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PMID: 19176526 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2alpha in regulating interfacial kinetics and binding and cellular function.

The Journal of biological chemistry ·Vol. 284 ·No. 14 ·2009-04-03 ·Pages 9596-611

Tucker DE, Ghosh M, Ghomashchi F, Loper R, Suram S, John BS, Girotti M, Bollinger JG, Gelb MH, Leslie CC

Abstract

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.

MeSH Terms
Animals Calcium/metabolism Catalytic Domain Cells, Cultured Culture Media, Serum-Free Enzyme Activation/drug effects Gene Expression Regulation Group IV Phospholipases A2/chemistry,genetics,metabolism Humans Kinetics Mice Mice, Knockout Mutation/drug effects Phosphorylation Protein Binding Protein Transport Tetradecanoylphorbol Acetate/analogs & derivatives,pharmacology
Chemicals
Culture Media, Serum-Free phorbolol myristate acetate Group IV Phospholipases A2 Tetradecanoylphorbol Acetate Calcium
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Tucker Dawn E
Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, Colorado 80206, USA.
Ghosh Moumita
Ghomashchi Farideh
Loper Robyn
Suram Saritha
John Bonnie St
Girotti Milena
Bollinger James G
Gelb Michael H
Leslie Christina C
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M807299200
Published
2009-04-03
Epub
2009-00-28
Pages
9596-611
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2666612
Subset
IM
Grants
NHLBI NIH HHS · P01 HL034303 · United States
NHLBI NIH HHS · HL50040 · United States
NHLBI NIH HHS · R01 HL050040 · United States
NHLBI NIH HHS · HL34303 · United States
NHLBI NIH HHS · R01 HL061378 · United States
NHLBI NIH HHS · HL61378 · United States
External Links
PubMed source DOI: 10.1074/jbc.M807299200 PMC Full Text View journal details
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