Phosphatidylinositol 4,5-bisphosphate decreases the concentration of Ca2+, phosphatidylserine and diacylglycerol required for protein kinase C α to reach maximum activity

Abstract

The C2 domain of PKCα possesses two different binding sites, one for Ca(2+) and phosphatidylserine and a second one that binds PIP2 with very high affinity. The enzymatic activity of PKCα was studied by activating it with large unilamellar lipid vesicles, varying the concentration of Ca(2+) and the contents of dioleylglycerol (DOG), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphadidylserine (POPS) in these model membranes. The results showed that PIP2 increased the Vmax of PKCα and, when the PIP2 concentration was 5 mol% of the total lipid in the membrane, the addition of 2 mol% of DOG did not increase the activity. In addition PIP2 decreases K0.5 of Ca(2+) more than 3-fold, that of DOG almost 5-fold and that of POPS by a half. The K0.5 values of PIP2 amounted to only 0.11 µM in the presence of DOG and 0.39 in its absence, which is within the expected physiological range for the inner monolayer of a mammalian plasma membrane. As a consequence, PKCα may be expected to operate near its maximum capacity even in the absence of a cell signal producing diacylglycerol. Nevertheless, we have shown that the presence of DOG may also help, since the K0.5 for PIP2 notably decreases in its presence. Taken together, these results underline the great importance of PIP2 in the activation of PKCα and demonstrate that in its presence, the most important cell signal for triggering the activity of this enzyme is the increase in the concentration of cytoplasmic Ca(2+).

Figure 1. Structure of PKCα C2 domain bound to Ca²⁺-POPS-PIP₂ in a quaternary complex.

The C2 molecule is shown in green. The three calcium ions are shown in yellow spheres, one of them bridging the protein with phosphatidylserine (PS) at the tip of the domain. The PIP₂ molecule is bound to the β3-β4 chains . PDB accession number 3GPE.

Figure 2. The dependence of PKCα activity on Ca²⁺ concentration.

The molar ratios of the lipid components of the vesicles used to activate the enzyme are shown. Ca²⁺ concentration was 200 µmol. SD calculated from 3 independent experiments.

Figure 3. The dependence of PKCα activity on the POPS molar percentage in the vesicles.

The molar ratios of the lipid components of the vesicles used to activate the enzyme are shown. Ca²⁺ concentration was 200 µmol. SD calculated from 3 independent experiments.

Figure 4. The dependence of PKCα activity on the DOG molar percentage in the lipid vesicles.

The molar ratios of the lipid components of the vesicles used to activate the enzyme are shown. Ca²⁺ concentration was 200 µmol. SD calculated from 3 independent experiments.

Figure 5. The dependence of PKCα activity on the PIP₂ molar percentage in the lipid vesicles.

The molar ratios of the lipid components of the vesicles used to activate the enzyme are shown. Ca²⁺ concentration was 200 µmol. SD calculated from 3 independent experiments.