CLONING AND EXPRESSION OF CYTOSOLIC PHOSPHOLIPASE A(2) (CPLA(2)) AND A NATURALLY-OCCURRING VARIANT - PHOSPHORYLATION OF SER505 OF RECOMBINANT CPLA(2) BY P42 MITOGEN-ACTIVATED PROTEIN-KINASE RESULTS IN AN INCREASE IN SPECIFIC ACTIVITY
Rd. Gordon et al., CLONING AND EXPRESSION OF CYTOSOLIC PHOSPHOLIPASE A(2) (CPLA(2)) AND A NATURALLY-OCCURRING VARIANT - PHOSPHORYLATION OF SER505 OF RECOMBINANT CPLA(2) BY P42 MITOGEN-ACTIVATED PROTEIN-KINASE RESULTS IN AN INCREASE IN SPECIFIC ACTIVITY, European journal of biochemistry, 238(3), 1996, pp. 690-697
Full-length cytosolic phopholipase A(2) (cPLA(2)) was cloned from U937
cells and polymorphonuclear leukocytes (PMNLs) while a naturally occu
rring variant of cPLA(2) which lacks residues Val473-Ala749 but has a
C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA(2)) was cloned from
PMNLs and mononuclear leukocytes. We were unable to clone DcPLA(2), f
rom U937 cells. When cPLA, and DcPLA(2) were expressed in insect cells
, both proteins were detected in cell lysates by SDS/PAGE as single ba
nds of apparent molecular masses 100 kDa and 57 kDa, respectively Full
-length cPLA(2) was active in cPLA, and lysophospholipase assays while
DcPLA(2) was inactive in both assays. cPLA(2) was phosphorylated stoi
chiometrically by p42 mitogen-activated protein (MAP) kinase in vitro
at a similar rate to other physiological substrates of this protein ki
nase and the major site of phosphorylation was identified by amino aci
d sequencing as Ser505, [P-32]Ser(P)505 in cPLA(2) was only dephosphor
ylated at a slow rate by mammalian tissue homogenates. Protein phospha
tases 2A, 2B and 2C all contributed significantly to the overall depho
sphorylation of cPLA(2). The phosphorylation of cPLA(2) by p42 MAP kin
ase correlated with an approximately 1.5-fold increase in specific enz
yme activity which was reversed by dephosphorylation.