To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (χ(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when χ(DO) < ~0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when χ(DO) > ~0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from χ(DO) ≃ 0.1 to χ(DO) ≃ 0.3, with the more cooperative transition growing at the expense of the broader one as χ(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when χ(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions χ(DO) ≃ 0.1 to χ(DO) ≃ 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35°C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing χ(DO) from χ(DO) 0.1 to χ(DO) ≃ 0.4, and activity remained high at higher DO contents. In contrast, at 2°C (a temperature below the main transition), PKC activity exhibited a maximum between χ(DO) ≃ 0.1 and χ(DO) ≃ 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.
Bibliographical noteFunding Information:
We thank Robert Doebler for assistance with the FTIR experiments, especially with regard to temperature control. We also thank Olga Cherti- hin, Prateek Gupta, Moira Resnick, and Betsy Vinton for their help during the purification of PKC isozymes and the subsequent kinase assays. This work was supported by grants from the National Institutes of Health, U.S. Public Health Service (ROI GM31184, ROI GM37658, and PO1 GM47525). AD and AH were supported in part by National Institutes of Health Research Service Awards T32 DK07320 and T32 DK07642, respectively. A preliminary account of these findings was presented at the 39th Annual Meeting of the Biophysical Society, 1995, San Francisco, CA (Dibble et al., 1995). This manuscript is an exten- sion of that work.