A A2-stimulating protein regulated by protein kinase C in Aplysia neurons.

We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica. A similar protein is present in rat cerebral cortex. This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC). It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20. Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity. The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with [3H]arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC). PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E. coli alkaline phosphatase. These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons.

Recent studies indicate that arachidonic acid and its metabolites might act as second messengers in nerve cells. Several neurotransmitters 11'13"2°'26"2s stimulate the release of arachidonic acid in nervous tissue. Application of arachidonic acid, as well as several of its metabolites formed through the 12-1ipoxygenase pathway, modulate ion channels in identified Aplysia neurons 2'7'21'23. Arachtdonic acid may also act as a retrograde messenger in long-term potentiation in the vertebrate hippocampus, an example of synaptic plasticity implicated in the formation of memory 4. Since receptor-mediated activity of phospholipase A 2 (PLA2) is thought to be the chief mechanism for releasing arachidonic acid from membrane phospholipids, a protein that regulates this enzyme in nervous tissue could alter synaptic efficacy and play a role in both short-term and long-term plasticity.
Although the mechanisms are not understood, several proteins affect the activity of PLA 2. Lipocortins are inhibitory, and it has been suggested that this inhibition is diminished when they are phosphorylated 1'6"16 '17. Stimulatory proteins have also been isolated from nonneural mammalian cells, an M r 28,000 PLAE-stimulating protein, PLAP 5"9'1° and a group of proteins called lipokinins 15. We describe the partial purification of a similar stimulatory protein from Aplysta central ganglia with somewhat different characteristics.
When crude homogenates from 100-200 Aplysia gangha (25-50 animals) were subjected to ion-exchange chromatography, an activity that stimulates porcine pancreatic PLA 2 was eluted from the column in 0.3 M NaCI. We desalted and concentrated the proteins in this fraction for analysis by size-exclusion HPLC. Activity was eluted as one component with M r 30,000 (Fig. 1A).
The increase in specific activity was 600-fold with recovery of all of the activity present in the initial homogenate. This high recovery may be misleading because inhibitory lipocortins, which are present in crude homogenates (ref. 8, and data not shown), are removed during the purification. The stimulation of the vertebrate PLA 2 observed was quite modest (Table I), and we would expect greater activity toward substrates other than E. coli membranes. We have not yet developed a convenient assay with Aplysia PLA 2 as enzyme or molluscan membranes as substrate. No intrinsic PLA2 activity was present in the purified fraction. A distinct component with intrinsic phospholipase or lysophospholipase activity was eluted with a mobility of M r 15,000 (data not shown). Fractionation of a homogenate from rat brain by ionexchange chromatography followed by size-exclusion HPLC yielded similar results (Fig. 1B). The PLA 2- Aplysta neural tissue and rat brain. With both Aplysm and rat, most of the actw]ty, which was assayed as described in the legend to Table  I , and then freeze-dried The dried protein was taken up in 0 5 ml of water for injection into a BioSll TSK-250 column (300 x 7 5 mm, Biorad, Richmond, CA). Protein was eluted at a flow rate of 1 ml/mln with Tns-HCl, 0 1 M (pH 6.8) and PMSE 10/tM Size was determined by reference to the elutlon of protein standards (bovine serum albumin, ovalbumln, carbonic anhydrase and a-lactalbumm, Sigma) B: Rat The activity in 0 4 g cortical slices from forebrain or hippocampus of male Wistar rats (200-300 g) with a razor blade and homogenized as above, was partially purified by the same procedure stimulating activity is not confined to the nervous system: Aplysta buccal muscle contams the protein with approximately the same specific activity (data not shown).
The protem has no effect on phospholipase C.  (Table I) The PLA2-stlmulating activity from rat brain was also destroyed by boiling and by treatment with trypsin (not shown).  stlmulatory protein, we estimate that each ganglion contains 0.50 ng (about 0.05% of total protein).

SDS-PAGE also showed that the partially purified
After developing the large-scale purification of the PLAE-stimulating protein with 100-200 ganglia as starting material, we adapted the protocol for 8 pleural-pedal ganglia which were homogenized in 0.3 ml of the homogenization buffer but without Tween 20. The 16,000 g supernatant was then centrifuged for 90 mln at 100,000 g and the pellet resuspended in 125/,l of homogenization buffer containing 0.05% Tween 20. The extract was then centrifuged for 2 min at 16,000 g, and the supernatant injected into the HPLC column (omitting the column chromatography step on DEAE Sephadex used in the large scale protocol). When the detergent was absent in the homogenization buffer, essentially all of the PLA 2activating activity sedimented with the 100,000 g pellet, but is readily extracted by brief exposure to 0.05% Tween 20.
We observed that ganglia from Aplysta which had been anesthetized by injection of isotonic MgCl 2 usually contained much less PLAe-stimulating actwity than did ganglia from stressed animals. Since the synaptic discharge that occurs during the dissection to remove the central nervous system can cause the translocation of protein kinase C (PKC) to membrane and its activation, we suspected that this activity might be regulated by protein phosphorylation. Sacktor et al. 24 showed that, after treatment with phorbol ester, fractionated neural membranes contain a major phosphorylated component with an Mr of 30,000. We now find that the activity in extracts is increased when intact Aplysia ganglia are incubated with PDBu (Fig. 3). An inactive phorbol is ineffective.
As would be expected if protein phosphorylation activates the PLAz-stimulating protein, we found that the activity from unanesthetized ganglia is greatly decreased after incubation for 30 min at 37 °C in 0.1 M Tris-HCl (pH 10) with E. coli alkaline phosphatase conjugated to agarose (Sigma). In two experiments, we found a 72% decrease in activity as compared to a 25% loss after incubations with agarose alone. In vitro phosphorylation of the partially purified protein with bovine PKC also leads to the labeling of an M r 30,000 component (Fig. 4).