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Physiologic regulation of protein kinase C activity requires its interaction with cellular membranes. We have recently shown that binding of the enzyme to plasma membranes is controlled by Ca2+, whereas enzyme activators, like phorbol esters, regulate both membrane binding and enzyme activity. Here we describe the factors which control the dissociation of protein kinase C from the plasma membrane. In the absence of phorbol esters, the dissociation reaction is rapid and is determined by varying the Ca2+ concentration between 0.1 and 1 microM. However, the presence of 4-beta-phorbol 12,13-dibutyrate greatly reduces enzyme release in response to Ca2+ depletion; removal of the phorbol ester itself permits efficient membrane-enzyme dissociation. The stabilization of the membrane-protein kinase C complex by phorbol esters can be reversed by ATP with an apparent Km for the nucleotide of 6.5 microM. The ATP effect requires MgCl2 and cannot be reproduced by other nucleotides or by a nonhydrolyzable analogue, suggesting that an ATP-dependent phosphorylation reaction may be involved. 4-beta-Phorbol 12,13-dibutyrate appears to stabilize membrane-enzyme association by reducing the apparent Km for Ca2+ to about 15 nM, whereas ATP reverses the phorbol ester effect by increasing the Km for Ca2+ to about 760 nM. Furthermore, the strong degree of negative cooperativity displayed by the Ca2+-dependent enzyme-membrane dissociation is consistent with the presence of multiple interacting Ca2+-binding sites on protein kinase C.

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly regulate surface transferrin receptors. Regulation of transferrin receptors by phorbol diesters involves receptor internalization in association with increased receptor phosphorylation (hyperphosphorylation). The intracellular mechanism of action of phorbol diester involves binding to and activation of the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Present studies comparing results obtained with whole cells and those from a cell-free system reconstituted from purified protein kinase C and transferrin receptor components have revealed that the transferrin receptor is phosphorylated by protein kinase C activated by phorbol esters. Following tryptic digestion and two-dimensional separation of phosphopeptides of phosphorylated transferrin receptors, two major and several minor phosphoserine-containing fragments are resolved. These fragments are identical whether transferrin receptor is phosphorylated in whole cells incubated with phorbol diesters or following phosphorylation of affinity immobilized transferrin receptor in the in vitro reconstitution system. Phosphoamino acid analysis of these fragments indicates that serine is the only amino acid phosphorylated in whole cells or in the cell-free system. In addition, colchicine is shown to inhibit in a dose-dependent manner phorbol diester-induced internalization but not hyperphosphorylation of the surface transferrin receptor in whole cells. This inhibition is specific for colchicine since inactive beta- and gamma-Lumicolchicine have no such effect, while taxol reverses the inhibition. These results indicate that the phorbol diester-mediated process of down-regulation of the surface transferrin receptor is associated with phosphorylation of the receptor by activated protein kinase C and requires an intact cytoskeleton to affect receptor internalization.