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Gonadotropin-Releasing Hormone Induction of Extracellular-Signal Regulated Kinase Is Blocked by Inhibition of Calmodulin

Department of Biomedical Sciences (M.S.R., S.P.B., J.X.), Cornell University, Ithaca, New York 14853; Department of Biomedical Sciences (A.M.N., T.A.F., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Molecular and Integrative Physiology (M.W.W.), University of Kansas Medical Center, Kansas City, Kansas 66160

Address all correspondence and requests for reprints to: Mark S. Roberson Ph.D., T3-004d Veterinary Research Tower, Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu.

Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca²⁺ in T3-1 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 -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.

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