Phosphorylation of cytosolic phospholipase A2 at serine 505 is dependent upon its phosphorylation at serine 515 in response to norepinephrine in vascular smooth muscle cells

Cytosolic phospholipase A 2 (cPLA 2 ) is activated by phosphorylation at S‐505 by ERK1/2. However, we have shown that rat brain CaMKII can phosphorylate recombinant cPLA 2 at S‐515 in vitro . The present study was conducted to determine if phosphorylation of cPLA 2 at S‐505 is mediated through its phosphorylation at S‐515 in rabbit aortic smooth muscle cells stimulated with norepinephrine (NE). We used specific phos‐S‐515 and phos‐S‐505 cPLA 2 antibodies and adenoviruses carrying ECFPcPLA 2 wt and its mutants S515A and S505A. NE 10 μM, increased phosphorylation of cPLA 2 at S‐515 followed by phosphorylation of ERK1/2 and further cPLA 2 S‐505 phosphorylation. KN‐93, an inhibitor of CaMKII but not KN‐92, attenuated phosphorylation of cPLA 2 at S‐515 and S‐505 and that of ERK1/2. U0126, an inhibitor of ERK1/2, reduced phosphorylation of cPLA 2 at S‐505 but not at S‐515. Furthermore, in cells transduced with Ad ECFPcPLA 2 wt, NE caused phosphorylation of cPLA2 at S‐515 and S‐505 and increased AA release, whereas S515A, S505A and S515A/S505A mutants reduced phosphorylation of cPLA 2 at S‐505 and S‐515 and ERK1/2 and AA release. These data suggest that phosphorylation and activation of cPLA 2 at S‐505 by ERK1/2 is mediated by its phosphorylation at S‐515 by CaMKII. This work was supported by NIH‐HLBI Grant 19134‐32.