Mechanism of Regulation of Group IVA Phospholipase A2 Activity by Ser727 Phosphorylation

Abstract

Although group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) has been reported to be phosphorylated at multiple Ser residues, the mechanisms by which phosphorylation at different sites regulates cPLA 2 ␣ activities are not fully understood. To explore the possibility that phosphorylation of Ser 727 modulates cellular protein-protein interactions, we measured the effect of Ser 727 mutations on the interaction of cPLA 2 ␣ with a reported cPLA 2 ␣-binding protein, p11. In vitro activity assays and membrane binding measurements by surface plasmon resonance analysis showed that a heterotetramer (A2t) of p11 and annexin A2, but not p11 or annexin A2 alone, directly binds cPLA 2 ␣ via Ser 727 , which keeps the enzyme from binding the membrane and catalyzing the phospholipid hydrolysis. Phosphorylation of Ser 727 disrupts this inhibitory cPLA 2 ␣-A2t interaction, thereby activating cPLA 2 ␣. Subcellular translocation and activity measurements in HEK293 cells cotransfected with cPLA 2 ␣ and p11 also showed that p11, in the form of A2t, inhibits cPLA 2 ␣ by the same mechanism and that phosphorylation of Ser 727 activates cPLA 2 ␣ by interfering with the inhibitory cPLA 2 ␣-A2t interaction. Collectively, these studies provide new insight into the regulatory mechanism of cPLA 2 ␣ through Ser 727 phosphorylation.

Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways

“…Phosphorylation at this site might promote the dissociation of a heterotetramer of cPLA2, Annexin A2, and p11 and increases cPLA2 enzymatic activity. 42 As direct regulators of intracellular calcium, we found the calcium release-activated calcium channel Orai1 being dephosphorylated (Thr295: ↓60 2nM ) and the inositol phosphate receptor regulator MRVI1 (IRAG) increasingly phosphorylated at Ser657 and Ser670 (↑10 2/5nM ). Both sites are known to be PKG targets preventing the release of Ca 21 from inositol phosphate sensitive stores and subsequent platelet aggregation.…”

Cytosolic phospholipase A2: physiological function and role in disease

“…Activation of cPLA 2 ␣ in vascular smooth muscle cells by calcium/calmodulin-dependent kinase II that phosphorylates S515 has been described ( 101 ). Phosphorylation of cPLA 2 ␣ on S727 by MAPK-interacting kinase does not enhance catalytic activity per se, but blocks its binding to an inhibitory complex in the cytosol composed of p11(S100A10)/annexin A2 ( 74,(102)(103)(104)(105) ( Fig. 1 ).…”

Activation of Cytosolic Phospholipase A2-α as a Novel Mechanism Regulating Endothelial Cell Cycle Progression and Angiogenesis

“…The role that serine phosphorylation plays in regulating both cPLA 2 ␣ activity and its release from the Golgi apparatus requires defining and could represent an important target for modulating phospholipid turnover. This is highlighted by the recent finding that phosphorylation of cPLA 2 ␣ on serine 727 disrupted binding to p11 and annexin A2 allowing the enzyme to access its phospholipid substrate (41). Another unique function of endothelial cells is their ability to proliferate, migrate, and differentiate to form new capillaries during angiogenesis.…”