Ro31-8220 inhibits protein kinase C to block the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells: p70 S6 kinase and MAPKAP kinase-1beta do not function downstream of PKC in activating PLD.
The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121-123] that Ro31-8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen-activated protein kinase-activated protein kinase-1beta, as well as of PKC. Here we show that the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase-1beta but is still completely blocked by Ro31-8220. We conclude therefore that p70S6k and MAPKAP kinase-1beta as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester-stimulated phospholipid turnover and that the inhibitory action of Ro31-8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI-3 kinase inhibitor, wortmannin, inhibits the phorbol ester-stimulated release of ethanolamine- but not choline-metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth-PLD is also regulated via a wortmannin-sensitive pathway.
Morreale
A
A
Department of Biology, University of York, UK.
Mallon
B
B
Beale
G
G
Watson
J
J
Rumsby
M
M
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002794:Choline / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019856:Ethanolamine / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D005910:Glioma; D007211:Indoles / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010739:Phospholipase D / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D020123:Sirolimus; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011090:Polyenes; D019856:Ethanolamine; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus; C064758:Ro 31-8220; D000077191:Wortmannin