Olivia L. May, Ph.D.

Phospholipase A₂ (PLA₂) catalyzes the hydrolysis of the middle (sn-2) ester bond of substrate phosphospholipids to release free fatty acids and lysophospholipids. The fatty acids may then be converted to various bioactive lipids. Transforming membrane fatty acids into powerful signaling mediators provides a means for cells to quickly (and transiently) respond to a battery of stimuli. PLA₂ enzymes are classically organized into four main types: secreted (sPLA₂), cytosolic (cPLA₂), Ca²⁺-independent (iPLA₂), and lipoprotein-associated (LpPLA₂ or PAF-AH). Of these, cPLA₂ is readily the most essential for eicosanoid biosynthesis since this ubiquitously-expressed enzyme shows specificity for phospholipids containing arachidonic acid.¹iPLA₂ is more involved in membrane homeostasis and energy metabolism. sPLA₂ is known to regulate extracellular phospholipids but is also expressed in macrophages and epithelial cells where they too may participate in lipid mediator generation. Lysosomal PLA₂ (LPLA₂) and adipose-specific PLA₂(AdPLA₂) represent two more recently identified PLAs whose classifications are based on specific location. While cPLA₂ is constitutively expressed, certain PLAs are activated in distinct cell populations to contribute to the supply of lipid mediator precursors needed for each varied function. The in vivo biological functions for each PLA₂ type continue to be surmised with the emergence of transgenic and knockout mouse strains, the use of specific inhibitors (see page 47 for PLA₂ inhibitors available from Cayman), and the analysis of human diseases caused by PLA₂ gene mutations. Many of these functions have been thoroughly detailed in a recent review by Murakami, et al., 2011.¹ This article will delve into the mechanics of activation and potential cooperation between the two intracellular PLA₂s: cPLA₂α and iPLA₂β.

cPLA₂ structure and function

While 6 isoforms have been identified in the cPLA₂ family, group IVA cPLA₂α remains the most extensively studied. It acts intracellularly, releasing arachidonic acid from perinuclear membrane phospholipids in response to calcium binding, and thus, initiating eicosanoid production. cPLA₂α has been shown to contribute to the airway anaphylactic response, rheumatoid arthritis, bone resorption, autoimmune encephalomyelitis, long term depression, cerebral ischemia/reperfusion, neurodegeneration, cancer, induction of parturition, platelet activation, gastrointestinal and renal tract protection, and negative regulation of muscle growth.¹ Beyond lipid mediator production, roles for cPLA₂α in Golgi transmembrane junction protein trafficking, NADPH oxidase activation in phagocytes, and inhibition of synaptic facilitation via caveolin-1 have emerged.¹

cPLA₂α activation

All cPLA₂ isoforms (except for cPLA₂γ) have a C2 domain at their N-terminal region (Figure 1). This domain is essential for perinuclear membrane translocation from the cytosol in response to calcium (Ca²⁺) and the subsequent release of arachidonic acid. Binding of two ions of Ca²⁺ to the C2 domain reduces the negative electrostatic potential of surface exposed Ca²⁺- binding loops. This enables a set of hydrophobic residues in the C2 domain, which are important for lipid specificity, to penetrate a phosphatidylcholine (PC)-rich membrane surface, thereby presenting the catalytic domain of cPLA₂ in a position where it can be activated (Figure 2). The catalytic domain contains a catalytic dyad consisting of Ser²²⁸and Asp⁵⁴⁹ that is located at the bottom of a deep active site channel (Figure 1). This narrow conduit is lined with hydrophobic residues and forms a pocket to which fatty acyl moieties of phospholipids bind. While the sn-2 ester bond of membrane phospholipids is under nucleophilic attack by Ser²²⁸, the catalytic center is activated by Asp⁵⁴⁹ when a change in conformation moves the loop, referred to as the lid region, covering the active site.²

Figure 1. cPLA₂α and iPLA₂β protein domains and cPLA₂α crystal structure. The first comprehensive structural model of iPLA₂β was proposed by Hsu et al., 2009.⁶ However, its crystal structure remains to be solved.

Beyond the initial jump start from calcium signaling to pry open the active site lid, MAPK phosphorylation at Ser⁵⁰⁵, which is located near a flexible interdomain linker that connects the C2 and catalytic domains, is required for sustained activation. Phosphorylation at Ser⁵⁰⁵induces a conformational change that promotes further membrane penetration of the hydrophobic residues and allows a patch of basic residues in the catalytic domain to specifically bind to PIP₂, which ultimately controls arachidonic acid release (Figure 2). Proximal to PIP₂binding, a certain set of basic residues in the C2 domain interact with ceramide-1-phosphate (C1P), which is thought to be required for agonist-induced translocation of cPLA₂α to the membrane. In all, these hydrophobic interactions reinforce cPLA₂α membrane contact even in the face of transient or submicromolar fluxes of Ca²⁺ availability. Hence, while initial association of cPLA₂α with the membrane requires Ca²⁺ activation of the C2 domain, the catalytic domain is capable of remaining attached to the membrane even after a decrease in calcium levels. Finally, maximum activation of cPLA₂α occurs when MAPK-activated kinases (MAPKAPKs) phosphorylate Ser⁷²⁷, disrupting an inhibitory interaction of Ser⁷²⁷ with p11/annexin A2 complexes that routinely prevent full binding of cPLA₂α to the membrane.

Figure 2. Ca²⁺-dependent, C2 domain-directed translocation of cPLA₂α to release arachidonic acid.

iPLA₂ structure and function

To date, 9 isoforms of iPLA₂ or patatin-like phospholipase domain-containing lipases (PNPLA) have been identified. Most of the members of this family, however, function as triglyceride lipases rather than phospholipases. Like cPLA₂, iPLA₂ also is located intracellularly, yet it is calcium independent. While iPLA₂ can generate lipid mediators from phospholipids, it acts on a diverse set of substrates including triglycerides and retinol esters. iPLA₂β (group VIA PLA₂ or PNPLA9) is the best described. It largely functions to regulate cell membrane homeostasis by participating in phospholipid remodeling through the deacetylation of phospholipids in the Land's cycle. A role for iPLA₂β has also been revealed in signaling leading to cell activation, proliferation, migration, and apoptosis. Additionally, iPLA₂β has been shown to be involved in regulating lipid metabolism, intracellular calcium homeostasis (which will be discussed further below), vascular contraction/relaxation, bone formation, sperm development, and glucose-induced insulin secretion.¹

iPLA₂β activation

iPLA₂β contains a binding site for calmodulin (CaM) in its C-terminus (Figure 1) that (along with an IQ motif ), forms a pocket enabling CaM to bind and inhibit iPLA₂β activity. The CaM-iPLA₂β complex forms in the absence of calcium, preventing iPLA₂β activity. When CaM is not present, the active site of iPLA₂β interacts with the CaM-binding domain leading to a catalytically active enzyme. How, then, is this Ca²⁺-independent phospholipase functional if inactive while bound to a calcium modulated protein? The answer is related to store-operated channels (SOC) and store-operated Ca²⁺ entry (SOCE).

SOC and SOCE are activated by depletion of endoplasmic reticulum (ER) Ca²⁺ stores (Figure 3). This depletion is detected by STIM1, a protein located in the ER membrane that binds calcium in the ER lumen, functioning as a low-affinity Ca²⁺ sensor. When ER calcium is depleted, STIM1 triggers a cascade of reactions that leads to the activation of the plasma membrane channel, Orai1 (CRACM1). Orai1 has been shown to form a SOC that is activated exclusively upon depletion of calcium. ³

Figure 3. Depletion of intracellular calcium stores triggers iPLA₂β-activation of store-operated Ca²⁺ entry (SOCE) channels and Ca²⁺ influx.

STIM1 expression is tightly coupled with the production of a Ca²⁺influx factor (CIF) that is generated in the ER when intraluminal calcium concentrations drop. CIF has been shown to displace inhibitory CaM from iPLA₂β.⁴ This would enable iPLA2ß to move from the cytosol to the membrane where it can generate lysophospholipids, that in turn activate Orai1-dependent SOC channels that allow Ca²⁺entry. Calcium influx is terminated when ER stores are refilled (as monitored by STIM1), terminating CIF production, which enables calmodulin to rebind to iPLA₂β.

cPLA₂α and iPLA₂β cooperation

Whereas the entry of extracellular Ca²⁺ through the SOCE pathway is crucial for cPLA₂α activation, iPLA₂β-mediated activation of SOCE may likely lie upstream of Ca²⁺-dependent activation of cPLA₂α. Moon et al,. 2008 have used iPLA₂β knockout mice to demonstrate that iPLA₂β is required for the initial phase of arachidonic acid release during calcium store depletion-induced Ca²⁺ entry or ionophore stimulation in aortic smooth muscle cells.⁵ Furthermore, only the late phase (and not the initial phase) of arachidonic release from wild type cells was shown to be reduced by inhibition of cPLA₂α.⁵ Thus, Ca²⁺influx initiated by iPLA₂β activation could potentially facilitate subsequent activation of cPLA₂α, enhancing arachidonic acid release. More research will be necessary in order to determine the temporal details of this proposed integration.

References

1. Murakami, M., Taketomi, Y., Miki, Y., et al.Prog. Lipid Res. 50(2), 152-192 (2011).

2. Burke, J.E. and Dennis, E.A. J. Lipid Res. 50, S237-S242 (2009).

3. Bolotina, V.M. J. Physiol. 586(13), 3035-3042 (2008).

4. Bolotina, V.M. and Csutora, P. Trends Biochem. Sci. 30(7), 378-387 (2005).

5. Moon, S.H., Jenkins, C.M., Mancuso, D.J., et al. J. Biol. Chem. 283(49), 33975-33987 (2008).

6. Hsu, Y.-H., Burke, J.E., Li, S., et al. J. Biol. Chem. 284(35), 23652-23661 (2009).

Two excellent reviews detailing emerging roles for sPLA₂:

Murakami, M. and Lambeau, G. Biochimie 95(1), 43-50 (2013).

Murakami, M., Taketomi, Y., Girard, C., et al.Biochimie 92(6), 561-582 (2010).