Abstract
Using a two hybrid system screen of a human cDNA library, we have found that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the carboxyl region of the 85-kDa cytosolic phospholipase A2 (cPLA2). p11 synthesized in a cell-free system interacts with cPLA2 in vitro. The p11-cPLA2 complex is detectable from a human bronchial epithelial cell line (BEAS 2B). Furthermore, p11 inhibits cPLA2 activity in vitro. Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA results in an increased PLA2 activity as well as an increased release of prelabeled arachidonic acid. This study demonstrates a novel mechanism for the regulation of cPLA2 by an S100 protein.
Highlights
The mammalian low molecular mass 14-kDa PLA2s mainly include two types, type I and type II
The plasmid containing the 1707-bp cytosolic phospholipase A2 (cPLA2) cDNA fragment corresponding to the COOH-terminal cPLA2 protein was designated as plasmid pGBT9cPLA2C
Two Hybrid Screen Indicated that p11 Binds to the C-terminal Region of cPLA2—In an effort to identify proteins that interact with either the NH2-terminal regulatory Ca21-binding domain or the COOH-terminal catalytic domain of the 85-kDa cPLA2 protein, different regions of the cPLA2 cDNA were cloned into pGBT9 vector to express the fusion proteins containing the common GAL4 DNA-binding domain and the NH2terminal or COOH-terminal fragment of the cPLA2 protein
Summary
Vol 272, No 27, Issue of July 4, pp. 17145–17153, 1997 Printed in U.S.A. p11, a Unique Member of the S100 Family of Calcium-binding Proteins, Interacts with and Inhibits the Activity of the 85-kDa Cytosolic Phospholipase A2*. Studies using antisense inhibition and selective inhibitors of different forms of PLA2s have demonstrated that the type II PLA2 participates in AA release and prostaglandin generation in the P388D1 macrophages (4 – 6) These findings have established an important role of the 14-kDa PLA2 for AA metabolism. Other protein kinases such as protein kinase C and cyclic AMP-dependent kinase (protein kinase A) cause phosphorylation of cPLA2 in vitro [12,13, 30] Both the calcium-dependent translocation mediated by the NH2-terminal Ca21-dependent lipid binding domain and phosphorylation of the COOH-terminal region play an important regulatory role in the activation of cPLA2 and regulation of AA release. The p11-cPLA2 complex was precipitated from a human bronchial epithelial cell line
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