Activation of Protein Kinase C by Coexisting Diacylglycerol-Enriched and Diacylglycerol-Poor Lipid Domains

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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...

Section: Discussionsupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Contributions to Maxima in Protein Kinase C Activation

Sando¹,

Chertihin²,

Owens

et al. 1998

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“…In an in vitro system Goldberg et al (57) found that both SAG and sn-1,2-dioleoylglycerol (DO) gave pronounced activation of PKC, which seemed to be related to the increased tendency of SAG and DO to form non-bilayer lipid domains in ternary mixtures of saturated PC and PS (58), whereas other DAGs (dioctanoylglycerol, dipalmitoylglycerol, and oleoyl-acetylglycerol) gave little or no activation. Further studies by these authors (59) showed that SAG and DO also gave conformational changes in the phosphatidylcholine head groups that correlated with the DAG-induced activation of PKC. Studies by others (55,56) with large unilamellar vesicles of saturated PC and PS also indicated that PKC activation may be related to the interphases between DO-rich and DO-poor domains in ternary mixtures with PC and PS.…”

Section: Discussionmentioning

confidence: 92%

Physical Properties of the Transmembrane Signal Molecule, sn-1-Stearoyl 2-Arachidonoylglycerol

Hindenes¹,

Nerdal²,

Guo³

et al. 2000

sn-1,2-Diacylglycerol (DAG), a key intermediate in lipid metabolism, activates protein kinase C and is a fusogen. Phosphoinositides, the main sources of DAG in cell signaling, contain mostly stearoyl and arachidonoyl in the sn-1 and -2 positions, respectively. The polymorphic behavior of sn-1-stearoyl-2-arachidonoylglycerol (SAG) was studied by differential scanning calorimetry, x-ray powder diffraction, and solid state magic angle spinning (MAS) 13 C NMR. Three ␣ phases were found in the dry state. X-ray diffraction indicated that the acyl chains packed in a hexagonal array in the ␣ phase, and the two sub-␣ phases packed with pseudo-hexagonal symmetry. In the narrow angle range strong diffractions of ϳ31 and ϳ62 Å were present. High power protondecoupled MAS 13

“…Lipid lateral domain heterogeneity has been related to PKC activation by DAG (19,42). To investigate the effects of alcohols on the phase behavior of the lipid bilayers, we used DSC and examined the DMPC/DMPS system with octanol and pentanol (Fig.…”

Section: Octanol and Pentanol Change The Phase Behavior And Induce Lamentioning

confidence: 99%

Lipid-dependent Activation of Protein Kinase C-α by Normal Alcohols

Shen¹,

Chertihin

Biltonen

et al. 1999

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Significant stimulation of protein kinase C-␣ (PKC␣) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED 50 ) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity.31 P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding.