Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C.
Phospholipase A2 (PLA2) is the enzyme regulating the release of arachidonic acid in most cell types. A high molecular mass, 85-kDa soluble form of PLA2 (cPLA2) has recently been identified, the activity of which is stably increased by stimulation of cells with hormones and growth factors. Growth factor stimulation of cells has been reported to result in increased phosphorylation of cPLA2 on serine residues, but the kinases mediating this effect have not been identified. We report here that human cPLA2 is phosphorylated in vitro by two growth factor-stimulated serine/threonine-specific kinases, p42 MAP kinase and protein kinase C (PKC). Phosphorylation of the cPLA2 enzyme by either kinase results in an increase in catalytic cPLA2-specific activity. Domains of the cPLA2 molecule have been expressed in Escherichia coli, and the fusion proteins purified. PKC and p42 MAP kinase give different patterns of phosphorylation of the recombinantly expressed cPLA2 fragments. p42 MAP kinase selectively phosphorylates the domain of cPLA2 containing a MAP kinase consensus sequence, whereas PKC phosphorylates sites in all three recombinantly expressed domains of the enzyme. Peptide mapping indicates that the site phosphorylated by p42 MAP kinase is different from those phosphorylated by PKC. The combined action of both of these kinases is likely to mediate the effects of growth factor stimulation on arachidonic acid release through the activation of cPLA2.
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
R A
RA
Nemenoff
S
S
Winitz
N X
NX
Qian
V
V
Van Putten
G L
GL
Johnson
L E
LE
Heasley
D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D005920:Glomerular Mesangium / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin; D013820:Thermolysin