Wayne B. Anderson

Hormone- and tumor promoter-induced activation or membrane association of protein kinase C in intact cells

Abstract The physiologic regulation of protein kinase C activity appears to be modulated by its interaction with cellular membranes. Tumor promoter- and hormone-induced stabilization of protein kinase C to a membrane fraction in intact cells apparently reflects activation of the enzyme. Thus, measurement of changes in membrane-associated protein kinase C in response to treatment of intact cells can provide insights into the mechanisms of hormone- and tumor promoter-mediated biological functions. TPA rapidly intercalates into the membrane lipid bilayer and is slowly metabolized, thus maintaining the kinase in stable association with the membrane. In contrast, diacylglycerol-mediated association of protein kinase C with membranes apparently is readily reversed. Thus, changes in protein kinase C distribution due to changes in diacylglycerol generation may be more difficult to assess because diacylglycerol-mediated binding may be reversed during the isolation of subcellular fractions. With the availability of polyclonal and monoclonal antibodies, it will be possible to determine directly with immunohistochemical techniques changes in the subcellular distribution of protein kinase C in response to hormones (J. F. Kuo, personal communication). Antibodies also may be employed to determine changes in the distribution of enzyme protein by immunoprecipitation. 20 Results of other studies indicate that the ratio of protein kinase C found in the cytosol and particulate fractions may be regulated, in part, by calcium. 4,21 Calcium (perhaps induced Ca 2+ flux from the extracellular medium) appears to be required for induced association of C kinase with the particulate fraction. It is possible that protein kinase C normally is present in cells in weak, calcium-dependent association (chelator sensitive) with membranes and that TPA intercalation, or diacylglycerol generation, at the membrane facilitates a high-affinity (chelator stable) interaction between protein kinase C and the membrane. Protein kinase C might be dissociated from the membrane by alteration in the membrane-binding site, by enhancing the metabolism of phospholipid or diacylglycerol required for binding, or by changing Ca 2+ levels. Tumor promoter-induced binding of C kinase appears to occur predominantly with plasma membranes. 3 However, results with NIH 3T3 cells indicate that TPA treatment also may promote an increase in protein kinase C activity found associated with a cell fraction containing nuclear and cytoskeletal components. Thus, assessment of the involvement of altered kinase C redistribution in mediating given biological responses may require the isolation of the various subcellular membrane fractions (i.e., nuclear, cytoskeletal, ribosomal, etc.). In summation, the described assays allow the design of experiments with intact cells to determine the possible involvement (activation) of protein kinase C in modulating the biological responses to hormones, tumor promoters, and other extracellular agents. That is, changes in the ratio of membrane to cytosol activities can serve as an index of activation of the enzyme within intact cells.

Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promoter periodate.

The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimulate kinase activity by Ca2+/phospholipid, and also by the loss of phorbol ester binding. This modification resulted in an increase in Ca2+/phospholipid-independent kinase activity (oxidative activation). However, at higher concentrations (greater than 100 microM) periodate also modified the catalytic domain, resulting in complete inactivation of PKC. The oxidative modification induced by low periodate concentrations (less than 0.5 mM) was completely reversed by a brief treatment with 2 mM dithiothreitol. In this aspect, the modification induced by periodate was different from that of the previously reported irreversible modification of PKC induced by H2O2. However, the inactivation of PKC induced by periodate at concentrations greater than 1 mM was not reversed by dithiothreitol. Among the phospholipids and ligands of the regulatory domain tested, only phosphatidylserine protected the regulatory domain from oxidative modification. In the presence of phosphatidylserine, the catalytic site was selectively modified by periodate, resulting in formation of a form of PKC that exhibited phorbol ester binding but not kinase activity. Both reversible and irreversible oxidative activation and inactivation of PKC also were observed in intact cells treated with periodate. Taken together these results suggest that periodate, by virtue of having a tetrahedral structure, binds to the phosphate-binding regions present within the phosphatidylserine-binding site of the regulatory domain and the ATP-binding site of the catalytic domain, and modifies the vicinal thiols present within these sites. This results in the formation of intramolecular disulfide bridge(s) within the regulatory domain or catalytic domain leading to either reversible activation or inactivation of PKC, respectively. Thus, oxidant mitogen/tumor promoters such as periodate may be able to bypass normal transmembrane signalling systems to directly activate pathways involved in cellular regulation.

Protein Kinase C ϵ Subcellular Localization Domains and Proteolytic Degradation Sites A MODEL FOR PROTEIN KINASE C CONFORMATIONAL CHANGES

Protein kinase C (PKC) ε has been found to have unique properties among the PKC isozymes in terms of its membrane association, oncogenic potential, and substrate specificity. Recently we have demonstrated that PKCε localizes to the Golgi network via its zinc finger domain and that both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKCε, a series of NIH 3T3 cell lines were created, each overexpressing a different truncated version of PKCε. The overexpressed proteins each were designed to contain an ε-epitope tag peptide at the COOH terminus to allow ready detection with an antibody specific for the tag. The subcellular localization of the recombinant proteins was analyzed by in vivo phorbol ester binding, immunocytochemistry, and cell fractionation followed by immunoblotting. Results revealed several regions of PKCε that contain putative subcellular localization signals. The presence either of the hinge region or of a 33-amino-acid region including the pseudosubstrate sequence in the recombinant proteins resulted in association with the plasma membrane and cytoskeletal components. The catalytic domain was found predominantly in the cytosolic fraction. The accessibility and thus the dominance of these localization signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation sites also were identified. The susceptibility of the overexpressed proteins to proteolytic degradation was dependent on the protein conformation. Based on these observations, a model depicting the interaction and hierarchy of the suspected localization signals and proteolytic degradation sites is presented.

Localization of serum-derived α2 macroglobulin in cultured cells and decrease after moloney sarcoma virus transformation

NRK cells and many other cultured fibroblasts were found to contain the protease inhibitor, alpha 2 macroglobulin (alpha 2M). This alpha 2M is present as a result of uptake of alpha 2M from the calf serum in the culture medium. Some of this alpha 2M is released back into the medium. In radiolabeling experiments with 14C-amino acids, no radioactivity was detected in intracellular or extracellular alpha 2M. Fluorescence microscopy of fixed cells using rhodamine-labeled antibodies indicated that alpha 2M is present in vesicular organelles different from primary lysosomes. Fluorescence microscopy of living cells shows that rhodamine-labeled alpha 2M (rhodamine-alpha 2M) is taken up into similar structures. Of the many cell lines examined, Moloney sarcoma virus-transformed cells had the lowest amounts of alpha 2M. Some of the effects of serum on the behavior of cultured cells could be a consequence of inhibition of cellular proteases by alpha 2M.

Susceptibility of protein kinase C to oxidative inactivation: Loss of both phosphotransferase activity and phorbol diester binding

Exposure of protein kinase C to low concentrations of either N‐chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF‐7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane‐associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E1 is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.

Rapid filtration assays for protein kinase C activity and phorbol ester binding using multiwell plates with fitted filtration discs.

In the conventional approach protein kinase activity and phorbol ester binding associated with protein kinase C (PKC) are measured by initially incubating samples in either test tubes or multiwell plates, followed by filtration of the terminated reaction mixture using either a manifold filtration device or a cell harvester. Here we report a method in which both the incubations and filtrations necessary for the determination of either protein kinase activity or phorbol ester binding are carried out in the same multiwell plate with fitted filtration discs made of polyvinylidene difluoride (Durapore membrane). Due to the very low binding of protein to these filters, there is no interference caused by these filters during the incubation period of the assays. The drawback with these filters compared to commonly used cellulose acetate membrane filters is that they retain less of the phosphate acceptor substrate histone H1 (only 15%) if filtered and washed with standard 5% trichloroacetic acid. However, this can be overcome by increasing the trichloroacetic acid concentration to 25% during filtration. For phorbol ester binding determinations, the samples are incubated with [3H]phorbol 12,13-dibutyrate in the microwells, the ligand bound PKC is adsorbed onto DEAE-Sephadex beads, and the beads then are filtered and washed in the same microwells. Furthermore, this multiwell filtration approach can also be adopted to previously described cytosolic phorbol ester receptor assays, which have the broader conditions for optimal binding to receptors. Durapore membrane filters are found to work well for punching into scintillation vials and there is complete recovery of the radioactivity retained with the filters. In the protein kinase assay the background radioactivity is very low (< 200 cpm) and in the phorbol ester binding assay the nonspecific binding is less than 1%. Thus, these low background values result in at least a fourfold increase in sensitivity for these assays. Since the incubations and filtrations are carried out in the same well without any transfer of the sample, the coefficient of variation in multiple determinations is found to be low. Furthermore, this method is rapid and more convenient for analyzing a larger number of samples than conventional methods which use test tubes, and it is less expensive to set up compared to the automated methods that use a cell harvester.

OXIDATIVE MODULATION OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN HUMAN FIBROBLASTS : POSSIBLE ROLE IN PSORIASIS

Previous studies have established that cyclic AMP-dependent protein kinase (PKA) activity, as well as 8-azido-[32P]-cAMP binding to the RI and RII regulatory subunits, are decreased in cells from psoriatic patients compared to cells from normal patients. Here we show that the exposure of normal human dermal fibroblasts in culture to hydrogen peroxide and to oxygen free-radical generating systems decreased PKA activity, as well as cyclic AMP binding to the RI and RII regulatory subunits, to levels similar to those observed with psoriatic fibroblasts. Likewise, treatment of normal cytosolic preparations of PKA, as well as purified bovine PKA II, in vitro with free radical generating systems also resulted in decreased PKA activity and 8-azido [32P]-cAMP binding to the RI and RII regulatory subunits. Further, treatment of psoriatic fibroblasts with free radical scavenging agents such as vitamins E and C, and mannitol, and also with superoxide dismutase, restored the ability of RI and RII to bind 8-azido-[32P]-cAMP toward normal levels. Western blot analysis showed that the protein levels of the RI and RII subunits are similar in normal and psoriatic fibroblasts, and that the amounts of RI and RII are not altered by treatment of the cells with free radical-generating systems. These results suggest that oxidative modification may serve as a mechanism to alter PKA activity in human cells, and that an altered oxidative state may be involved in mediating the decrease in PKA activity and cyclic AMP binding noted in cells from psoriatic patients.

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts.

Two different methods were used to study directly α‐thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo‐[su3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+‐dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris‐ and bisphosphate. Basal phospholipase C activity was stimulated up to 3‐fold by GTP or GTP‐γ‐S. Of the two mitogens, α‐thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only α‐thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, α‐thrombin but not EGF potentiated GTP‐γ‐S‐dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP‐binding protein couples α‐thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2‐phosphodiesterase activity.

Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus

Abstract Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K m ATP or a change in the kinetics of Mg ++ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.