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 x 50 A at low mol % PS to 75 x 75 A 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.
Highlights
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
Recent experiments in a DMPC/DMPS/DAG system revealed a peak in PKC activity at DAG mol fractions that corresponded to a mixture of compositionally distinct lipid domains as determined by differential scanning calorimetry [32, 33]
Potential explanations for the decrease in activity at high mol % DAG included (a) some requirement for interface regions between domains that would be maximal under maximal domain coexistence conditions; (b) a possible dilution of PKC multimers or PKC-substrate aggregates as an optimal domain is increased; and (c) the decrease in mol % PS that would occur as DAG was increased at the expense of PC and PS in those studies
Summary
(Received for publication, September 3, 1998, and in revised form, September 24, 1998). Formation of two-dimensional crystals of PKC on lipid monolayers 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 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. Goldberg and Zidovetski [31] observed similar maxima as a function of DAG in unsaturated systems and noted a correlation between increasing activity and increasing frustration of the bilayer, but decreasing activity as bilayers were disrupted to form a HexII phase All of these lipid systems are complex, and the increase of one component is always at the expense of another or accompanied by a change in total lipid concentration.
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