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

Abstract

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 proteintyrosine 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 GnRHactivated 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-(DGK-). Using reciprocal co-immunoprecipitation we show that there is a significant elevation of the association between catalytically active c-Src with DGK-in both HEK293 cells and murine gonadotrope L␤T2 cells. Employing lipid kinase assays we have shown that the catalytic activity of DGK-is significantly heightened in both HEK293 and L␤T2 cells by GnRH. In addition, we demonstrate that the activation of DGK-exerts a functional role in the murine gonadotrope L␤T2 cell line. Elevated expression of DGK-resulted in a shortening of the time scale of ERK activation in these cells suggesting a potential role of endogenous DGK-in controlling the induction of LH␤ transcription by ERK1/2.Gonadotropin-releasing hormone (GnRH) 1 is the central regulator of the reproductive system and was first isolated as a decapeptide from mammalian hypothalami. GnRH released from the hypothalamus activates heptahelical G protein-coupled receptors (GPCRs) specific for GnRH on pituitary gonadotrope cells and mediates the secretion of the gonadotropins, luteinizing (LH) and follicle-stimulating hormone. This process is controlled, in part, by the elevation of intracellular calcium and the activation of protein kinase C (for review see Ref. 1). GnRH initiates both of these processes via the activation of phospholipase C-␤, which hydrolyzes phosphatidylinositol bisphosphate forming inositol trisphosphate and diacylglycerol (DAG). These two metabolites generate the intracellular signals required for gonadotropin secretion thus facilitating consistent reproductive function. Unlike most other GPCRs the Type I GnRH receptor does not possess an intracellular carboxyl terminus, which is typically involved in autoregulation of the receptors signaling activity (for review see Ref.2). Therefore upon stimulation the Type I GnRH receptor chronically activates phospholipase C-␤. It has, however, been demonstrated that there is a regulatory process upon GnRH-mediated signaling at the level of inositol trisphosphate receptor activation and subsequent intracellular calcium mobilization (3). The protracted GnRH receptor stimulation causes a physical downregulation of the intracellular...

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Lck-Dependent Tyrosine Phosphorylation of Diacylglycerol Kinase α Regulates Its Membrane Association in T Cells

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Identification and Characterization of a Novel Human Type II Diacylglycerol Kinase, DGKκ

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