Effects of isoflurane on the expressed Cav2.2 currents in Xenopus oocytes depend on the activation of protein kinase Cδ and its phosphorylation sites in the Cav2.2α1 subunits.

The effects of isoflurane on the modulation of two neuronal voltage-gated calcium channels (Ca(v); Ca(v)2.1 and 2.2) by protein kinase C (PKC) isozymes ßII, ε or δ and their combination were examined. Ca(v)2.1α1 or Ca(v)2.2α1 with ß1b and α2δ subunits were expressed in Xenopus oocytes and the currents (I(Ba)) were recorded by two-electrode voltage clamp. Isoflurane (0.70 mM) decreased both Ca(v)2.1 and 2.2 currents by 20-35% and also caused translocation of PKCδ to the membrane. Compared to the wild type (WT), isoflurane caused greater inhibition of Ca(v)2.2 currents in the absence of stimulatory PKC sites (Thr-422, Ser-1757, Ser-2108, Ser-2132) and in the presence of inhibitory PKC site (Ser-425). In contrast, isoflurane caused less inhibition of I(Ba) in the oocytes expressing S425A, the inhibitory site mutant, compared to WT. PKCδ by itself did not modulate Ca(v)2.2 currents, but potentiated these currents in the presence of isoflurane. PKCε increased Ca(v)2.2 currents either alone or in combination with isoflurane. Ca(v)2.1 currents were not modulated by phorbol-12-myristate, 13-acetate (PMA) or acetyl-ß-methylcholine (MCh), activators of PKC. Yet the presence of isoflurane caused PMA (but not MCh) to enhance Ca(v)2.1 currents. PKCßII and PKCε isozymes activated by PMA, did not alter Ca(v)2.1 currents. However, in the presence of isoflurane, these two isozymes together potentiated Ca(v)2.1 currents. The variable responses of Ca(v)2.1 currents to PKCßII and PKCε and Ca(v)2.2 currents to PKCδ in the presence of isoflurane may be due to increased affinity or accessibility of these isozymes to their Ser/Thr PKC sites of Ca(v)α1 subunits.

Site-specific regulation of CA(V)2.2 channels by protein kinase C isozymes betaII and epsilon.

Ca(v)2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I-II linker and C-terminus of the alpha(1) 2.2 subunit. Here we show that PMA enhancement of Ca(v)2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC betaII or PKC epsilon isozymes, as shown previously for Ca(v)2.3 currents, and that microinjection of PKC betaII or PKC epsilon isozymes in the oocytes expressing the WT Ca(v)2.2 channels increases the basal barium current (I(Ba)). The I-V plot shows a large increase in current amplitude with PKC betaII and PKC epsilon isozymes with only a small shift in the peak I(Ba) in the hyperpolarizing direction. The potentiation of Ca(v)2.2 currents by microinjection of PKC betaII and PKC epsilon isozymes was not altered by the inhibition of G proteins with GDPbetaS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of alpha(1) 2.2 subunit revealed that PKC betaII or PKC epsilon isozymes (but not PKC alpha or delta) can provide full enhancement through the stimulatory site (Thr-422) in the I-II linker but that PKC epsilon is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC betaII or epsilon at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC betaII also enhances Ca(v)2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC epsilon provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Ca(v)2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.

Activation-dependent degradation of protein kinase C eta.

Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKC eta, we expressed the novel PKCs eta and θ and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKC eta, but not wild type or kinase-dead PKC eta, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKC eta with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKC eta was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKC eta. Treatment with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal and lactacystin led to accumulation of PKC eta proteolytic products and potentially ubiquitinated forms. While wild type PKC eta localizes mostly to the detergent-soluble fraction of the cell, a significant portion of full-length constitutively active PKC eta and of kinase-dead, active conformation PKC eta were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKC eta also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKC eta in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKC eta accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome. Oncogene (2000) 19, 4263 - 4272

Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.

Lipid-dependent activation of protein kinase C-alpha by normal alcohols.

Significant stimulation of protein kinase C-alpha (PKCalpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED(50)) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. (31)P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding.

Contributions to maxima in protein kinase C activation.

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.

Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells.

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.

Influence of lipid on the structure and phosphorylation of protein kinase C alpha substrate peptides.

The structure and phosphorylation of two protein kinase C (PKC) alpha substrate peptides were investigated in varying lipid systems using enzyme activity assays and circular dichroism (CD) spectroscopy. The alpha-peptide, which exhibits the typical PKC alpha substrate motif and is based on the pseudosubstrate region of PKCalpha, was phosphorylated to a similar extent in bovine brain phosphatidylserine vesicles or diheptanoylphosphatidylcholine (PC7) micelles (both with 5 mol % 1,2-dioleoyl-sn-glycerol), whereas neuromodulin (NM)-peptide, which does not exhibit this motif by virtue of its primary structure, was phosphorylated to a much lesser extent in the PC7 micellar system. CD spectra of the peptides indicated that NM-peptide underwent a dramatic structural change in the presence of dimyristoylphosphatidylserine (DMPS) vesicles, whereas spectra acquired in PC7 micelles were similar to those acquired in buffer alone. No significant structural change was observed in the alpha-peptide in the presence of either lipid. PKC activity assays conducted with a series of NM-peptides successively substituted with nitroxide spin labels at each residue position suggested that several residues distal to the phosphorylation site are necessary for substrate recognition. The effect of these substitutions is not consistent with the binding of the NM-peptide to PKC in an extended structure, but is consistent with the binding of this peptide in a helical conformation. Furthermore, the docking of a helical NM-peptide to the substrate binding site of PKC suggests that the interaction is energetically feasible. These results suggest that PKC may recognize some non-linear substrate motifs and that lipid binding may convert a protein into a better PKC substrate.

Two-dimensional crystals of protein kinase C.

Three two-dimensional (2D) crystal forms of protein kinase C (PKC) alpha and three of PKC delta have been grown on lipid monolayers composed of dioleoylphosphatidylcholine: dioleoylphosphatidylserine: (45:50:5 molar ratio). In the absence of DO, two additional 2D crystals of PKC delta are seen, suggesting that the presence of diolein (DO) alters the conformation of intact PKC at the lipid surface. Reconstructions of electron micrographs of these eight lattices show good reproducibility and indicate that several are appropriate for three-dimensional reconstruction to 20 A resolution.

Selective up-regulation of protein kinase C eta in phorbol ester-sensitive versus -resistant EL4 mouse thymoma cells.

Stimulation of sensitive EL4 mouse thymoma cells (s-EL4) with phorbol esters results in production of interleukin 2 (IL-2), adherence to a plastic substrate, and growth inhibition, whereas a phorbol ester-resistant variant (r-EL4) fails to respond. Previous studies revealed substantially decreased expression of protein kinase C (PKC) epsilon in the r-EL4 versus s-EL4 cells. This work has been extended to examine the more recently described PKC isozymes. Western and Northern analyses revealed a marked decrease in PKC eta and theta in r-EL4 as compared to s-EL4 cells. Treatment of these lines with phorbol ester for 24 h resulted in down-regulation of all PKC isozymes examined except PKC eta, which was up-regulated in the s-EL4 cells at the time of maximal IL-2 production. Two newly isolated EL4 clones, resistant to phorbol ester-induced growth inhibition but still exhibiting the phorbol ester-induced adherence and IL-2 production, both expressed PKC eta and theta. Collectively, these observations suggest a dissociation of growth inhibition from adherence and IL-2 production pathways and a potential role for PKC eta in the latter.

A role for protein kinase CbetaI in the regulation of Ca2+ entry in Jurkat T cells.

T cell activation leading to cytokine production and cellular proliferation involves a regulated increase and subsequent decrease in the intracellular concentration of Ca2+ ([Ca2+]i). While much is understood about agonist-induced increases in [Ca2+]i, less is known about down-regulation of this pathway. Understanding the mechanism of this down-regulation is critical to the prevention of cell death that can be the consequence of a sustained elevation in [Ca2+]i. Protein kinase C (PKC), activated by the diacylglycerol produced as a consequence of T cell receptor engagement, has long been presumed to be involved in this down-regulation, although the precise mechanism is not wholly clear. In this report we demonstrate that activation of PKC by phorbol esters slightly decreases the rate of Ca2+ efflux from the cytosol of Jurkat T cells following stimulation through the T cell receptor or stimulation in a receptor-independent manner by thapsigargin. On the other hand, phorbol ester treatment dramatically reduces the rate of Ca2+ influx following stimulation. Phorbol ester treatment is without an effect on Ca2+ influx in a different T cell line, HSB. Down-regulation of PKCbetaI expression by 18-h phorbol ester treatment is associated with a loss of the response to acute phorbol ester treatment in Jurkat cells, suggesting that PKCbetaI may be the isozyme responsible for the effects on Ca2+ influx. Electroporation of an anti-PKCbetaI antibody, but not antibodies against PKCalpha or PKCgamma, led to an increase in the rate of Ca2+ influx following stimulation. Taken together, these data suggest that PKCbetaI may be a component of the down-regulation of increases in [Ca2+]i associated with Jurkat T cell activation.

Activation of protein kinase C by coexisting diacylglycerol-enriched and diacylglycerol-poor lipid domains.

To test the hypothesis that activation of protein kinase C (PKC) is related to the interface between coexisting diacylglycerol- (DAG-) enriched and DAG-poor phases, the thermotropic phase behavior of the ternary mixtures dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO), DMPC/DMPS/1-palmitoyl-2-oleoylglycerol (PO), and DMPC/DMPS/dimyristoylglycerol (DM) was analyzed and compared with the ability of the lipid mixtures to support PKC activity. Differential scanning calorimetry (DSC) was used to monitor the gel-to-liquid crystalline phase transition as a function of the mole fraction of DO (chiDO), PO (chiPO), or DM (chiDM) in DMPC/DMPS (1:1) multilamellar vesicles (MLVs) and of chiDO in large unilamellar vesicles (LUVs). The addition of DAG at low mole fractions gave rise to the appearance of two or more overlapping transitions. The phase boundaries of the ternary mixtures deduced from the partial phase diagrams were chiDO = approximately 0.10 and approximately 0.3 for DMPC/DMPS/DO, chiPO = approximately 0.05 and approximately 0.4 for DMPC/DMPS/PO, and chiDM = approximately 0.025 and approximately 0.5-0.6 for DMPC/ DMPS/DM. Above these mole fractions of DAG, the transitions again became very sharp. The ability of the lipid mixtures to support activity of PKC alpha and PKC eta was examined below and above the gel-to-liquid crystalline phase transition. In the gel phase, PKC activity went through a maximum as a function of increasing mole fraction of each DAG and was restricted to lipid compositions in which coexisting phases were observed. Maximal activity decreased with increasing saturation of the DAG. In the fluid state, maximal PKC activity was shifted to higher DO mole fractions and the peak was much broader. Collectively, these data support a role for both the presence and nature of interface between compositionally distinct domains in activation of PKC.

Deficient tyrosine phosphorylation of c-Cbl and associated proteins in phorbol ester-resistant EL4 mouse thymoma cells.

Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.

Activation of protein kinase C alpha and delta by bile acids: correlation with bile acid structure and diacylglycerol formation.

The feedback repression of cholesterol 7alpha-hydroxylase transcriptional activity and mRNA levels by taurocholate (TCA) occurs via a protein kinase C (PKC)-dependent signal. To determine whether bile acids could activate PKC indirectly via generation of diacylglycerol (DG), their effects on DG levels in primary cultures of rat hepatocytes were determined using a DG kinase assay. To determine whether bile acids might activate PKC isozymes more directly, their effects on PKC alpha and delta purified from baculovirus expression systems were examined in phosphatidylserine/phosphatidylcholine/Triton X-100 (PS/PC/TX) mixed micelles. Addition of tauroursodeoxycholate (TUDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) (50 microM) to the cells rapidly (15 min) increased DG content in cultured rat hepatocytes to 105%, 155%, and 130%, respectively, as compared to untreated control cultures. Addition of TCA increased PKC alpha specific activity with EC50 of approximately 400 nM; maximal activity was observed with 5 microM. Other taurine-conjugated bile acids (5 microM) increased PKC alpha specific activity (pmol/min/microg protein) in proportion to their relative hydrophobicity: PS/PC/TX 17 +/- 2; + TUDCA 29 +/- 18; + TCA 68 +/-13; + TDCA 166 +/- 21; and, taurochenodeoxycholate 178 +/- 20 (P vs. PS/PC/TX = 0.54, 0.019, 0.002, and 0.001, respectively); unconjugated bile acids gave similar results (r2 for activity vs. hydrophobicity index 0.59). Taurine-conjugated bile acid interaction enthalpies, as determined by dimyristoyl-phosphatidylcholine chromatography, were more highly correlated (r2 = 0.96) with PKC alpha activation than with the hydrophobicity index. TCA also stimulated the activity of purified PKCdelta with EC50 of approximately 150 nM and maximally (2.7-fold) at 1 microM. Free and taurine-conjugated bile acids (1 microM) increased PKCdelta activity according to their hydrophobicity index (r2 = 0.89) and interaction enthalpies (r2 = 0.96).

Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation.

To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.

Activation of protein kinase C by lysophosphatidic acid: dependence on composition of phospholipid vesicles.

Lysophosphatidic acid (LPA) has attracted recent attention as a major serum-derived regulator implicated in responses to vascular injury and inflammation, in tumour invasiveness and in neuronal signalling and remodelling. Although the possibility of a specific G-protein-coupled LPA receptor protein has been suggested, characterization of such a receptor is lacking. Since LPA can activate protein kinase C (PKC) pathways in many cells and PKC activators mimic many LPA effects, the possibility of more direct LPA effects on PKC was investigated. Phosphatidylcholine (PC)/phosphatidylserine (PS)/diacylglycerol (DAG) lipid vesicles of defined acyl chain composition were used to activate the enzyme. At total concentrations of saturated PC/PS + DAG vesicles (2-3 mM) that provided maximal PKC activation, 1-10 mol % [18:1]-LPA led to a further approx. 2-fold activation of PKC alpha. At lower lipid concentrations, a greater increase was observed with LPA concentrations up to 16-20 mol %. Higher concentrations of LPA were inhibitory. The LPA activation of PKC was dependent on the presence of DAG, PS and Ca2+. [18:1]-Lysophosphatidylcholine produced similar PKC activation in PC/PS/DAG vesicles. [14:0]-LPA was less effective, and longer-chain saturated lysolipids were ineffective. In unsaturated PC/PS vesicles, very little to no effect of LPA was discernable. These results suggest that physiologically or pathologically relevant concentrations of LPA can contribute to PKC activation depending on the composition of the lipid membrane. We hypothesize that LPA may affect the formation of lipid domains that are recognized by the enzyme.

Protein kinase C in cyclic stretch-induced nerve growth factor production by urinary tract smooth muscle cells.

Cyclic stretch of cultured urinary tract smooth muscle cells has been used to mimic some of the events that occur with bladder obstruction. The stretch stimulus induces production of nerve growth factor (NGF), which has been implicated in changes in bladder innervation. Stretch-induced NGF production was blocked by actinomycin. Involvement of protein kinase C (PKC) in the stretch-induced NGF production is strongly suggested by the following observations. Phorbol ester activators of PKC mimicked the stretch response as did platelet-derived growth factor (PDGF), which acts, in part, through generation of endogenous diacylglycerols. Both stretch- and PDGF-induced NGF production were blocked by prolonged incubation with phorbol ester to downregulate PKC. Western blot analysis confirmed partial downregulation of the Ca(2+)-dependent PKC-alpha and PKC-beta 1 and near complete downregulation of the Ca(2+)-independent PKC isozymes delta, epsilon, and zeta. The involvement of PKC in transducing a physical stimulus (stretch) into a biochemical response (NGF production) has implications for novel types of therapeutic intervention in ailments such as bladder obstruction.

Rapid tyrosine phosphorylation of an 85,000 M(r) protein after phorbol ester stimulation of EL4 thymoma cells.

Early signalling events between protein kinase C (PKC) activation and lymphokine transcription were compared between phorbol ester-sensitive and -resistant EL4 cell lines which do or do not respond with interleukin 2 (IL2) production, respectively. The earliest event detected in the sensitive cell line was a dramatic increase in the tyrosine phosphorylation of an 85,000 M(r) protein (p85; 30 s), followed by mobility shifts of raf-1, mitogen-activated protein kinase kinase (MEK), mitogen-activated protein (MAP) kinase, lck and ZAP-70 (within 5 min). In contrast, p85 was not detected in the resistant cell line and lck and raf-1 mobility shifts exhibited delayed kinetics. Both vanadate and okadaic acid blocked the phorbol ester-stimulated p85 tyrosine phosphorylation in the sensitive cell line, suggesting that a phosphatase activity downstream of PKC activation may be required for p85 tyrosine phosphorylation. Characterization of p85 and its regulation should help elucidate some of the earliest events in this PKC pathway.

Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C.

The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family.