The transition from quiescence (G 0 ) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at ϳ10 min, peaking at ϳ3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated ϳ3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until ϳ4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G 0 3 G 1 transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle.

The enzyme CTP:phosphocholine cytidylyltransferase (CT) binds reversibly to membranes and is active only in its membrane-bound form. Membrane lipid composition influences the equilibrium between its soluble and membrane-bound forms. Whereas the enzyme is not activated by phosphatidylcholine (PC) vesicles, it is activated by PC vesicles that have been oxidized with HClO(4) [Drobnies, A. E., et al. (1998) Biochim. Biophys. Acta 1393, 90-98]. Here we explore the mechanism of activation of CT by a PC oxidized with lipoxidase. Multilamellar vesicles (MLVs) containing > or =5 mol % oxidized 1-palmitoyl-2-arachidonoylPC (PAPC) progressively activated the enzyme, which was fully activated by 25 mol % oxidized PC. The effect of oxidized PAPC on lipid order was investigated by (2)H NMR, using MLVs containing PAPC perdeuterated on the palmitoyl chain. Spectral depaking generated order parameter profiles along the sn-1 chain. The average order parameter (S(CD)) in the plateau region at 37 degrees C decreased from 0.18 to 0.15 with increasing percent of oxidized PAPC (0-25%). The change in S(CD) was even greater near the end of the palmitoyl chain. CT activation was inversely related to lipid order. The major component of the lipoxidase-oxidized PAPC was purified and characterized by mass spectrometry and NMR. This component, 1-palmitoyl-2-(11,15-dihydroxy)eicosatrienoylPC (dihydroxyPAPC), incorporated into PAPC MLVs, also stimulated CT activity and reduced the lipid order parameter. Both effects were reversed by egg sphingomyelin. We propose that CT activation by oxidized PAPC is mediated by effects on lipid packing perturbations. This is the first study to report the effects of a purified oxidized PC on the orientational order along the acyl chain and to correlate the lipid disordering of the oxidized PC with the activation of a membrane-associated regulatory enzyme.

Protein kinase C (PKC) and CTP:phosphocholine cytidylyltransferase (CT) are two examples of enzymes that are regulated by reversible binding to membranes, and this binding is influenced by membrane physical properties. CT activation by oxidized phosphatidylcholines was recently demonstrated and was linked to the acyl chain disordering effect of the oxidized species (Biochemistry 38, 15606). In this paper, we compare the responses of PKC and CT to an oxidized PC, and investigate the physical properties of lipid bilayers that modulate the activity of these enzymes. We show that 1-palmitoyl, 2-(11,15 dihydroxy) eicosatrienoyl PC (diOH-PAPC) caused less of an increase in the temperature of the lamellar to hexagonal II transition (T(H)) of an unsaturated PE, compared to its parent, PAPC. Using a polarity-sensitive interfacial probe, we also found evidence to suggest that this oxidized PC increases interfacial packing pressure. We found that whereas diOH-PAPC activates CT, it inhibits PKC relative to the parent PAPC. The activities of both CT and PKC are known to increase in the presence of non-lamellar forming lipids. The greater activating effect of diOH-PAPC compared with PAPC, is consistent with a stimulation of the activity of CT by negative curvature strain. However, this is not the case with PKC, for which we suggest that surface packing pressure is of prime importance.

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