Related Experiment Videos
Transmembrane signaling: tumor promoter distribution
1Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0130.
Abstract:
Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.
Insights
The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) localizes to the inner leaflet of cell membranes. This finding supports TPA
Area of Science:
- Cell biology
- Biochemistry
- Molecular pharmacology
Background:
- Diacylglycerol activates protein kinase C (PKC) in transmembrane signaling.
- The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics diacylglycerol, activating PKC and phosphorylating membrane proteins in human erythrocytes.
- The precise location of TPA within plasma membranes is not well understood, hindering molecular modeling.
Purpose of the Study:
- To determine the transbilayer distribution of TPA within the human red cell plasma membrane.
- To provide direct evidence for TPA's localization to inform its mechanism of action.
Main Methods:
- Utilized membrane-splitting techniques based on freeze-fracture of planar cell monolayers.
- Quantified the transmembrane partitioning of radiolabeled [3H]TPA.
- Assessed TPA association with the red cell plasma membrane under conditions of stimulated PKC-mediated phosphorylation.
Main Results:
- Over 90% of [3H]TPA associated with the human red cell plasma membrane.
- Following bilayer splitting, two-thirds of the TPA partitioned to the cytoplasmic leaflet.
- This represents the first direct topographic localization of TPA in a biological membrane.
Conclusions:
- TPA preferentially associates with the cytoplasmic leaflet of the plasma membrane.
- This localization supports the hypothesis that TPA activation of PKC requires its presence on the cytoplasmic side of the membrane.