Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin.
Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone alpha-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.
Roberson
Mark S
MS
Department of Biomedical Sciences, Cornell University, T3-004d Veterinary Research Tower, Ithaca, New York 14853, usa. msr14@cornell.edu
Bliss
Stuart P
SP
Xie
Jianjun
J
Navratil
Amy M
AM
Farmerie
Todd A
TA
Wolfe
Michael W
MW
Clay
Colin M
CM
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D017930:Genes, Reporter; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D013449:Sulfonamides; D007987:Gonadotropin-Releasing Hormone; C063302:KN 62; C017967:W 7; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D008156:Luciferases; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D002118:Calcium