Influence of the Physical State of the Membrane on the Enzymatic Activity and Energy of Activation of Protein Kinase C R † Antonia M. Jime ´nez-Monreal, Francisco J. Aranda, Vicente Micol, ‡ Pilar Sa ´nchez-Pin ˜era, Ana de Godos, and Juan C. Go ´mez-Ferna ´ndez* Departamento de Bioquı ´mica y Biologı ´a Molecular “A”, Facultad de Veterinaria, UniVersidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain ReceiVed December 29, 1998; ReVised Manuscript ReceiVed March 16, 1999 ABSTRACT: The activation of protein kinase C R was studied by using a lipid system consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) (molar ratio 4:1) and different proportions of 1-palmitoyl-2-oleoyl-sn-glycerol (POG). The phase behavior of the lipidic system was characterized by using differential scanning calorimetry and 31 P NMR, and a phase diagram was elaborated. The results suggested the formation of two diacylglycerol/phospholipid complexes, one at 15 mol % of POG and the second at 30 mol % of POG. These two complexes would define the three regions of the phase diagram: in the first region (concentrations of POG lower than 15 mol %) there is gel-gel immiscibility at temperatures below that of the phase transition between C 1 and pure phospholipid, and a fluid lamellar phase above of the phase transition. In the second region (between 15 and 30 mol % of POG), gel-gel immiscibility between C 1 and C 2 with fluid-fluid immiscibility was observed, while inverted hexagonal H II and isotropic phases were detected by 31 P NMR. In the third region (concentrations of POG higher than 30 mol %), gel-gel immiscibility seemed to occur between C 2 and pure POG along with fluid-fluid immiscibility, while an isotropic phase was detected by 31 P NMR. When PKC R activity was measured, as a function of POG concentration, maximum activity was found at POG concentrations as low as 5-10 mol %; the activity slighty decreased as POG concentration was increased to 45 mol % at 32 °C (above T c ) whereas activity did not change with increasing concentrations of POG at 5 °C (below T c ). When the activity was studied as a function of temperature, at different POG concentrations, and depicted as Arrhenius plots, it was found that the activity increased with increasing temperatures, showing a discontinuity at a temperature very close to the phase transition of the system and a lower activation energy at the upper slope of the graph, indicating that the physical state of the membrane affected the interaction of PKC R with the membrane. Protein kinases C (PKCs) 1 are a family of enzymes constituted by at least 12 isoenzymes which are involved in many cellular processes and which are major receptors of tumor promoters such as bryostatin and phorbol esters (see refs 1-3 for comprehensive reviews on PKCs). Of the different PKC isoenzymes, it is PKC R that will receive attention in this paper. This isoenzyme, which belongs to the classic type, is activated by the diacylglycerols generated by phospholipases after cell stimulation, and also by phos- phatidylserine and Ca 2+ . It is thought that membrane structure may be important in determining the activation of PKC R (3, 4). There is a great deal of information suggesting that membrane structure is modulated by the presence of DAGs. For example, it has been shown that DAGs may produce membrane structural changes in membranes such as lateral phase separations (5-10), nonbilayer phases (6, 7, 11-13), and dehydration of the membrane interface (13, 14). Sig- nificantly, the dehydration produced by DAG has more drastic effect on phosphatidylserine than on phosphatidyl- choline (13). These effects may be responsible for the facilitating effect of membrane fusion (15-19) and perhaps for the activation not only of PKC but also of other enzymes such as phospholipases (20-22), CTP phosphocholine cyti- dyltransferase (23), and tyrosine kinase (24). On the other hand, several suggestions have been made concerning the modulation of PKC activity by DAGs. It has been suggested that PKC activity increases with the tendency of lipids to form nonbilayer phases, as could be the case in regions of high bilayer curvature produced by molecules, such as diacylglycerols or phosphatidylethanolamines, which † This work was supported by Grants PB95-1022 and PB96-1107 from Direccio ´n General de Ensen ˜anza Superior (Spain). * To whom correspondence should be addressed. Tel: +34- 968364766; Fax: +34-968364147. E-mail: jcgomez@fcu.um.es. ‡ Present address: Centro de Biologı ´a Molecular y Celular. Univer- sidad Miguel Herna ´ndez. C/ Mono ´var s/n. E-03206 Elche, Alicante, Spain. 1 Abbreviations: DAGs, diacylglycerols; 1,2-DMG, 1,2-dimyristoyl- sn-glycerol; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPS, 1,2-dimyristoyl-sn-glycero-3-phosphoserine; 1,2-DPG, 1,2-dipalmitoyl- sn-glycerol; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSC, differential scanning calorimetry; ∆σ, chemical shift anisotropy; EGTA, ethylene glycol-bis(-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; EDTA, ethylendiaminetetraacetic acid; PC, phosphatidylcholine; PKC, protein kinase C; 31 P NMR, 31 P-nuclear magnetic resonance; POG, 1-palmitoyl-2-oleoyl-sn-glycerol; POPC, 1-palmitoyl-2-oleoyl-sn-glyc- ero-3-phosphocholine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3- phos- pholserine. 7747 Biochemistry 1999, 38, 7747-7754 10.1021/bi983062z CCC: $18.00 © 1999 American Chemical Society Published on Web 05/26/1999