Diacylglycerol kinase ι regulates Ras guanyl-releasing protein 3 and inhibits Rap1 signaling

Abstract

To study the physiological function of diacylglycerol (DAG) kinase (DGK ), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGK . DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGK inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGK bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGK by crossing wild-type or DGK -deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGK -deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGK regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGK , which is structurally similar to DGK , inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK. D iacylglycerol (DAG) is a potent activator of several signaling proteins, many of which, when abnormally active, can contribute to the initiation or promotion of cancer (1, 2). Several enzymes can metabolize signaling DAG, but the major route is thought to be by its phosphorylation, a reaction that produces phosphatidic acid and is catalyzed by the DAG kinases (DGKs) (reviewed in ref. 2). DAG exerts its effects by activating classical and novel protein kinase C isoforms, as well as other proteins including the Ras guanyl nucleotide-releasing proteins (RasGRPs) (reviewed in refs. 3-5). Although the transforming effects of DAG have been attributed to activation of PKCs, identification of the RasGRP family suggested that DAG could also transform cells by directly activating RasGRPs, which in turn could lead to excess Ras signaling. The four known RasGRP proteins are RasGRP1 [calcium DAG guanine exchange factor II (CalDAG-GEFII)], RasGRP2 (CalDAG-GEFI), RasGRP3 (CalDAG-GEFIII), and RasGRP4 (reviewed in ref. 5). Each RasGRP activates either Ras or Rap1, except RasGRP3, which is unique because it facilitates exchange for both .Activating mutations in Ras are found in a number of tumors (reviewed in ref. 11). Using a mouse model, Chin et al. (12) showed that Ras expression was an absolute requirement for melanoma tumor maintenance, and other studies have demonstrated a role for Ras in metastasis (reviewed in ref. 13). Together, these data clearly show that abnormally active Ras contributes to the maintenance and progression of many types of cancer.Rap1-dependent signaling is not a...

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

“…Considering the substrate preference of DGKε toward the arachidonoyl-containing DG, it is presumed that DGKε is intimately involved in resynthesis and/or turnover of PIP 2 after the stimulation. It is known that DGKι binds and regulates RasGRP3 and predominantly reduces Rap1 activation (Regier et al 2005). This, together with the fi nding that DGKι -KO mice develop fewer tumors in response to a phorbol ester or after wounding (Regier et al 2005), suggests that deleting DGKι in mice may reduce the tumor formation in Ras-dependent manner.…”

“…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.…”

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

“…Thus, it appears that DGK␦ regulates PKCs and consequently modulates EGFR and insulin signaling. It is important to note, however, that DAG affects numerous proteins in addition to cnPKCs (35) and that DGKs regulate some of these DAG targets (8,32,33). So some of the effects caused by deleting DGK␦ might also be mediated through non-PKC DAG targets, a possibility that will be important to explore.…”

“…Together, these data suggested that DGK␦ reduces DAG levels and consequently inhibits PKC activity. DGKs regulate specific signaling events through their interactions with DAG-activated proteins such as the PKCs (8,26,32,33). There are two splice variants of DGK␦; to test whether either of them could bind PKCs, we coexpressed each splice variant with each of the DAG-activated PKC isotypes (␣, ␦, , and ) found in keratinocytes and then immunoprecipitated the DGK.…”

“…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).…”