Ser515 phosphorylation-independent regulation of cytosolic phospholipase A2alpha (cPLA2alpha) by calmodulin-dependent protein kinase: possible interaction with catalytic domain A of cPLA2alpha.
Calmodulin (CaM)-dependent protein kinase (CaM kinase) is proposed to regulate the type alpha of cytosolic phospholipase A(2) (cPLA(2)alpha), which has a dominant role in the release of arachidonic acid (AA), via phosphorylation of Ser515 of the enzyme. However, the exact role of CaM kinase in the activation of cPLA(2)alpha has not been well established. We investigated the effects induced by transfection with mutant cPLA(2)alpha and inhibitors for CaM and CaM kinase on the Ca(2+)-stimulated release of AA and translocation of cPLA(2)alpha. The mutation of Ser515 to Ala (S515A) did not change cPLA(2)alpha activity, although S228A and S505A completely and partially decreased the activity, respectively. Stimulation with hydrogen peroxide (H(2)O(2), 1 mM) and A23187 (10 microM) markedly released AA in C12 cells expressing S515A and wild-type cPLA(2)alpha, but the responses in C12-S505A, C12-S727A, and C12-S505A/S515A/S727A (AAA) cells were reduced. In HEK293T cells expressing cPLA(2)alpha, A23187 caused the translocation of the wild-type, the every mutants, cPLA(2)alpha-C2 domain, and cPLA(2)alpha-Delta397-749 lacking proposed phosphorylation sites such as Ser505 and Ser515. Treatment with inhibitors of CaM (W-7) and CaM kinase (KN-93) at 10 microM significantly decreased the release of AA in C12-cPLA(2)alpha cells and C12-S515A cells. KN-93 inhibited the A23187-induced translocation of the wild-type, S515A, AAA and cPLA(2)alpha-Delta397-749, but not cPLA(2)alpha-C2 domain. Our findings show a possible effect of CaM kinase on cPLA(2)alpha in a catalytic domain A-dependent and Ser515-independent manner.
Shimizu
Masaya
M
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan.
Nakamura
Hiroyuki
H
Hirabayashi
Tetsuya
T
Suganami
Akiko
A
Tamura
Yutaka
Y
Murayama
Toshihiko
T
D019943:Amino Acid Substitution; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D001596:Benzylamines / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054513:Group IV Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
D016718:Arachidonic Acid; D001596:Benzylamines; D000001:Calcimycin; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017931:DNA Primers; D004791:Enzyme Inhibitors; D054513:Group IV Phospholipases A2; D007476:Ionophores; D011994:Recombinant Proteins; D012694:Serine; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides; C017967:W 7; C516389:PLA2G4A protein, human