Novel phorbol ester-binding motif mediates hormonal activation of Na+/H+ exchanger.

Protein kinase C (PKC) is considered crucial for hormonal Na(+)/H(+) exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we report that rather than PKC, direct binding of PEs/diacylglycerol to the NHE1 lipid-interacting domain (LID) and the subsequent tighter association of LID with the plasma membrane mainly underlies NHE1 activation. We show that (i) PEs directly interact with the LID of NHE1 in vitro, (ii) like PKC, green fluorescent protein (GFP)-labeled LID translocates to the plasma membrane in response to PEs and receptor agonists, (iii) LID mutations markedly inhibit these interactions and PE/receptor agonist-induced NHE1 activation, and (iv) PKC inhibitors ineffectively block NHE1 activation, except staurosporin, which itself inhibits NHE1 via LID. Thus, we propose a PKC-independent mechanism of NHE1 regulation via a PE-binding motif previously unrecognized.

Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/pi interaction.

Phorbol ester-type tumor promoters such as indolactam-V (IL-V, 1) bind to the C1 domains of protein kinase C (PKC) isozymes. A more convenient method to investigate the interaction between each tumor promoter and PKC C1 domain is needed. Focusing on our recent finding that the indole ring of IL-V is involved in the CH/pi interaction with Pro-11 of the PKCdelta-C1B domain, we developed new fluorescent probes (2-4) from IL-V by forming a pyrroloindazole ring. Compound 2 without a substituent at the pyrroloindazole ring bound most strongly to PKC C1 domains with a potency similar to IL-V, but its fluorescent intensity was the weakest of any of the probes. Although the binding affinity of 3 with a methyl group was significantly weaker than that of IL-V, 4 with a trifluoromethyl group showed moderate affinity and the most potent fluorescence intensity. The fluorescence intensity and emission maxima of 4 changed significantly when bound to the PKCdelta-C1B peptide in both the presence and absence of phosphatidylserine. These results suggest that 4 could be a useful probe for analyzing the interaction of tumor promoters with PKC C1 domains.

Phorbol esters: structure, biological activity, and toxicity in animals.

Phorbol esters are the tetracyclic diterpenoids generally known for their tumor promoting activity. The phorbol esters mimic the action of diacyl glycerol (DAG), activator of protein kinase C, which regulates different signal transduction pathways and other cellular metabolic activities. They occur naturally in many plants of the family Euphorbiacaeae and Thymelaeaceae. The biological activities of the phorbol esters are highly structure specific. The phorbol esters, even at very low concentrations, show toxicological manifestations in animals fed diets containing them. This toxicity limits the use of many nutritive plants and agricultural by-products containing phorbol esters to be used as animal feed. Therefore, various chemical and physical treatments have been evaluated to extract or inactivate phorbol esters so that seed meals rich in proteins could be used as feed resources. However, not much progress has been reported so far. The detoxifying ability has also been reported in some molluscs and in liver homogenate of mice. Besides, possessing antinutritional and toxic effects, few derivatives of the phorbol esters are also known for their antimicrobial and antitumor activities. The molluscicidal and insecticidal properties of phorbol esters indicate its potential to be used as an effective biopesticide and insecticide.

Protein kinase C translocation by modified phorbol esters with functionalized lipophilic regions.

Several novel phorbol esters were prepared with polar functional groups terminating their C12 and/or C13 acyl chains. Designed to be inhibitory protein kinase C (PKC) ligands, these phorbol analogues contain various polar functional groups (amide, ester, carboxylic acid, or quaternary ammonium salt) to prevent membrane insertion of the PKC-phorbol ester complex. All phorbol derivatives were synthesized with use of diterpene starting materials obtained from croton oil, the seed oil of Croton tiglium. The ability of these derivatives to recruit PKC to the lipid bilayer-a usual requirement for enzyme activation-was determined by using a sucrose-loaded vesicle assay. Phorbol 12-octanoate-13-acetate derivatives translocate PKC-betaII to increasing degrees as the functionality on the C12 ester becomes more hydrophobic. Likewise, PKC translocation by carboxylic acid-containing phorbol esters was dependent upon length and saturation of the hydrocarbon tether. The most promising PKC inhibitors had short carboxylic acids capping their C12 and C13 acyl chains, since these compounds did not recruit PKC to any appreciable extent.

Dramatic switching of protein kinase C agonist/antagonist activity by modifying the 12-ester side chain of phorbol esters.

A dramatic switching of PKC agonist and antagonist activity was observed by modification of the hydrophilicity of the 12-ester side chain of phorbol. Thus, phorbol ester 4 that contains a glycol at the 12-ester chain demonstrated a pure and significant antagonist ability of PKC; however, 3 that contains an alkanol at the 12-ester chain demonstrated a potent PKC agonist activity. On the basis of the structural difference between 3 and 4 and results of the partition assay in the Hela cell/PBS buffer system, we propose that 4 acts as a translocation poison of the PKC-phorbol ester complex. The approach of controlling the agonist/antagonist activity of phorbol esters by the nature (i.e., hydrophilicity, charge, and rigidity, etc.) of the 12-ester chain may be very useful for developing selective PKC inhibitors and a potential pharmaceutical compound for anticancer therapies.

Structural determinants of phorbol ester binding in synaptosomes: pharmacokinetics and pharmacodynamics.

The present study used structurally distinct phorbol esters to investigate the relationship between their pharmacokinetics of binding to protein kinase C (PKC) in rat brain cortex synaptosomes, their affinity for PKC in synaptosomes and ability to enhance noradrenaline release from rat brain cortex. Affinity binding studies using [3deoxyphorbol 13-tetradecanoate (dPT)=PDB&z. Gt;12-deoxyphorbol 13-acetate (dPA)=phorbol 12,13-diacetate (PDA). In intact synaptosomes PDB, dPA and PDA rapidly displaced bound [3H]PDB whereas PMA and dPT were comparatively slow. However, the displacement rates for all the phorbol esters were equally rapid in synaptosomal membranes or synaptosomes permeabilised with Staphylococcus alpha-toxin. These results suggest that the lipophilic phorbol esters (dPT and PMA) are slower to displace [3H]PDB binding because they are hindered by the plasma membrane. In brain cortex slices it was found that the rate of displacement of [3H]PDB binding was closely correlated with the degree of elevation of transmitter noradrenaline release. Thus kinetic characteristics may determine biological responses and this may be particularly evident in events which occur rapidly or where there is fast counter-regulation.

Protein kinase C imaging using carbon-11-labeled phorbol esters: 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate as the potential ligand for positron emission tomography.

Protein kinase C plays a crucial role in signal transduction for a variety of biologically active substances which activates cellular functions and their proliferation. The actions are closely related to both normal and abnormal functions in the nervous system. Tumor-promoting phorbol esters can substitute for diacylglycerols which are important ligands that bind to protein kinase C. Three typical phorbol esters, phorbol 13-[1-11C]butyrate, phorbol 12,13-[1-11C]dibutyrate and 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate, were synthesized by using [11C]ethylketene with a high specific activity (186GBq/mumol). Their in vivo autoradiograms demonstrated a heterogenous distribution in rat brain. 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate was particularly suited for in vivo use due to its nontumor-promoting activity and its ready permeability to the blood-brain barrier. High optical density was observed in the cortex, amygdala and hippocampus. The in vivo binding properties of this compound to protein kinase C were confirmed by in vivo displacement studies with unlabeled 12-deoxyphorbol 13-isobutyrate-20-butyrate and unlabeled phorbol 12,13-dibutyrate. This suggests that 12-deoxyphorbol 13-isobutyrate-20-[1-11C] butyrate has a specific binding affinity for protein kinase C.

Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts.

Transformed cells differ from normal cells in that they fail to respond to normal signals for regulation of growth and differentiation. This disordered signal transduction probably contributes to maintenance of the transformed phenotype. Several lines of evidence suggest that changes in the Ca2(+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), may be important for transformation. To determine the role of PKC in transformation, we compared the levels and subcellular distribution of total phorbol ester receptors and PKC in normal and SV40-transformed rat embryo fibroblasts (REF52 cells). We also used our alpha-PKC (Type 3)-specific monoclonal antibodies to compare alpha-PKC content and regulation. We found no differences in quantity or subcellular distribution of PKC in 100,000 x g soluble and pelletable fractions. Downmodulation, which represents a feedback loop for limiting PKC activity, occurs to the same extent in both cell types. A major difference between the normal and transformed cells was revealed by immunofluorescence of alpha-PKC. In normal cels, alpha-PKC is tightly associated with the cytoskeleton and appears to be organized into focal contacts because it colocalizes with talin. In contrast, in SV40-REF52 cells, alpha-PKC is not tightly associated with the cytoskeleton and does not colocalize with talin. The difference in subcellular localizations correlates with a loss of two alpha-PKC-binding proteins in the transformed cells. These results indicate that inappropriate subcellular location of alpha-PKC may contribute to maintenance of the transformed phenotype.