title: Calcium-independent binding to interfacial phorbol esters causes protein kinase C to associate with membranes in the absence of acidic lipids.
abstract: The mechanism of interaction of phorbol esters with conventional protein kinase Cs was addressed by examining the direct binding of this class of activators to protein kinase C beta II. Binding measurements reveal that the major role of phorbol esters is to increase the affinity of protein kinase C for membranes by several orders of magnitude. The relative increase depends linearly on the mole fraction of phorbol esters in membranes, with the potency illustrated by the finding that 1 mol% phorbol 12-myristate 13-acetate (PMA) increases protein kinase C's membrane association by approximately 4 orders of magnitude. For comparison, diacylglycerol (DG), which also activates protein kinase C by increasing the enzyme's membrane affinity, is 2 orders of magnitude less effective than PMA in altering protein kinase C's membrane affinity. The remarkably high-affinity interaction with phorbol esters allowed us to measure the direct binding of protein kinase C to PMA in neutral membranes and, thus, to evaluate the effect of Ca2+ on the phorbol ester interaction in the absence of Ca2+ effects on the enzyme's interaction with acidic lipids. Changing the Ca2+ concentration over 5 orders of magnitude had no effect on the direct interaction of protein kinase C with PMA immobilized in phosphatidylcholine membranes. Thus, the Ca(2+)-binding site for membrane association and the phorbol ester-binding site do not interact allosterically. Lastly, a method that does not have the limitations of the Scatchard plot for analysis of amphitropic proteins was used to determine the dissociation constant of protein kinase C from phorbol esters: expressed relative to membrane lipids, the dissociation constant is 1.5 x 10(-5) mol %. In summary, our data reveal that (1) the direct binding of protein kinase C to phorbol esters, in the absence of interactions with acidic lipids, provides a major contribution to the free energy change involved in the association of protein kinase C with membranes and (2) this interaction is not regulated by Ca2+.
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla 92093-0640, USA.
authors.forename: M
authors.initials: M
authors.lastname: Mosior
authors.forename: A C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D008081:Liposomes / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium