Expression of a catalytically inactive form of diacylglycerol kinase α induces sustained signaling through RasGRP

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Abstract
Regulating the generation and clearance of lipid second messengers, such as diacylglycerol (DAG), is critical for the correct propagation of intracellular signaling pathways. DAGK type alpha acts as a negative modulator of the DAG levels generated during T cell activation, which is initiated by triggering of the endogenous T cell receptor (TCR), as well as by stimulation of an ectopically expressed human muscarinic type 1 receptor. Here we show that stimulation of either of these receptors causes rapid, transient membrane translocation of the recently discovered Ras guanyl nucleotide release protein (RasGRP), followed by activation of mitogen-activated protein kinase (MAPK). When cells expressing a catalytically inactive form of DAGKalpha were analyzed, however, similar agonist stimulation resulted in sustained signaling through RasGRP and MAPK. Biochemical analysis showed that expression of kinase-dead diacylglycerol kinase a (DGKalpha) led to a greater, more sustained, DAG accumulation following receptor stimulation. These results suggest that, in T cells, agonist-stimulated DAG generation is the key factor controlling activation of the Ras/MAPK signaling pathway through membrane translocation of RasGRP. Moreover, we demonstrate that through the modulation of the intracellular level of agonist-stimulated DAG, DGKalpha alters Ras activation and downstream signaling dramatically, a process of utmost importance for sound immunological function.
Funding Information
  • Ministerio de Ciencia y Tecnología
  • Comunidad de Madrid