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Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.

To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23-24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P0 (Golgi) and myelin basic proteins (more direct).

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