Gonadotropin-releasing Hormone-induced Activation of Diacylglycerol Kinase-ζ and Its Association with Active c-Src

Gonadotropin-releasing hormone (GnRH)-induced receptor activation has been demonstrated to entrain a wide variety of signaling modalities. Most signaling pathways are concerned with the control of serine, threonine, or tyrosine-protein kinases, however, in the current article we demonstrate that in both a model cell line and in gonadotropes, GnRH additionally mediates the activation of lipid-directed kinases. We have shown that there is a functional connection between protein-tyrosine kinase modulation and lipid kinase activation. In HEK293 cells stably expressing the Type I mammalian GnRH receptor, we employed a proteomic approach to identify novel protein binding partners for GnRH-activated c-Src. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry we identified a GnRH-induced association between c-Src and the lipid kinase, diacylglycerol kinase-ζ (DGK-ζ). Using reciprocal co-immunoprecipitation we show that there is a significant elevation of the association between catalytically active c-Src with DGK-ζ in both HEK293 cells and murine gonadotrope LβT2 cells. Employing lipid kinase assays we have shown that the catalytic activity of DGK-ζ is significantly heightened in both HEK293 and LβT2 cells by GnRH. In addition, we demonstrate that the activation of DGK-ζ exerts a functional role in the murine gonadotrope LβT2 cell line. Elevated expression of DGK-ζ resulted in a shortening of the time scale of ERK activation in these cells suggesting a potential role of endogenous DGK-ζ in controlling the induction of LHβ transcription by ERK1/2.