Role of Phosphorylation and Basic Residues in the Catalytic Domain of Cytosolic Phospholipase A2α in Regulating Interfacial Kinetics and Binding and Cellular Function

Abstract

Group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA 2 ␣ (IMLF ؊/؊ ) were reconstituted with wild type and cPLA 2 ␣ mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA 2 ␣ translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA 2 ␣ to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA 2 ␣ to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF ؊/؊ expressing phosphorylation site (S505A) and PIP 2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA 2 ␣, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA 2 ␣ reduced activation by PIP 2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA 2 ␣ hydrolytic activity.

Critical role of cPLA2 in Aβ oligomer-induced neurodegeneration and memory deficit

“…Increase in free AA released by cPLA 2 in response to A␤ oligomers could therefore be involved in synaptic dysfunctions. Moreover, a fraction of cPLA 2 binds to Golgi apparatus in response to calcium and its activity is increased by Ser 505 phosphorylation (Tucker et al, 2009). Previous reports showed that cPLA 2 activity modifies the transport of several proteins between Golgi vesicles and plasma membranes in nonneuronal cell types (Choukroun et al, 2000;Regan-Klapisz et al, 2009).…”

The molecular basis of ceramide-1-phosphate recognition by C2 domains

“…HEPES, pH 7.4, 10 mM, containing 0.16 M KCl and 500 nM Ca 2+ , where and catalytic domain, respectively. Although the cationic site in the catalytic domain is promiscuous in anionic lipid binding ( 21,23,26 ), C1P is the only membrane-embedded anionic lipid that has been shown to increase membrane affi nity of the cPLA 2 ␣ C2 domain ( 19 ). Thus, it is thought that C1P acts as a coincidence detector ( 27 ) to promote the membrane docking of the C2 domain through elongating the membrane residence time ( 19,24,28 ).…”

Phospholipase A2IVα Regulates Phagocytosis Independent of Its Enzymatic Activity

“…The role of the phosphorylation of PLA 2 IV␣ in induction of membrane translocation is not fully defined, although a requirement for PLA 2 IV␣ phosphorylation for its full enzymatic activation has been reported by different groups (15,21,22). Serine phosphorylation on PLA 2 IV␣ (Ser-505 or Ser-727) is mediated mainly by the mitogen-activated protein kinases (MAPKs) ERK1/2, and by the stress kinases p38 and JNK, and this has been shown to increase the intrinsic enzymatic activity of PLA 2 IV␣ (23,24).…”

“…However, this has been revised through more recent studies that have shown that the cationic ␤-groove of the C2 domain can bind to ceramide 1-phosphate on internal membranes (13). Furthermore, the cationic cluster of the PLA 2 IV␣ catalytic domain (Lys-488, Lys-541, Lys-543, and Lys-544) can bind to anionic phospholipids, although the requirement for this interaction for membrane translocation or regulation of enzymatic activity is still under debate (14,15). The majority of these studies have documented translocation of PLA 2 IV␣ to internal cell membranes, such as the nuclear envelope, Golgi complex, and endoplasmic reticulum, whereby PLA 2 IV␣ contributes to the structure and function of these cell compartments (16 -18).…”