Contributions to Maxima in Protein Kinase C Activation

Abstract

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 ؋ 50 Å at low mol % PS to 75 ؋ 75 Å at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.Protein kinase C (PKC) 1 (1), which constitutes a family of structurally related kinases, is defined by the phospholipid dependence of its activity, with all isozymes activated by acidic phospholipids, preferably phosphatidylserine (PS), and all but PKC and PKC/ further stimulated by diacylglycerols (DAG) or phorbol esters. The originally identified ␣, ␤, and ␥ family members also require calcium binding to the C2 domain, which is distinct in these isozymes (for reviews, see Refs. 2-4). Although the role of some lipid components is becoming better defined, variable results in different systems leave many questions unanswered.Structural analyses of DAG and phorbol ester analogues (5-7) and analysis of PKC deletions and mutations argue for specific phorbol ester and DAG binding to the Cys-rich, zinccontaining C1 domain (8 -11). X-ray fluorescence (12), NMR (13), and x-ray crystallography (14) have revealed the structure of this domain. Binding of phorbol ester does not alter the conformation, but helps to present a hydrophobic surface on the domain that could facilitate insertion into the membrane to a depth of 7-8 Å (14). The two versions of the C1 domain, C1A and C1B, present in most PKC isozymes have structural differences (reviewed in Ref. 15) that may account for greatly differing affinities of fluorescent phorbol e...

Bile acids stimulate PKCα autophosphorylation and activation: role in the attenuation of prostaglandin E1-induced cAMP production in human dermal fibroblasts

“…These authors proposed that the decreased activity observed at high lipid concentrations was due to the dilution of PKC dimers or higher aggregates with greater kinase activity. However, in contrast to the present study, the decrease in PKC activity was associated with a decreased autophosphorylation (54). of CDCA in the absence or presence of 5 g PS.…”

“…This latter observation could explain, at least in part, the results in the present study that the increased phosphorylation of PKC␣ by CDCA, although not leading to a direct activation of this kinase, increases the affinity and, in turn, the activation of PKC␣ by PMA. Sando et al (54) have observed a correlation between autophosphorylation and activation of PKC with a lipid-dependent, normally distributed activation pattern. These authors proposed that the decreased activity observed at high lipid concentrations was due to the dilution of PKC dimers or higher aggregates with greater kinase activity.…”

Regulation of PKCα Activity by C1-C2 Domain Interactions

“…This is consistent with a concentration-dependent equilibrium between monomeric and dimeric forms of PKC␣. Evidence supporting the notion that PKC may be active in PKC-PKC and/or PKC-substrate complexes has also been provided elsewhere (55)(56)(57)(58)(59)(60). Furthermore, evidence that PKC may become active upon self-association in the cellular environment was presented recently based on the observation of PKC␣ dimers in lysates derived from murine B82L fibroblasts treated with calcium ionophore, phorbol ester, or epidermal growth factor (60).…”

Characterization of the Interaction of Ingenol 3-Angelate with Protein Kinase C

“…ingenol 3-angelate could not cause the same degree of association of the C1b domain with lipid that is induced by DAG or PMA. The basis of the decrease in association of the C1b domain at high ligand concentrations is uncertain, but optimal concentrations of different activators for maximal activation have been reported previously (52,53), suggesting submaximal effects at higher ligand concentrations. It clearly does not appear to reflect a major change in the composition of the bulk lipid phase, because the incorporation of the ligand into the liposome in the absence of receptor was modest (see Fig.…”