Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2alpha in regulating interfacial kinetics and binding and cellular function.
Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA(2)alpha (IMLF(-/-)) were reconstituted with wild type and cPLA(2)alpha mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA(2)alpha translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA(2)alpha to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA(2)alpha to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF(-/-) expressing phosphorylation site (S505A) and PIP(2) binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA(2)alpha, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA(2)alpha reduced activation by PIP(2) but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA(2)alpha hydrolytic activity.
Tucker
Dawn E
DE
Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, Colorado 80206, USA.
Ghosh
Moumita
M
Ghomashchi
Farideh
F
Loper
Robyn
R
Suram
Saritha
S
John
Bonnie St
BS
Girotti
Milena
M
Bollinger
James G
JG
Gelb
Michael H
MH
Leslie
Christina C
CC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation; D054513:Group IV Phospholipases A2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000187:drug effects; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
D016895:Culture Media, Serum-Free; C016658:phorbolol myristate acetate; D054513:Group IV Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium