Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500-to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.The recent advent of diacylglycerol as the endogenous activator ofprotein kinase C has propelled diacylglycerol into the limelight as an important second messenger, coupling receptor-mediated hydrolysis of phosphatidylinositols to activation of protein kinase C. It has been known for some time that diacylglycerol is produced as a result of the hydrolysis of phosphatidylinositols by phospholipase C (1-3). Many reports (4-8) indicate that protein kinase C can translocate from cytosol to membranes as a result of the formation of diacylglycerol and the increased cytosolic calcium concentration resulting from inositol trisphosphate formation. However, diacylglycerol is rapidly converted to phosphatidate by diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) (9, 10). Like protein kinase C, diacylglycerol kinase requires phospholipid for catalytic function (11, 12). Moreover, because diacylglycerol kinase is at least partially responsible for the half-life of diacylglycerol, it may play an important role in regulating the coupling of agonist-induced generation of membrane diacy...

Section: Discussionmentioning

confidence: 99%

Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system.

Besterman¹,

Pollenz²,

Booker³

et al. 1986

“…The specificity of the activation process is considerable, with only a narrow range of unmodified diacylglycerols in the S configuration being active (5)(6)(7)(8)(9). We recently proposed (10) a structural hypothesis concerning the nature of the diacylglycerol-binding region in PKC.…”

mentioning

confidence: 99%

The pharmacophore of debromoaplysiatoxin responsible for protein kinase C activation.

Kong

Kishi

Rando

1991

Protein kinase C is physiologically activated by 1,2-diacyl-sn-glycerol in the S configuration. The enzyme is also powerfully activated by structurally diverse tumor promotors. A model has been developed that demonstrates how the various tumor promotors and diacylglycerols can all be accommodated by the same binding site of the kinase. One prediction of this model concerns the structural nature of the pharmacophore in the tumor promotor debromoaplysiatoxin. This prediction is realized by synthesizing the analogs with the deduced pharmacophore and demonstrating that they are potent activators of protein kinase C. These rindings provide strong experimental support for our structural model of protein kinase C activation.Protein kinase C (PKC) is an important regulatory enzyme involved in the control of diverse biological phenomena (1, 2). Under quiescent conditions, the enzyme is located in the cytoplasm, where it remains catalytically inactive (2). The enzyme is activated, in a transient fashion, when 1,2-diacylsn-glycerols in the S configuration are produced in the membrane by the action of a specific phospholipase C (2, 3). This latter enzyme cleaves inositol (poly)phospholipids to generate diacylglycerols and (poly)phosphoinositols (2, 4). The liberated diacylglycerol binds to the regulatory domain of PKC and activates PKC by increasing its affinity for calcium ions to the steady-state physiological range (2).The specificity of the activation process is considerable, with only a narrow range of unmodified diacylglycerols in the S configuration being active (5-9). We recently proposed (10) a structural hypothesis concerning the nature of the diacylglycerol-binding region in PKC. Our major concern was to resolve how the same effector binding site in PKC can specifically recognize both diacylglycerols and the structurally diverse tumor promotors (10-13). We proposed the structural correlation among PKC activators summarized in Fig. 1 (10). We have demonstrated (14) that PKC activation is also stereospecific with respect to the second chiral center of 3-methylated 1,2-diacyl-sn-glycerols. Furthermore, the absolute configuration at C2 and C3 of the active 3-methylated diacylglycerols is the same as the absolute configuration at C29 and C30 of the naturally occurring tumor promotor debromoaplysiatoxin (DAT) (Fig. 1) (10). These observations support our structural hypothesis and forge a structural link between tumor promotors and diacylglycerols for PKC activation. In this report, this structural link is further explored by synthesizing and testing two classes of the hybrids of diacylglycerols and aplysiatoxins. It is demonstrated that the structural predictions made by our hypothesis are realized in both classes of hybrids and that the 3,4-dihydroxybutyrate moiety found in aplysiatoxins is the minimally essential pharmacophore of this class of tumor promotors. MATERIALS AND METHODSMaterials. Bovine phosphatidylserine was obtained from Avanti Polar Lipids. 1,2-Dioleoyl-sn-glycerol and histone type VS were fr...

“…Diacylglycerols are also membrane-oriented (7) second messengers participating in signal transduction across membranes (8,9). The biologically active form of diacylglycerols are the membrane-bound 1,2-diacyl-sn-glycerols (10). The general structure of saturated 1,2-diacyl-sn-glycerols has been reported (11) and the crystal structure of 1,2-dilauroyl-sn-glycerol (12) and 1,2-dipalmitoyl-sn-glycerol (13) have been solved.…”

mentioning

confidence: 99%

Single crystal structure of a mixed-chain triacylglycerol: 1,2-dipalmitoyl-3-acetyl-sn-glycerol.

Goto¹,

Kodali²,

Small³

et al. 1992

The mixed chain triacylglycerol 1,2-dipalmitoyl-3-acetyl-sn-glycerol was synthesized and its crystal structure was determined to a rinal reliability factor (R) of 0.11. Two molecules are present in the monoclinic unit cell: space group P21; a = 5.375(1), b = 8.286(2), c = 42.96(1) i; 13 = 93.30(2)°, V = 1910 A3, p = 1.065 g/cm3, and jt = 5.7 cm-'. The structure is a trilayer: a bilayer of palmitate chains packed in the (3 mode (T//) and an interdigitated monolayer of acetates. The glycerol backbone and acetate extend roughly linearly from the sn-i chain. The sn-2 chain bends around the C-2 carbon to lie next to the sn-i chain. Analysis of the torsion angles indicate that the glycerol conformation of 1,2-dipalmitoyl-3-acetyl-sn-glycerol is markedly different from single acid triacylglycerols and from 1,2-diacyl-sn-glycerols but very similar to 1,2-dimyristoyl-sn-glycero-3-phosphocholine.Acylglycerols are ubiquitous and highly prevalent biological molecules. Triacylglycerols are the major form of lipids stored in tissues of both plants and animals. Although their physical properties such as polymorphic forms, melting and transition temperatures, volumes, and viscosity are generally known (1-3), specific detailed crystal structures are largely lacking. Only two structures on single-chain triacylglycerols, tricaprin by Jensen and Mabis in 1963 (4) and trilaurin by Larsson in 1964 (5), have been published. A single complex triacylglyceride structure, 2-(11-bromoundecanoyl)-1,1'-dicaprin, was published (6), but its structure, especially the conformation of the glycerol and the chain orientation, are quite similar to the structure of single-chain triacylglycerols (4, 5). Our understanding of the structure and polymorphism of acylglycerols would be greatly enhanced by knowing the crystal structures of some of the forms of mixed-chain triacylglycerols. Diacylglycerols are intermediates in the metabolism of triacylglycerols and phospholipids. Diacylglycerols are also membrane-oriented (7) second messengers participating in signal transduction across membranes (8, 9). The biologically active form of diacylglycerols are the membrane-bound 1,2-diacyl-sn-glycerols (10). The general structure of saturated 1,2-diacyl-sn-glycerols has been reported (11) and the crystal structure of 1,2-dilauroyl-sn-glycerol (12) and 1,2-dipalmitoyl-sn-glycerol (13) have been solved. We have synthesized the three acetyl derivative of 1,2-dipalmitoyl-sn-glycerol and defined its crystal structure.$ The structure of this triacylglycerol with an acetate in position 3 differs considerably around the glycerol backbone from 1,2-diacylglycerols (12-14) or simple acid triacylglycerols such as trilaurin (4, 5). The glycerol conformation is, however, quite similar to one of the two conformers of dimyristoyl phosphatidylcholine (15). 11 MATERIALS AND METHODS 1,2-Dipalmitoyl-3-acetyl-sn-glycerol (PP2) was synthesized as described by Kodali et al. (16). After several trials of crystallization, a fairly good crystal with dimensions of 0.02 x 0.06 x 0.5 ...