Phorbol ester-regulated oligomerization of diacylglycerol kinase δ linked to its phosphorylation and translocation

Abstract

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKδ containing a sterile α-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures. Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKδ formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization. Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKδ. Furthermore, we found that DGKδ was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKδ mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester. A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester, i.e. oligomer dissociation, phosphorylation, and translocation. We confirmed that tumor-promoting phorbol esters did not directly bind to DGKδ. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKδ and that DGKδ is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.