Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C.

R.A Nemenoff,S Winitz,N.X Qian,V Van Putten,G.L Johnson,L.E Heasley

doi:10.1016/s0021-9258(18)53948-x

R.A Nemenoff, S Winitz + Show 4 more

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https://doi.org/10.1016/s0021-9258(18)53948-x

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Abstract

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.

Highlights

(cPLA2) has recently been identified, the activity of lipase C activation and production of inositol trisphosphate which is stably increased by stimulation of cells with [2]
A number of hormone and growth factor serinelthreonine kinases have been identified. One such candidate proteinkinase, is p42 MAP kinase, which is a member of a family of kinases designated ERKs [14,15,16]. p42 MAP kinase has been shown to be activated by polypeptide growth factors [17] and phorbol myristate acetate (PMA) [18],as well as hormones acting through G-protein coupled receptors [19, 20]; activation requires phosphorylation of the enzyme on both tyrosine and threonineresidues
To determine if cPLA, was regulated by phosphorylation, we initially established whether purified cPLAz was a substrate for either protein kinase C or p42 MAP kinase

Summary

Introduction

(cPLA2) has recently been identified, the activity of lipase C activation and production of inositol trisphosphate which is stably increased by stimulation of cells with [2]. AVP, EGF, and Phospholipase Az (PLA2),’ which hydrolyzes membrane PMA all stimulatep42 MAP kinase activity. Peptide Mapping-Purified fragment B was phosphorylated with purified PKC or p42 MAP kinase.

Results

Conclusion