB/SF, induces malignant cell transformation in rat-1A cells.

We have established Rat-1A cell lines constitutively expressing c-Fos and the two products of the fosB gene, FosB and its short form, FosB/SF. The expressed proteins in the different stable transfectants have been characterized by immunofluorescence and immunoprecipitation analysis. Our results demonstrate that constitutive expression of FosB, like the constitutive expression of c-Fos and, to a lesser extent, FosB/SF, results in cells that grow to increased saturation densities and have the ability to grow in an anchorage-independent manner. Most important is the finding that expression of these proteins augments the tumorigenic potential of Rat-1A cells. These results show that both forms of FosB have a stimulatory effect on cell proliferation.

Production of platelet-activating factor is a component of the angiotensin II-protein kinase C activation pathway in bovine adrenocortical cells.

Lyso-platelet-activating factor (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) enzyme activity was characterized for the first time in bovine adrenocortical tissue. It was found to be associated with the microsomal membrane fraction, in which it exhibited a specific activity of 0.4 nmol/min per mg of protein and catalytic properties similar to those described in other cell types. The adrenocortical acetyltransferase activity was increased by 2-3-fold on incubation of the preparation with purified protein kinase C (PKC) under phosphorylating condition. This activation was optimal after 5 min of incubation and paralleled an increase in PKC-catalysed 32P incorporation into microsomal proteins. Both acetyltransferase activation and protein phosphorylation were dependent on the presence of Ca2+ and phospholipids, and were blocked in the presence of the potent PKC inhibitor H-7. In the intact adrenocortical cell, angiotensin II and a potent phorbol ester (phorbol 12-myristate 13-acetate) were able to rapidly induce an increase in the biosynthesis of PAF, which was mostly released into the extracellular medium. These data suggest that bovine adrenocortical lyso-PAF acetyltransferase may be regulated by a PKC-dependent activation pathway, whereas no evidence for an additional adrenocorticotropin/cyclic AMP-dependent stimulation process was obtained in this cell type. Bovine adrenocortical cell membrane preparations were shown to possess high-affinity PAF-binding sites (Kd approximately 0.5 nM). Altogether, these observations suggest that PAF production and release may play a role in the autocrine or paracrine control of adrenocortical cell activation.

Expression of protein kinase C isoforms in various steroidogenic cell types.

Different endocrine, steroidogenic cell types were examined for their content in protein kinase C (PKC) subtypes I, II and III. The expression of the PKC isoforms was assayed following high-performance liquid chromatography separation and characterization using a set of antipeptide antibodies specific for each enzyme isotype. Bovine and rat adrenocortical cells, as well as porcine Sertoli cells expressed only the type III PKC. By contrast, Leydig cell expressed both the isotypes I, II and II at similar levels. Taking into account the biological effect observed in these various cell types upon PKC activation, it may be suggested that the type III iso-PKC is involved in the steroidogenic activation pathways, whereas the expression of the types I, II, and III, only in Leydig cells, may contribute to a different array of cross-talk regulation pathways in these cells.

Activation of protein kinase C in lipid monolayers.

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.

Immunocharacterization of beta- and zeta-subspecies of protein kinase C in bovine neutrophils.

The isoforms present in a crude preparation of bovine neutrophil protein kinase (PKC) were identified by immunodetection with antibodies directed against specific sequences of bovine and rat brain PKC isozymes. The major isoform of bovine neutrophil PKC was identified as beta-PKC and the minor one as zeta-PKC.

Neurohormonal control of calcium sensitivity of myofilaments in rat single heart cells.

To investigate the changes in the properties of cardiac contractile proteins due to neurohormonal stimulation, different agonists were applied to single cells isolated from rat ventricle. Cells were then rapidly skinned by Triton X-100, and force was recorded after gluing the cells to a strain gauge. The skinned cells had mechanical properties very similar to those described for thin trabeculas. Tension-pCa relations were highly reproducible from one cell to another, with sarcomere length fixed at 2.1 microns. The application of alpha 1-adrenergic and muscarinic agonists, which increase the turnover of phosphatidylinositol, for 5 minutes before skinning the cells increased the sensitivity of the myofilaments to calcium, as indicated by a leftward shift of the tension-pCa relation, whereas beta-adrenergic stimulation induced a rightward shift. The increase in calcium sensitivity was also evoked by protein kinase C activators such as 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate but not by protein kinase C itself or by purinergic agonists, although the latter also increased the turnover of phosphatidylinositol. Incubation of the skinned cells with phosphatase reversed the alterations in calcium sensitivity induced by previous agonist stimulation of the intact cells. In conclusion, this study demonstrates a potentially influential mechanism for the physiological regulation of cardiac muscle contractility.

Differential inhibition of protein kinase C subtypes.

Catalytic properties of protein kinase C isoforms purified from rat brain and bovine adrenocortical tissues were examined. The results showed that known inhibitors of PKC activity such as gossypol and H-7 were active on all the three isolated enzyme isoforms with similar IC50 values. However, whereas the type III brain isozyme activity was not affected by a preincubation with phosphatidylserine (PS), the same treatment resulted in a virtually complete loss of the type I and II isoform activities within 4 min at 30 degrees. This kinase inactivation caused by PS preincubation was prevented in the presence of ATP-Mg2+ or its competitive inhibitor H-7. These findings indicate that the type III isoform can clearly be distinguished from the other members of the PKC family by this specific property. This approach was used to confirm the characterization of the single form of PKC detected in bovine adrenocortical tissue as a type III isotype. This specific behavior toward phosphatidylserine suggests that the molecular organization of the phospholipid sensitive, regulatory domain of the PKC isoform III with regard to its catalytic site and thus its mechanism of activation may differ from that of other PKC isotypes.

A single form of protein kinase C is expressed in bovine adrenocortical tissue, as compared to four chromatographically resolved isozymes in rat brain.

Protein kinase C purified to apparent homogeneity from rat brain was resolved into four active moieties following chromatography over a hydroxyapatite high resolution system. By contrast, the same procedure applied to bovine adrenocortical protein kinase C revealed that a single protein kinase C isoform could be detected in this tissue, with a chromatographic behavior identical to that of one of the brain isoenzymes. Although the isolated protein kinase C isozymes were all activated to various degrees in the presence of phospholipids and calcium, quantitative differences were observed in their catalytic properties, especially with regard to their sensitivity to diacylglycerol and TPA and to their relative affinity for different protein substrates. These observations confirmed at the protein level the heterogeneity of protein kinase C predicted on the basis of cDNA cloning studies. They also suggest that the expression of a specific set of protein kinase C isoenzyme(s) in a given cell type deserves further attention, since it may reflect a functional significance with regard to the regulation of specific cellular processes.