Localization and function of cytosolic phospholipase A2alpha at the Golgi

Abstract

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha, Group IVA phospholipase A(2)) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA(2)alpha translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme's calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA(2)alpha translocation to the Golgi. However, other features of cPLA(2)alpha regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA(2)alpha C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA(2)alpha is targeted. Increasing evidence strongly suggests that cPLA(2)alpha plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA(2)alpha regulates the transport of tight junction and adherens junction proteins through the Golgi to cell-cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA(2)alpha affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA(2)alpha deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA(2)alpha. Thus, there is continued need for studies employing highly specific cPLA(2)alpha antagonists in addition to genetic deletion of cPLA(2)alpha in mice.

Fig. 1. Proposed mechanism of cPLA₂α localization and function on the Golgi

The N-terminal C2 domain of cPLA₂α is attached to a large catalytic domain that contains the catalytic site Ser/Asp dyad, and the sites phosphorylated by MAPKs (Ser505) and MAPK-interacting kinases (Ser727). The hydroxyl group of Ser727 interacts with p11/Annexin A2 complexes maintaining cPLA₂α in an inactive state. Phosphorylation of Ser727 causes disassociation of the bulky p11/Annexin A2 complex allowing the calcium-dependent interaction of cPLA₂α with the Golgi membrane. Calcium binding to the cPLA₂α C2 domain reduces the negative electrostatic potential of the surface exposed CBLs allowing the surrounding hydrophobic residues (green) in CBL1 and CBL3 to penetrate the membrane. The basic residues (R57/K58/R59) (yellow) in the C2 domain of cPLA₂α form the proposed site for interaction with C-1-P. Calcium-dependent binding of cPLA₂α to the Golgi positions the catalytic domain on the membrane, which is stabilized by interaction of Trp464 (red) in the catalytic domain with the membrane. There is evidence that association of cPLA₂α with the Golgi is influenced by changes in cholesterol content. Phosphorylation at Ser505 increases the hydrolytic activity of cPLA₂α on the membrane perhaps by promoting a conformational change due to its proximity to the flexible linker that connects the catalytic and C2 domains. A patch of basic residues (K488/K541/K543/K544) (teal) in the catalytic domain also regulates the ability of cPLA₂α to release arachidonic acid from the Golgi. These residues are necessary for activation of cPLA₂α in vitro by polyphosphoinositides, however, the endogenous anionic components in the Golgi that interact with this basic site have not been identified. Therefore the ability of cPLA₂α to release arachidonic acid (AA) and form lysophospholipids in the Golgi involves increases in calcium, phosphorylation and interaction of basic residues with anionic components in the membrane. Lysophospholipids generated at the rims of the Golgi cisternae by cPLA₂α are thought to induce positive membrane curvature for formation of tubules that connect the Golgi stacks and promote intra-Golgi transport. Surface representation of the x-ray crystal structure of cPLA₂α (PDB: 1CJY) was made using PYMOL.