Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKCθ. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers. The calculated EC(50) values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.     

Lipid activators of protein kinase C

Among the many reported lipid activators of protein kinase C only those of high affinity can be considered true physiological effectors, at present the tumor promoters, e.g., phorbol esters; 1,2-diacyl-sn-glycerols; and phosphatidylinositol 4,5-bisphosphate. Many other compounds (including arachidonic acid) are activators at high, unphysiological concentrations only, and they seem to be sterically unsuited for bonding to the enzyme. Such pseudo-activators possibly act by scrambling the structure of the regulatory moiety of the kinase.     

Diacylglycerols as activators of protein kinase C

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.     

Structural analogies between protein kinase C activators

Phorbol esters and diacylglycerols activate protein kinase C but specific structural parameters appear to be required for the enzyme activation. We have analyzed the conformation of potent and not potent diacylglycerols and phorbol esters. The orientation of the CH20H group at C3 of 1,2 diolein is remarkably similar to that of the same group at C-20 of 4 beta phorbol didecanoate and crucial for potency in activating the enzyme. Our data suggest that the new conformational approach here described could be used to rationally design specific inhibitors preventing the effects of tumor promoters and to predict the structure of potential tumor promoters.     

Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C

Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.     

Regulation of protein kinase C activity by lipids

Protein kinase C is activated by the simultaneous presence of phospholipid, a diglyceride, and Ca2+. Under physiological conditions the activity of the enzyme is regulated by the availability of diglycerides, which are the products of phosphoinositide hydrolysis. The phospholipid-kinase interactions appear not to be of a highly specific nature. Phosphatidylserine (PS) is presumed to be the endogenous lipid that interacts with the kinase, but other acidic lipids can substitute. On the other hand, the kinase-diglyceride interactions are highly specific in nature, as would be expected of a physiological regulator. These interactions are stereo-specific and stoichiometric with respect to diglyceride. The specificity is directed toward the glycerol backbone and hydrophilic oxygen moieties of the diglyceride. The removal of one or more of the oxygen atoms or the addition of a single methyl group to the glycerol backbone virtually abolishes the activity of a putative diglyceride activator. The extreme specificity of the kinase toward the diglycerides, however, must be contrasted with the abilities of structurally diverse tumor promotors and irritants to activate the kinase. Specific small-molecule antagonists of protein kinase C have yet to be developed. The small-molecule antagonists that have been developed so far have been relatively nonspecific cationic lipids that appear to function by interfering with the interaction between the acidic phospholipids and Ca2+.     

Diacylglycerols as activators of protein kinase C (Review)

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.     

Regulation fo protein kinase C activity by various lipids

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracelluular communication.Abbreviations used (PK-C) Protein kinase C - (cAMP-PK) cAMP dependent protein kinase - (DAG) diacylglycerol - (PtdSer) phosphatidylserine - (InsP ₃) inositol 1,4,5-trisphosphate - (PtdIns 4,5-P₂) inositol 4,5 bisphosphate - (FFA) free fatty acid - (MBP) myelin basic protein - (ATP) adenosine triphosphate - (GTP) guanine triphosphate - (TPA) 12-tetradecanoylphorbol-13-acetate - (EGF) epidermal growth factor - (PDGF) platelet derived growth factor - (NeuNAc) and N-acetylneuraminic acid     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Phosphorylation of T cell membrane proteins by activators of protein kinase C

Activation of the enzyme protein kinase C (PKC) plays an important role in T cell activation. We investigated the phosphorylation of CD2, CD3, CD4, CD5, CD7, CD8, CD28 (Tp44), CD43 (sialophorin, gp115), and LFA-1 after incubation of human PBMC with the (PKC) activator PMA. These proteins were chosen for their role in transmembrane signal transduction (CD2, CD3, CD5, CD28, CD43), cell-cell interaction and adhesion (CD2, CD4, CD8, and LFA-1), or involvement in immunodeficiency states (CD43, CD7). CD5, CD7, CD43, and the alpha-chain of LFA-1 were found to be constitutively phosphorylated. PMA induced rapid hyperphosphorylation of CD5, CD7, and CD43, but not of the LFA-1 alpha-chain, and induced the phosphorylation of CD3, CD4, CD8 and of the LFA-1 beta-chain. PMA did not cause the phosphorylation of CD2 and CD28. PMA-induced phosphorylation was partially inhibited by the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride. Finally, the T cell activator Con A, which binds to the CD3/TCR complex was shown to induce a profile of protein phosphorylation similar to that observed with PMA. We conclude that PKC-mediated phosphorylation of T cell Ag may represent an important regulatory mechanism that governs the process of T cell activation.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Stimulation of protein phosphorylations in frog rod outer segments by protein kinase activators. Suppression of light-induced changes in membrane current and cGMP by protein kinase C activators

Addition of protein kinase C activators to electropermeabilized frog rod photoreceptors enhances the phosphorylation of proteins with molecular masses of 54, 24, 19, 17, 12, and 11 kDa. The latter two correspond to components I and II, which are also phosphorylated by cyclic nucleotide-dependent protein kinase. Stimulation of phosphorylation by the protein kinase C activator oleoylacetylglycerol (OAG) is half-maximal at 7.7 microM OAG and is reduced by the protein kinase C inhibitor H-7. In contrast with earlier observations, no effects of calcium, calmodulin, or insulin on protein phosphorylations are observed. We find evidence for only three protein kinases in rod outer segments: a protein kinase C-like activity, cAMP-dependent protein kinase, and rhodopsin kinase. With the exception of components I and II, the substrate proteins for each kinase are distinct. Treatment of intact rods with OAG decreases the amplitude of the photoresponse and dark levels of cGMP up to 40%, as well as depressing the light-stimulated decrease in cGMP levels. These effects are observed between 0.1 and 1 microM OAG. The data suggest that OAG-sensitive reactions may modulate pathways that support the light response.     

Biphasic regulation of macrophage attachment by activators of cyclic adenosine monophosphate-dependent kinase and protein kinase C

A method is described that enabled us to study the adhesiveness of J-774 murine macrophages. Cell attachment was stimulated by activators of kinase C (i.e., phorbol esters) as well as kinase A (cyclic adenosine monophosphate; cAMP). This novel effect of cAMP was observed when its levels were increased via receptor triggering (prostaglandin E1, beta-adrenergic agonists), activation of Ns (cholera toxin), or inhibition of phosphodiesterase (Ro 20-1724) or when the kinase was directly activated by Br8-cAMP. The simultaneous treatment with kinase A and kinase C activators at the time of attachment resulted in a partially additive response. On the other hand, preincubation of the cells in suspension with one of the activators rendered them refractory to subsequent stimulation at the onset of the adhesion assay, whatever agent was used. Such a refractoriness was also observed in cells preincubated with oleoyl-acetyl-glycerol (OAG). On the other hand, when added at the time of attachment, this near-physiological activator of kinase C evoked a biphasic response: the early stimulation of cell attachment was followed by an accelerated rate of "detachment." In conclusion, kinase C and kinase A play a role in the sequence of events leading to cell adhesion. The cross desensitization observed is distal and takes place at or beyond the kinase step.     

Decreased sulfation of cellular chondroitin sulfate in response to activators of protein kinase C

The sulfation of cellular chondroitin sulfate in human promyelocytic leukemia HL60 cells was inhibited by a number of phorbol diesters, which concurrently induced differentiation into monocytic cells. Inhibition was dependent on concentration, and was 90% complete at 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), the most active ester. Maximal effects were seen within 2-4 hours following initiation of treatment. The degree of inhibition observed correlated well with the ability of the esters to induce differentiation, and with their reported affinity for a "receptor", identified as protein kinase C associated with certain lipids. Chondroitin sulfation was also inhibited in cells treated with sn-1,2-dioctanoylglycerol, a lipid which is considered to be an endogenous activator of protein kinase C. Our findings therefore indicate that monocytic differentiation of HL60 cells occurs subsequent to reduced glycosaminoglycan sulfation via activation of the calcium-activated, phospholipid-dependent protein kinase.     

Rapid modulation of tumor necrosis factor membrane receptors by activators of protein kinase C

Tumor necrosis factor membrane receptors are rapidly down-regulated upon treatment of activated T lymphocytes with various activators of protein kinase C. Loss of binding-capacity was half maximal after 2 min. incubation in 10 ng/ml of phorbol 12-myristate 13-acetate. A similar modulation could be induced with either the calcium ionophore A 23187 or the protein kinase C activator 1-oleyl-2-acetyl glycerol, whereas 1,2-diolein and dibutyryl cAMP were ineffective. Protein kinase C inhibitor H7 antagonizes the phorbol ester-induced TNF receptor modulation. These data suggest an important role of protein kinase C in the control of TNF responsiveness by regulation of TNF binding-capacity possibly via direct phosphorylation of specific receptor proteins.     

Arachidonic acid,protein kinase C activators and bud formation in Hydra vulgaris

1. The effect of the protein kinase C (PKC) activators verrucosin B (VB), 1,2-sn-dioctanoylglycerol (diC₈) and phorbol-12-myristate-13-acetate (PMA), of arachidonic acid (AA) and of substances interfering with its release, re-uptake and metabolism was studied in Hydra vulgaris.2. All PKC activators potently inhibited bud formation, VB and PMA being 10,000 × more potent than diC⁸. VB effect was maximal already after 10 min incubation with hydra and persisted at 24 hr incubations.3. AA and substances inhibiting its re-uptake from cell membrane or its metabolism also inhibited bud formation, whereas oleyl-oxyethyl-phosphorylcholine (OOPC), an inhibitor of phospholipase A₂, potently induced bud formation.4. The findings described herein suggest a role for both PKC activation and AA in the inhibition of bud formation in H. vulgaris.