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 is often highly expressed in epithelial malignancies and likely has an active role in tumor development. But how it promotes tumorigenesis is not clearly defined. Recent evidence suggests that this may involve transactivation of the epidermal growth factor receptor through Eprostanoid receptors, but reports differ about the mechanism by which this occurs. We found that Eprostanoid receptors 2-4, but not 1, transactivated the epidermal growth factor receptor. This required metalloproteinase activity, leading to release of growth factors from the cell surface. Both transforming growth factor-α and amphiregulin were released in response to overexpression of cyclooxygenase-2, but betacellulin and heparin-binding EGF-like growth factor were not. The metalloproteinase tumor necrosis factor-α converting enzyme was required for proteolytic release of transforming growth factor-α. We also found that addition of epidermal growth factor receptor ligands to HEK293 cells induced cyclooxygenase-2 expression, suggesting that by activating epidermal growth factor receptor signaling, cyclooxygenase-2 potentially creates a self-perpetuating cycle of cell growth. Consistent with this, inhibition of cyclooxygenase-2 reduced growth of epidermal growth factor receptor overexpressing MCF-10A breast epithelial cells in threedimensional culture.

Purpose Lung cancer is a leading cause of cancer deaths and efforts are underway to identify novel therapies to treat these tumors. Diacylglycerol kinase η (DGKη), an enzyme that phosphorylates diacylglycerol to form phosphatidic acid, has been shown to modulate MAPK signaling downstream of EGFR, which is an oncogenic driver in some lung cancers. Since mutations in EGFR and K-Ras are common in lung cancer, we hypothesized that limiting the function of DGKη would attenuate oncogenic properties of lung cancer cells. Methods We determined the expression levels of DGKη in a mouse models of mutant EGFR and K-Ras lung cancer and in human lung cancer cell lines with activating mutations in either EGFR or K-Ras. We also tested the effects of shRNA-mediated depletion of DGKη in lung cancer cells and tested if DGKη depletion augmented the effects of afatinib, a new generation EGFR inhibitor. Results DGKη was expressed in malignant epithelium from mice with mutant EGFR or K-Ras lung cancer. It was also expressed in human lung cancer cell lines with EGFR or K-Ras mutations. Depleting DGKη in lung cancer cell lines, harboring mutant EGFR, reduced their growth on plastic and in soft agar and also augmented the effects of afatinib, an EGFR inhibitor. DGKη depletion also reduced growth of one of two lung cancer cell lines that harbored mutant K-Ras. Conclusions Our data indicate that DGKη is a potential therapeutic target in lung cancers, especially those harboring EGFR mutations. Our findings warrant further studies to examine the effects of limiting its function in vivo.

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase ␦ (DGK␦), a lipid kinase that terminates diacylglycerol signaling. In DGK␦-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the deubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase C␣ (PKC␣), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGK␦-deficient cells. Moreover, the effects of PKC␣ were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGK␦ and PKC␣ modulate the levels of ubiquitinated EGFR through Akt and USP8. The EGFR2 is an important cancer target that is often abnormally active in human tumors. Its role in carcinogenesis has been firmly established in numerous animal models of cancer. For example, mice harboring mutations in the tumor suppressor adenomatous polyposis coli gene develop 50 -90% fewer polyps when EGFR signaling is disrupted either genetically (1) or through the use of EGFR inhibitors (1-3). Its importance in tumorigenesis makes it critical that we understand in more detail the mechanisms by which EGFR can be modulated so that we can identify additional avenues to inhibit this important signaling pathway.Upon binding to its ligands the EGFR activates phospholipase C enzymes that generate diacylglycerol (DAG), an important lipid second messenger that recruits and can activate numerous signaling proteins, including PKC enzymes. DAG is metabolized by a family of proteins called the diacylglycerol kinases (DGKs), which consume DAG in a spatially discrete manner that allows some DGK isoforms to regulate specific DAG target proteins. DGK␦ is a type II DGK with two alternatively spliced products, DGK␦1 and DGK␦2, that differ at their amino termini (4). This alternative splicing does not alter their DGK activity, but appears to affect their sub-cellular localization (4). We disrupted the DGK␦ gene in mice and found that DGK␦ null mice displayed a phenotype almost identical to that of EGFR null mice: the pups were born with open eyelids, developed respiratory difficulty, and died within 24 h after birth (5). This phenotype was specific to DGK␦: other DGK knock-out mice (␣, ⑀, , , and ) did not display these defects (Refs. 5-9 and data not shown). In DGK␦ null mice, we found that EGFR expression was red...