Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA

Abstract

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-di0leoyl-~sn-glycerol is observed in the absence of added Ca2 '. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca2 +-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glyceroI in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-.~n-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar Craction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2 + to high affinity sites on the enzyme.Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2 ' . There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.Protein kinase C (PKC) plays an important role in cell regulation and is the receptor for the phorbol ester tumor promoters. This protein kinase was first identified as a calcium-dependent and phospholipid-dependent enzyme [ l]. The phospholipid requirement for the enzyme is specific for anionic lipids with phosphatidylserine being most effective [2] but phosphatidic acid [2 -41, cardiolipin [3] and phosphatidylinositol [2] are also active. Calcium binds to PKC only in the presence of phospholipid [5] and the binding affinity for Ca2+ is increased with more phosphatidylserine in the membrane [6]. Diacylglycerols markedly increase the sensitivity of PKC to activation by Ca2+ in the presence of phospholipid [7]. In contrast to phosphatidylserine, we find that addition of 1,2-dioleoyl-sn-glycerol (Ole,Gro) to phosphatidic acid causes a calcium-induced inhibition, rather than activation, of the Correspondence to

Mechanism of Interaction of Protein Kinase C with Phorbol Esters

“…Similarly, because Ca 2ϩ and PS act allosterically (24), considerably less Ca 2ϩ is required to achieve the same level of binding to PMA-containing membranes compared with DG-containing membranes. Indeed, the affinity for PS, in the presence of DG, is sufficiently high that significant association occurs in the absence of Ca 2ϩ (24,36). Thus, the chelator resistance reflects the extraordinarily high affinity interaction with PMA-containing membranes.…”

Protein kinase C and phospholipase D: intimate interactions in intracellular signaling

“…In addition, several groups have described PA-responsive protein kinases that to date remain unidentified but that have been determined as separate from known members of the PKC superfamily [125][126][127]. Early in vitro studies investigating lipid regulation of the classical PKC isoenzymes demonstrated an ability of PA to substitute for PS in the activation of PKC [28,123,124]. Importantly, the potency and specificity of PA substitution for PS were dependent upon whether the assay was performed with Triton X-100 mixed micelles or with sonicated lipid vesicles containing lipid bilayers [28,128].…”

“…2). Although the physiological function of PA per se is not well understood, several in vitro and in vivo targets of PA have been identified, including PIP 5-kinase [119], PI-PLCb [120], Raf-1 [121], protein phosphatase 1 [122] and PKC itself [28,123,124]. In addition, several groups have described PA-responsive protein kinases that to date remain unidentified but that have been determined as separate from known members of the PKC superfamily [125][126][127].…”

G Protein Activation Stimulates Phospholipase D Signaling in Plants.

“…Although the lack of conviction about DAG signaling in plants is not an argument for the importance of PtdOH, its acceptance as a signal in animal cells, together with our demonstration of its biological activity in C. eugametos, means that it should now receive more attention from plant cell biologists. In animal cells, no consensus picture has yet formed about how PtdOH operates, but a number of recent reports describe a nove1 type of protein kinase that is specifically activated by this lipid ( Figure 1OB; Bocckino et al, 1991;Epand et al, 1992;Nakanishi and Exton, 1992;Stasek et al, 1993;Khan et al, 1994;Limatola et al, 1994). Another potentia1 site of action appears to be the PLC signaling cascade itself, because PtdOH has been found to activate PtdlnsP 5-kinase and PLC specifically (Jackowski and Rock, 1989;Kroll et al, 1989;Moritz et al, 1989;Hashizume et al, 1992;Jacob et al, 1993;Jones and Carpenter, 1993), in this way amplifying the PLC signaling cascade ( Figure 1OB).…”