Protein kinase inhibitor H7 blocks the induction of immediate-early genes zif268 and c-fos by a mechanism unrelated to inhibition of protein kinase C but possibly related to inhibition of phosphorylation of RNA polymerase II.
1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7) has often been used in combination with protein kinase inhibitor (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) (HA1004) to assess the contribution of protein kinase C (PKC) to cellular processes, including the induction of gene expression. This use of H7 and HA1004 is based upon the fact that H7 inhibits PKC more potently than HA1004 in in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Here we describe experiments that show that this interpretation is not correct with regard to the induction of two immediate-early genes, zif268 and c-fos, in PC12D cells. In these studies we confirmed that H7, but not HA1004, potently blocks the induction of zif268 and c-fos mRNA by nerve growth factor, carbachol, phorbol ester, Ca2+ ionophore, or forskolin. Surprisingly, however, H7 has no effect on the ability of these agents to activate mitogen-activated protein kinase (MAPK), an upstream activator of zif268 and c-fos gene expression. H7 also does not inhibit preactivated MAPK in vitro. Taken together, these results suggest that H7 blocks gene expression by acting at a site downstream from MAPK. H7 has previously been shown to block transcription in vitro by blocking the phosphorylation of the carboxyl-terminal domain of RNA polymerase II (Yankulov, K., Yamashita, K., Roy, R., Egly, J.-M., and Bentley, D. L.(1995) J. Biol. Chem. 270, 23922-23925). In this study, we show that pretreating PC12D cells with H7, but not with HA1004, significantly reduces levels of phosphorylated RNA polymerase II in vivo. These results suggest that H7 blocks gene expression by inhibiting the phosphorylation of RNA polymerase II, a step required for progression from transcription initiation to mRNA chain elongation.
Kumahara
E
E
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan. kumahara@m.u-tokyo.ac.jp
Ebihara
T
T
Saffen
D
D
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D017781:Genes, Immediate-Early / Q000235:genetics*; D016762:Genes, fos / Q000235:genetics*; D017874:Immediate-Early Proteins; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D009414:Nerve Growth Factors; D010703:Phorbol Esters; D012333:RNA, Messenger; D014157:Transcription Factors; C080501:1-(5-isoquinolinylsulfonyl)-3-methylpiperazine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D012319:RNA Polymerase II