Phorbol ester-regulated oligomerization of diacylglycerol kinase delta linked to its phosphorylation and translocation.
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 DGKdelta containing a sterile alpha-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 DGKdelta 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 DGKdelta. Furthermore, we found that DGKdelta was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKdelta 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 DGKdelta. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKdelta and that DGKdelta is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.
Imai
Shin-ichi
S
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan.
Sakane
Fumio
F
Kanoh
Hideo
H
D000818:Animals; D001704:Biopolymers / Q000737:chemistry*; D015153:Blotting, Western; D019556:COS Cells; D002850:Chromatography, Gel; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D010766:Phosphorylation; D011233:Precipitin Tests; D021381:Protein Transport; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D020798:Two-Hybrid System Techniques
D001704:Biopolymers; D019852:Diacylglycerol Kinase; D013755:Tetradecanoylphorbol Acetate