Diacylglycerol kinase δ regulates protein kinase C and epidermal growth factor receptor signaling

Abstract

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGK␦, we disrupted its gene in mice and found that DGK␦ deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGK␦-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGK␦ knockout cells, indicating that DGK␦ regulates EGFR by modulating PKC signaling. D iacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (1, 2). DAG, the substrate of the DGK reaction, is a key intracellular signaling factor that activates PKCs, Ras guanyl nucleotidereleasing proteins, and some transient receptor potential channels (3, 4). DAG also recruits a number of proteins to membrane compartments, including the chimaerins, PKD, and the Munc13 proteins (3). Its effects on numerous and diverse targets underscores the importance of DAG signaling and indicates that DAG affects a broad array of signaling events. Because the consumption of DAG by DGKs is thought to attenuate these actions, the DGK reaction is biologically important and likely regulates numerous DAG signaling pathways.Mammalian DGKs differ in their structures, patterns of tissue expression, and catalytic properties. Ten of them have been identified and are classified into five subtypes based on their structural motifs (1, 2, 5, 6). Their structural diversity and distinct expression patterns indicate that each isoform may perform a different biological function. Supporting functional diversity, the DGK knockout mice that have been studied to date have distinct phenotypes, including resistance to seizures in DGK knockout mice (7), attenuated Ras signaling in DGK knockouts (8), and hyperactive T cell signaling in DGK -deficient mice (9).As a type II DGK, DGK␦ has a characteristic pleckstrin homology domain and a sterile ␣-motif domain (Fig. 1A). To determine its biological function, we generated mice with a targeted mutation of the DGK␦ gene. Our data indicate an important role for DGK␦ in modulating PKC and EGF receptor (EGFR) signaling. Results and DiscussionWe used mice to make a targeted deletion of the N-terminal portion of the DGK␦ catalytic domain (see Fig. 7, which is published as supporting information on the PNAS web site). Southern blot analysis of tail DNA confirmed proper insertion of the targeting vector (data not shown), and RT-PCR demonstrated absence of DGK␦ mRNA in homozygous mutant cells (Fig. 7C). Also confirming DGK␦ gene inactivation, DGK␦ protein was absent in knockout keratinocyte and dermal fibroblast cell lysates (Fig. 7D). Deleting DGK␦ did not significantly affect mRNA expression of other DGKs in brain tissue (Fig. 7E). Finally, to establish the expression pattern of DGK␦ in WT mice...

Cyclooxygenase-2 (COX-2) Negatively Regulates Expression of Epidermal Growth Factor Receptor and Causes Resistance to Gefitinib in COX-2–Overexpressing Cancer Cells

“…Several reports have shown that activated JNK positively regulates EGFR expression via c-Jun activation (27)(28)(29), and we investigated whether COX-2 regulates JNK activity, resulting in EGFR down-regulation in the tested cancer cells. Overexpressed COX-2 specifically activated JNK compared with control cells (Fig.…”

“…Several studies reported that c-Jun transcription factor, a substrate of JNK, positively regulates transcription of the EGFR gene (27)(28)(29). Therefore, we investigated whether JNK is involved in COX-2-induced EGFR down-regulation.…”

Lipid Messenger, Diacylglycerol, and its Regulator, Diacylglycerol Kinase, in Cells, Organs, and Animals: History and Perspective

“…DGKδ -KO mice have eyelids open at birth and die soon because of the respiratory diffi culty (Crotty et al 2006). The study on isolated cells reveals that DGKδ -defi ciency reduces EGF receptor (EGFR) protein expression and activity.…”

“…Functional analyses of DGK isozymes have been performed on knockout mice of DGKs, including DGKα (Olenchock et al 2006), DGKζ (Zhong et al 2003), DGKδ (Crotty et al 2006), DGKε (Rodriguez de Turco et al 2001), and DGKι (Regier et al 2005). DGKα and DGKζ are implicated in T cell receptor (TCR) signaling, especially T cell anergy.…”

Novel One-step Immunoassays to Quantify α-Synuclein

“…Perhaps most intriguing of these is DGKQ, due to its identification in genome-wide association studies as a PD risk factor. The lipid kinase family of DGKs catalyzes the conversion of diacylglycerol to phosphatidic acid and thereby regulate events downstream of these two lipid signals in part by antagonizing PKC activation by diacylglycerol (44,45). DGKQ activity in turn is inhibited by GTP-bound RhoA (46).…”