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Departments of Pharmacology, Physiology and Radiology, School of Medicine, Department of Biomedical Engineering, College of Engineering, Wayne State University, Detroit, Michigan; Decision and Information Sciences Division, Argonne National Laboratory, Argonne, Illinois; Marine Biological Laboratory, Woods Hole, Massachusetts
1 Marine Biological Laboratory, Woods Hole, MA
The calcium signal required for nuclear envelope breakdown (NEB) and mitosis arises from a calcium-regulatory subset of the perinuclear endoplasmic reticulum (pn-ER) (1–4); further, this pre-NEB calcium signal is encoded in space-time and evoked by leukotriene B4 (LtB4) (4, 5), a downstream product of phospholipase A2 (PLA2). In fact, a calcium-independent phospholipase A2 (iPLA2) activity (6, 7), revealed by in vivo imaging with a confocal microscope (5), is also associated with the pn-ER and occurs just before the pre-NEB calcium signal (3, 5). If this iPLA2 activity is blocked, both the pre-NEB calcium signal and NEB itself are inhibited (5). We have also reported that phosphofructokinase (PFK) is present on that same calcium-regulatory subfraction of pn-ER (which we designated as subfraction B; 2, 5). PFK activity is known to be modulated by its downstream metabolic products; thus, this activity is up-regulated by AMP and ADP, and is down-regulated by ATP in excess over ADP, by ATP analogues with a ß-
phosphodiester bond that cannot be hydrolyzed (e.g., AMP-PCP), and by citrate. Finally, we previously demonstrated that the pn-ER subfraction that exhibits ATP-dependent calcium uptake activity (e.g., 2) has a non-mitochondrial creatine kinase activity that rapidly established and maintained an excess of ATP over ADP under conditions used in that study (8).
This set of related activities, all associated with the pn-ER, are reminiscent of various mechanisms involving a rapid and reversible co-regulatory interaction between iPLA2 and PFK, mediated by glycolytic products. Such mechanisms have been observed in cardiac muscle (9, 10), rat parotid secretory cells (11), pancreatic islet cells and pancreatic tumor cells (12, 13), and control of radiation repair (14). We therefore hypothesized that a reversible interaction between iPLA2 and PFK, modulated by the metabolic products of PFK activity, would also provide a metronome-like mechanism that would, in turn, control the series of biochemical events (3, 4, 5) leading to the pre-NEB calcium signal, NEB, and mitosis. To test this hypothesis, we have used, as a model system, pn-ER subfractions isolated from first and second cell cycle prophase cells obtained from the sand dollar (Echinaracnius parma). First, we assayed these subfractions for the effects of calcium levels on PLA2, and then we determined the effect of metabolic products of PFK on iPLA2 activity.
The pn-ER subfractions were isolated from prophase mitotic apparatus (MA) from first and second cell cycle cells, as previously described (2, 3, 6). Briefly, the prophase MA-associated pn-ER subfractions were resolved using ultracentrifugation at 140,000 x g on sucrose density gradients. Four pn-ER subfractions were collected at the interfaces between 0.25 M and 1.0 M sucrose, 1.0 M and 1.3 M sucrose, 1.3 M and 1.5 M sucrose, and 1.5 M and 2.0 M sucrose; these subfractions are designated A, B, C and D, respectively. The sucrose solutions were buffered with 100 mM KCl, 10 mM NaCl, 30 mM imidazole, 5 mM MgCl2; and pH was adjusted to 7.20.
The assay for PLA2 activity was significantly modified from that described previously (7). The reactions were performed in 100 mM KCl, 10 mM NaCl, 30 mM imidazole, 5 mM MgCl2, 4 mM CHAPS, 30% (v/v) glycerol, pH 7.20, with a reaction volume of 500 µl; the substrate stock solution was 100 mM 1-hexadecyl-2-arachidonylthio-2-deoxy-sn-glycero-3-phosphorylcholine (arachidonyl-thio-PC) at 12° C. The reactions were stopped by the addition of 10 µl of a solution containing 1.0 mg/ml bromoenol lactone, 25 mM 5,5'-dithio-bis(2-nitrobenzoic acid, 500 mM EGTA, pH 7.20. A minimum of triplicate assays was performed on eight different pn-ER preparations; the mean values are presented. The standard deviations from the mean of PLA2 activity, measured for each subfraction at each calcium concentration, did not exceed 4.5% of the mean values. The effect of calcium was assessed by including CaCl2 in the reaction solutions to make a series of final reaction concentrations between 0 and 500 mM CaCl2 (Fig. 1A). The data for each pn-ER subfraction were expressed as specific activity per milligram protein as a function of calcium concentration in the reaction milieu (Fig. 1A).
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When no calcium was added, all pn-ER subfractions exhibited nearly identical levels of specific activity for PLA2 (Fig. 1A). Therefore, this PLA2 activity, which is independent of added calcium, is defined as an iPLA2 activity (8). Addition of 10 nM calcium to the reaction milieu decreased the PLA2 activity in each pn-ER subfraction, relative to iPLA2. In 30 nM calcium, the PLA2 activity in subfraction B was stimulated 3.5-fold relative to iPLA2 activity, but was reduced in subfractions A and C and only slightly elevated in D relative to the level of iPLA2. In the presence of 50 nM calcium, the PLA2 activity in all subfractions was again lower than that for iPLA2. In 100 nM calcium, PLA2 activities in subfraction B and D were stimulated 1.7-fold and 2-fold, respectively; but the activity was again lower in subfractions A and C. In 300 nM calcium, PLA2 activity declined in each subfraction relative to values for iPLA2 activity. Finally, in 500 nM calcium, activities for subfractions A and B were roughly one-third that of iPLA2, while values for subfractions C and D were similar to those observed for iPLA2 activities in those subfractions. In summary, clear increases in calcium-dependent PLA2 (cPLA2) activities, relative to iPLA2 activities, were observed in subfraction B in 30 nM and 100 nM calcium, and in D in 100 nM calcium. As for the other fractions, substantial reductions in cPLA2 activity relative to iPLA2 activities in all subfractions, were observed at 10 nM, 50 nM and 300 nM calcium.
Among downstream metabolic products of PFK, citrate increased iPLA2 activity nearly 1.7-fold over control values (Fig. 1B). In contrast, AMP and ADP, known up-regulators of PFK activity, decreased iPLA2 activity to 10% and 84.9% of control values, respectively. In addition, we observed that addition of ADP and creatine phosphate (and thus ATP), and AMP-PCP resulted in marked elevations of iPLA2 activity. The iPLA2 activity for ADP + creatine phosphate was about 1.4-fold greater than controls; the iPLA2 activity for AMP-PCP was about 2-fold greater than controls (Fig. 1B).
We have found, in this study, that the pn-ER of prophase MA contains both calcium-independent (iPLA2), and calcium-dependent PLA2 (cPLA2) activities (Fig. 1A). Addition of calcium to 30 nM or 100 nM appears to stimulate PLA2 activity in pn-ER subfraction B. Addition of calcium to 100 nM also appears to stimulate PLA2 activity in pn-ER subfraction D. Our discovery of a 30 nM cPLA2 activity in pn-ER subfraction B may indicate a type of calcium-modulated PLA2 activity not previously reported. Our finding of a 100 nM cPLA2 activity is consistent with the requirement for 80 to 100 nM calcium for cPLA2 in other systems (7).
The reciprocal modulation of iPLA2 by metabolic by-products of PFK supports our notion that an interaction between iPLA2 and PFK could regulate perinuclear iPLA2. The reduction of iPLA2 activities upon exposure to AMP or ADP may represent a modulator-induced disassociation of iPLA2 and PFK that had remained associated during the isolation of the MA and the preparation of the pn-ER. The pattern of responses is consistent with a transient regulatory interaction of PFK and iPLA2 (Fig. 1C).
These results confirm and extend our earlier observations that the pn-ER exhibits an iPLA2 activity that is necessary for the pre-NEB calcium signal and NEB. These results, taken together with our earlier modeling efforts (4, 5) further suggest that a potential regulatory interaction between iPLA2 and PFK may be used to coordinately regulate iPLA2 activity, the production of arachidonic acid, LtB4 and thus calcium signals, and glycolysis (4, 5). Moreover, this interaction would also impact the pentose phosphate pathway, and control of glutathione red/ox state (4, 5). This would provide an essential level of feedback regulation both within and among the metabolic pathways that have been described as the integrated metabolic network present on the calcium regulatory subfraction of the pn-ER (4, 5).
At a whole cell scale, such a mechanism would involve rapid and extensive changes in metabolite levels, which are not observed. However, evidence for this regulatory mechanism has been observed to operate on vesicles within microdomains (very restricted spaces), where levels of calcium and other metabolites can vary over several orders of magnitude of concentration in a millisecond timescale (e.g., 3, 15, 16, 17). Conditions that favor PFK activity would contribute to the disassociation of PFK from iPLA2 (Fig. 1C, left), while increases in local levels of downstream products of PFK activity that exert feedback inhibition on PFK would promote reassociation of PFK with iPLA2, thereby activating iPLA2 activity, and thus leading to a calcium signal. Localized decrease in ATP levels, due to calcium pump or calcium-dependent kinase activities, would deplete local ATP levels, and thus activate PFK, which in turn, would inhibit iPLA2 activity (Fig. 1C, right).
The metabolically modulated interaction between iPLA2 and PFK would also provide a limit oscillator on individual vesicles within microdomains that would coordinately control calcium signals and glucose-6-phosphate metabolism. Such a mechanism would be switched by localized changes in levels of ATP or citrate or other modulators of PFK activity. Thus, a coded localized calcium signal could be evoked and terminated by changes in the level of metabolites within an individual microdomain. The combination of iPLA2, 30 nM cPLA2, and 100 nM cPLA2 activities may provide the mitotic cell with an amplitude-selective notch filter mechanism for producing arachidonic acid, LtB4 and a space-time coded calcium signal. Such a mechanism would provide a "metronome" type switch, dependent upon local metabolic conditions, that would regulate localized space-time encoded calcium signals during a brief, defined period in the cell cycle, as exemplified by the pre-NEB calcium signal (1–5).
Research grant support by NSF (MCB-99082680) and Wayne State University is gratefully acknowledged. Thanks are extended to Drs. John R. Hummel, George D. Pappas, Howard Rasmussen, Raoul F. Reiser, and Stephen M. Wolniak for their helpful comments and advice during these studies. We also thank the anonymous reviewers for their helpful suggestions in the refinement of the manuscript.
Literature Cited
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