Barry R. Ganong

1,2-Diacylglycerols do not potentiate the action of phospholipases A2 and C in human platelets

1,2-Diacylglycerol has recently been reported to potentiate the ability of phospholipases A and C to hydrolyze phospholipids in a cell-free system. The present study has been undertaken to investigate whether 1,2-diacylglycerol can also perform this function in intact cells using the platelet as a test system. Exogenous 1-oleoyl-2-acetyl-glycerol ( OAG ) and 1,2- didecanoylglycerol , at concentrations sufficient to produce maximal phosphorylation of a 40,000 dalton protein, caused no significant formation of [3H]inositol phosphates and [32P]phosphatidic acid (products of phospholipase C activation) or [14C]arachidonic acid metabolites and lysophosphatidyl[3H]inositol (products of phospholipase A2 activation). These data therefore imply that 1,2-diacylglycerols do not potentiate the actions of phospholipases A2 and C in intact platelets at concentrations that are physiologically relevant.

Synthesis of cell-permeant diacylglycerol analogs for structure-function analysis of protein kinase C and other enzymes

Publisher Summary Protein kinase C is a multifunctional protein kinase whose in vitro activity is dependent on phospholipid, calcium, and phorbol esters or diacylglycerols. It is also a high-affinity phorbol ester receptor that is nearly ubiquitous in mammalian cells and presumably mediates the biological effects of this class of potent tumor promoters. Diacylglycerols generated by receptor-dependent phosphoinositide degradation are endogenous activators of protein kinase C, functioning as second messengers in hormone signal transduction. This hypothesis is supported by demonstrations that a synthetic diacylglycerol––l-oleoyl-2-acetylglycerol (OAG)––induced biological responses in a variety of cell types. Structural analogs of diC 8 are prepared to provide insight into the molecular mechanism of protein kinase C activation in vitro , to identify inhibitors, and to identify nonmetabolizable activators of protein kinase C in cells. The chapter discusses the uses of diC 8 analogs.

Regulation of Protein Kinase C by Lipid Cofactors

Hormone-stimulated biological responses in a number of tissues and cell types are associated with turnover of inositol phospholipids. This phenomenon was first observed over 30 years ago by Hokin and Hokin (1953) and, since then, has been demonstrated in a wide variety of systems (reviewed by Michell, 1975; Berridge, 1984; Hirasawa and Nishizuka, 1985; Hokin, 1985). The significance of phosphatidylinositol turnover remained a mystery until two relatively recent discoveries helped clarify the role of inositol phosphatides in transmembrane signaling.

Phospholipid Bilayer Assembly: Facilitated Transmembrane Movement of Phosphatidylcholine

The transmembrane movement of phospholipids is a fundamental step in membrane assembly and intracellular lipid sorting. Phospholipid synthesis occurs on the cytoplasmic surface of the endoplasmic reticulum. Translocation to the lumenal surface is required for assembly of the bilayer. Studies employing a spectrophotometric assay to measure the transmembrane movement of phosphatidylglycerol and diacylglycerol analogs in phospholipid vesicles revealed half-times of greater than 8 days and less than 15 seconds, respectively. The hypothesis that rat liver microsomes possess a “PC flippase” was tested by determining whether sn 1,2-dibutyroylphosphatidylcholine (diC4PC) was transported into the lumen. This compound was chosen for study because it retains the polar head group, the portion of PC unable to spontaneously move across the membrane. Since diC4PC is water soluble, standard transport methods could be applied. DiC4PC uptake into the microsomal lumen was saturable, time dependent and proportional to the amount of microsomes employed. An intact permeability barrier was required. DiC4PC transport was inhibited by structural analogs (but not sn-2,3-diC4PC) and was inhibited by proteases and protein modification reagents. Transport of diC4PC was not observed across PC vesicles or red cell membranes where PC movement is slow. The transport of diC4PC is a protein facilitated process. It is inferred that the same system functions in PC translocation in the assembly of the endoplasmic reticulum bilayer.

Protein Kinase C Regulation by Diacylglycerols: Structure-Function Relationships and Mechanism

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids, diacylglycerols, and diacylglycerol analogues. Triton X-100 micelles containing 8 mol% phosphatidylserine (PS) and 2.5 mol% sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1. Protein kinase C activity was almost totally dependent on diC18:1 in the mixed micellar assay. At 8 mol% PS, diC18:1 stimulated maximally at 1 mol%. At 2.5 mol% diC18:1, PS did not activate until 3 mol% and then did so cooperatively with maximal stimulation occurring at 6–8 mol%. Molecular sieve chromatography demonstrated that monomeric protein kinase C interacts with Triton X-100 micelles in a PS and Ca2+ dependent manner. Interpretations follow: 1) a single molecule of diC18:1 activates monomeric protein kinase C; 2) a phospholipid bilayer is not required; 3) four or more molecules of PS are required. In addition, several diacylglycerol analogues were synthesized to determine the exact structural features required for activation. The data suggest that both carbonyls of the oxygen esters and the 3-hydroxyl are required. A model of protein kinase C activation by PS and diacylglycerol was formulated.