Activation of conventional and novel protein kinase C isozymes by different diacylglycerol molecular species

Abstract

A variety of diacylglycerol (DG) molecular species are produced in stimulated cells. Conventional (α, βII and γ) and novel (δ, ε, η and θ) protein kinase C (PKC) isoforms are known to be activated by DG. However, a comprehensive analysis has not been performed. In this study, we analyzed activation of the PKC isozymes in the presence of 2-2000 mmol% 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- or 18:0/22:6-DG species. PKCα activity was strongly increased by DG and exhibited less of a preference for 18:0/22:6-DG at 2 mmol%. PKCβII activity was moderately increased by DG and did not have significant preference for DG species. PKCγ activity was moderately increased by DG and exhibited a moderate preference for 18:0/22:6-DG at 2 mmol%. PKCδ activity was moderately increased by DG and exhibited a preference for 18:0/22:6-DG at 20 and 200 mmol%. PKCε activity moderately increased by DG and showed a moderate preference for 18:0/22:6-DG at 2000 mmol%. PKCη was not markedly activated by DG. PKCθ activity was the most strongly increased by DG and exhibited a preference for 18:0/22:6-DG at 2 and 20 mmol% DG. These results indicate that conventional and novel PKCs have different sensitivities and dependences on DG and a distinct preference for shorter and saturated fatty acid-containing and longer and polyunsaturated fatty acid-containing DG species, respectively. This differential regulation would be important for their physiological functions.

Keywords: Activation; DG, diacylglycerol; DGK, diacylglycerol kinase; Diacylglycerol; Isoform; MBP, myelin basic protein fragment 4-14; Molecular species; PKC, protein kinase C; Protein kinase C; TPA, 12-O-Tetradecanoylphorbol-13-acetate; cPKC, conventional protein kinase C; nPKC, novel protein kinase C.

Fig. 1

Effects of different DG molecular species on PKCα activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2.The results are the means±SD of four independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity. The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005), and among the DG molecular species (^#P<0.05).

Fig. 2

Effects of different DG molecular species on PKCβΙΙ activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2. The results are the means ± SD of three independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity. The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005).

Fig. 3

Effects of different DG molecular species on PKCγ activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2. The results are the means±SD of five independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity. The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005), and among the DG molecular species (^#P<0.05).

Fig. 4

Effects of different DG molecular species on PKCδ activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2. The results are the means±SD of four independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity. The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005), and among the DG molecular species (^#P<0.05, ^##P<0.01).

Fig. 5

Effects of different DG molecular species on PKCε activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2. The results are the means±SD of four independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity. The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005), and among the DG molecular species (^#P<0.05).

Fig. 6

Effects of different DG molecular species on PKCη activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2. The results are the means±SD of four independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity.

Fig. 7

Effects of different DG molecular species on the activation of PKCη expressed in COS-7 cells. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs, as described in Section 2. The activities of vector-transfected cells were subtracted. The results are the means±SD of three independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG). The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005), and among the DG molecular species (^#P<0.05, ^##P<0.01).

Fig. 8

Effects of different DG molecular species on PKCθ activation. Lipid vesicles were prepared with different DG molecular species (16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- and 18:0/22:6-DG) and PKC activity toward MBP was determined in vesicles as a function of increasing concentrations of DGs as described in Section 2. The results are the means ± SD of three independent experiments. The left axis shows the relative activity compared to the control (0 mmol% DG) and the right axis shows the specific activity. The data are significantly different from the control, 0 mmol% DG (*P<0.05, **P<0.01, ***P<0.005), and among the DG molecular species (^#P<0.05, ^##P<0.01).