Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.
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References
1.
TophamMK, and EpandRM: Mammalian diacylglycerol kinases: Molecular interactions and biological functions of selected isoforms. Biochim Biophys Acta, 2009; 1790:416–424.
2.
GotoK, HozumiY, NakanoT, SainoSS, and KondoH: Cell biology and pathophysiology of the diacylglycerol kinase family: Morphological aspects in tissues and organs. Int Rev Cytol, 2007; 264:25–63.
3.
SakaneF, ImaiS, KaiM, YasudaS, and KanohH: Diacylglycerol kinases: Why so many of them?. Biochim Biophys Acta, 2007; 1771:793–806.
4.
GotoK, WatanabeM, KondoH, YuasaH, SakaneF, and KanohH: Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain. Brain Res Mol Brain Res, 1992; 16:75–87.
5.
YanagisawaK, YasudaS, KaiM, ImaiS, YamadaK, YamashitaT, JimbowK, KanohH, and SakaneF: Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-induced apoptosis of human melanoma cells through NF-kappaB activation. Biochim Biophys Acta, 2007; 1771:462–474.
6.
HasegawaH, NakanoT, HozumiY, TakagiM, OginoT, OkadaM, IsekiK, KondoH, WatanabeM, MartelliAM, and GotoK: Diacylglycerol kinase zeta is associated with chromatin, but dissociates from condensed chromatin during mitotic phase in NIH3T3 cells. J Cell Biochem, 2008; 105:756–765.
7.
NakanoT, HozumiY, GotoK, and WakabayashiI: Localization of diacylglycerol kinase epsilon on stress fibers in vascular smooth muscle cells. Cell Tissue Res, 2009; 337:167–175.
8.
BaldanziG, PietronaveS, LocarnoD, MerlinS, PorporatoP, ChianaleF, FilighedduN, CantelmoAR, AlbiniA, GrazianiA, and PratM: Diacylglycerol kinases are essential for hepatocyte growth factor-dependent proliferation and motility of Kaposi's sarcoma cells. Cancer Sci, 2011; 102:1329–1336.
9.
NakanoT, HozumiY, GotoK, and WakabayashiI: Involvement of diacylglycerol kinase gamma in modulation of iNOS synthesis in Golgi apparatus of vascular endothelial cells. Naunyn Schmiedebergs Arch Pharmacol, 2012; 385:787–795.
10.
NakanoT, HozumiY, IwazakiK, OkumotoK, IsekiK, SaitoT, KawataS, WakabayashiI, and GotoK: Altered expression of diacylglycerol kinase isozymes in regenerating liver. J Histochem Cytochem, 2012; 60:130–138.
11.
HozumiY, AkimotoR, SuzukiA, OtaniK, WatanabeM, and GotoK: Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland. Cell Tissue Res, 2015; 362:295–305.
12.
JonesDR, D'SantosCS, MeridaI, and DivechaN: T lymphocyte nuclear diacylglycerol is derived from both de novo synthesis and phosphoinositide hydrolysis. Int J Biochem Cell Biol, 2002; 34:158–168.
13.
OlenchockBA, GuoR, CarpenterJH, JordanM, TophamMK, KoretzkyGA, and ZhongXP: Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat Immunol, 2006; 7:1174–1181.
14.
SanjuanMA, JonesDR, IzquierdoM, and MeridaI: Role of diacylglycerol kinase alpha in the attenuation of receptor signaling. J Cell Biol, 2001; 153:207–220.
15.
SchaapD, de WidtJ, van der WalJ, VandekerckhoveJ, van DammeJ, GussowD, PloeghHL, van BlitterswijkWJ, and van der BendRL: Purification, cDNA-cloning and expression of human diacylglycerol kinase. FEBS Lett, 1990; 275:151–158.
16.
FujiiY, KanekoM, NeyazakiM, NogiT, KatoY, and TakagiJ: PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin. Protein Expr Purif, 2014; 95:240–247.
