Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.
We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.
Hefner
Y
Y
Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Borsch-Haubold
A G
AG
Murakami
M
M
Wilde
J I
JI
Pasquet
S
S
Schieltz
D
D
Ghomashchi
F
F
Yates
J R
JR
Armstrong
C G
CG
Paterson
A
A
Cohen
P
P
Fukunaga
R
R
Hunter
T
T
Kudo
I
I
Watson
S P
SP
Gelb
M H
MH
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D012694:Serine; C491395:MKNK1 protein, human; D017346:Protein Serine-Threonine Kinases; D010741:Phospholipases A; D054467:Phospholipases A2