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Identification of the Phosphorylation Sites of Cytosolic Phospholipase A2 in Agonist-stimulated Human Platelets and HeLa Cells
The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 P-phosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional ph...
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Published in: | The Journal of biological chemistry 1998-02, Vol.273 (8), p.4449-4458 |
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Main Authors: | , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 P-phosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic
phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA 2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets
and HeLa cells stimulated with interferon-α or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa
stress-activated protein kinase (p38 mapk ), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets.
An additional p38 mapk inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA 2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation
sites on cPLA 2 expressed in a baculovirus/insect cell system to cPLA 2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide
mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which
has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA 2 in different mammalian cells stimulated with multiple agonists. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.273.8.4449 |