Evans John H, Fergus Daniel J, Leslie Christina C
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.
BMC Biochem. 2002 Oct 8;3:30. doi: 10.1186/1471-2091-3-30.
The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear.
MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126.
These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2.
85 kDa的胞质型磷脂酶A2（cPLA2）介导MDCK细胞中花生四烯酸（AA）的释放。尽管钙和丝裂原活化蛋白激酶调节cPLA2，但cPLA2易位和磷酸化与丝裂原活化蛋白激酶（MAPK）激活及AA释放之间的相关性尚不清楚。
U0126对MEK1的抑制作用抑制了ATP和离子霉素诱导的AA释放。这与ERK激活的抑制直接相关，但与cPLA2在Ser505位点的磷酸化无关，ERK抑制仅部分抑制该位点的磷酸化。当用茴香霉素激活p38维持cPLA2在Ser505位点的化学计量磷酸化时，仍可观察到U0126对AA释放的抑制作用。野生型cPLA2以及含有S505A或S727A突变的cPLA2对ATP和离子霉素的反应中向高尔基体的易位动力学相似，且不受U0126影响。
这些结果表明，MEK1/ERK途径的抑制降低了cPLA2水解膜磷脂的能力，且活性降低与cPLA2的磷酸化和向膜的易位无关。结果还表明，在磷酸化位点Ser505和Ser727发生突变的cPLA2与野生型cPLA2具有相似的易位动力学。
