Mechanism of Interaction of Protein Kinase C with Phorbol Esters

Abstract

A variety of approaches have been employed to demonstrate that the interaction of protein kinase C ␤II with phorbol ester-containing membranes is reversible, is not accompanied by significant insertion of the protein into the hydrophobic core of the membrane, and is qualitatively similar to the interaction with diacylglycerol (DG). First, we show that under conditions when protein kinase C is bound with equal affinity to membranes containing either DG or phorbol myristate acetate (PMA), increasing ionic strength causes a similar reduction in membrane binding. The similar sensitivity to ionic strength indicates that the forces mediating the binding of protein kinase C to PMA are not significantly different from those mediating the binding to DG. At sufficiently high concentrations of PMA and relatively low concentrations of phosphatidylserine, the binding of protein kinase C to membranes became markedly less sensitive to ionic strength, suggesting that under these conditions direct non-electrostatic interactions with PMA dominate over electrostatic interactions with the lipid headgroups. Importantly, regardless of the strength of the interaction with PMA, protein kinase C exchanges between vesicle surfaces: protein kinase C bound first to phorbol ester-containing multilamellar vesicles exchanged to large unilamellar vesicles upon addition of an excess surface area of the latter. Lastly, the enzyme's intrinsic tryptophan fluorescence was not quenched by bromines located at various positions in the hydrophobic core of the membrane. In contrast, the enzyme's tryptophan fluorescence was significantly quenched by probes positioned at the membrane surface. In summary, our results are consistent with protein kinase C binding reversibly to PMA-or DG-containing membranes primarily via interactions at the membrane interface. Generation of diacylglycerol (DG)1 in the plasma membrane is the key signal in activating most members of the lipidregulated family of protein kinase C isozymes (1, 2). This lipid second messenger has recently been shown to regulate protein kinase Cs function by dramatically increasing the enzyme's affinity for phosphatidylserine (PS)-containing membranes (3-5). The resulting high affinity membrane interaction leads to a conformational change that activates the enzyme: specifically, the autoinhibitory pseudosubstrate domain of the molecule (6) is removed from the active site, thus allowing substrate binding and catalysis (7,8).Phorbol esters are functional analogues of DG: they compete for the same binding site on the molecule as DG (9), and they can replace DG in activating the enzyme (10, 11). Unlike DGs, which are metabolized within minutes (12, 13), phorbol esters are long lived in the cell. As a result, these molecules have proved invaluable in activating protein kinase C in situ, and a plethora of studies over the past decade have described the phorbol ester-dependent translocation of protein kinase C to cellular membranes (11,14). However, several reports have questioned whether phorbol esters r...

Bioactivity of phytochemicals in some lesser-known plants and their effects and potential applications in livestock and aquaculture production systems

“…At least six phorbol esters are present in jatropha seeds (Haas and Mittelbach, 2000). The phorbol esters are reported to mimic the action of diacyl glycerol, an activator of protein kinase C, which regulates different signal transduction pathways (Mosior and Newton, 1995). Interference with the activity of protein kinase C affects a number of processes including, phospholipid and protein synthesis, enzyme activities, DNA synthesis, phosphorylation of proteins, cell differentiation and gene expression.…”

How Mannheimia haemolytica defeats host defence through a kiss of death mechanism

“…For example, phorbol myristate acetate (PMA), a mimetic of diacylglycerol that activates the protein kinase C [50,132,136], increases the expression of LFA-1 from normal bovines but not from BLAD animals, because the mutant CD18 is no more able to bind any CD11. These findings indicate that the expression of CD18 by bovine neutrophils is a dynamic system, capable of rapidly responding to inflammatory stimuli by increasing surface expression of CD18 [33,62].…”

“…4). DAG is also known to activate the protein kinase C (PKC) family in a variety of cell systems [12,132,136]. Phorbol esters, such as phorbol myristate acetate (PMA), can substitute for DAG in activating PKC [132] in a prolonged action, since phorbol esters are not readily metabolised.…”

“…DAG is also known to activate the protein kinase C (PKC) family in a variety of cell systems [12,132,136]. Phorbol esters, such as phorbol myristate acetate (PMA), can substitute for DAG in activating PKC [132] in a prolonged action, since phorbol esters are not readily metabolised. PMA rapidly induces NF-κB translocation into the nucleus [104] and increases the expression of LFA-1 from bovine neutrophils, leading to enlarge the disease by increasing the number of LKT binding sites (Fig.…”

Historical review of research on protein kinase C in learning and memory

“…In this respect it is worth noting that the suggestion that prolonged translocation of PKC from the soluble to an integral membrane protein form plays an important role in memory processes (as described in section 3.3) has recently been questioned. The doubt is primarily based on the observation of rapid redistribution of phorbol ester-induced membrane-associated PKC back to the cytosol after the dissociation of the applied phorbol esters in vivo (Szallasi et al, 1994;Mosior and Newton, 1995).…”