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Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505. 1995

K Abdullah, and W A Cromlish, and P Payette, and F Laliberté, and Z Huang, and I Street, and B P Kennedy
Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Merck Frosst Canada Inc., Pointe Claire-Dorval, Quebec.

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010741 Phospholipases A Phospholipases that hydrolyze one of the acyl groups of phosphoglycerides or glycerophosphatidates.
D010768 Phosphoserine The phosphoric acid ester of serine. Serine Phosphate,Phosphorylserine,Seryl Phosphate,Phosphate, Serine,Phosphate, Seryl
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002522 Chlorocebus aethiops A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research. African Green Monkey,Cercopithecus aethiops,Cercopithecus griseoviridis,Cercopithecus griseus,Cercopithecus pygerythrus,Cercopithecus sabeus,Cercopithecus tantalus,Chlorocebus cynosuros,Chlorocebus cynosurus,Chlorocebus pygerythrus,Green Monkey,Grivet Monkey,Lasiopyga weidholzi,Malbrouck,Malbrouck Monkey,Monkey, African Green,Monkey, Green,Monkey, Grivet,Monkey, Vervet,Savanah Monkey,Vervet Monkey,Savannah Monkey,African Green Monkey,Chlorocebus cynosuro,Green Monkey, African,Green Monkeys,Grivet Monkeys,Malbrouck Monkeys,Malbroucks,Monkey, Malbrouck,Monkey, Savanah,Monkey, Savannah,Savannah Monkeys,Vervet Monkeys
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013755 Tetradecanoylphorbol Acetate A phorbol ester found in CROTON OIL with very effective tumor promoting activity. It stimulates the synthesis of both DNA and RNA. Phorbol Myristate Acetate,12-Myristoyl-13-acetylphorbol,12-O-Tetradecanoyl Phorbol 13-Acetate,Tetradecanoylphorbol Acetate, 4a alpha-Isomer,12 Myristoyl 13 acetylphorbol,12 O Tetradecanoyl Phorbol 13 Acetate,13-Acetate, 12-O-Tetradecanoyl Phorbol,Acetate, Phorbol Myristate,Acetate, Tetradecanoylphorbol,Myristate Acetate, Phorbol,Phorbol 13-Acetate, 12-O-Tetradecanoyl,Tetradecanoylphorbol Acetate, 4a alpha Isomer

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