Discovery and analysis of dovel lipid signal transduction complexes

二酰基甘油（DAG）激酶（DGK）通过磷酸化DAG生成PA来调节DAG和磷脂酸（PA）两种信号脂质的平衡。迄今为止，已经鉴定出10种哺乳动物DGK同工酶。出乎我们的意料，最近的研究表明，DGK同工酶在多种信号转导途径中发挥着关键作用。最近，我们发现DGKa在几种人类黑色素瘤细胞系中表达，但在非癌性黑色素细胞中不表达。有趣的是，野生型DGKa的过表达，而不是其激酶死亡突变体的过表达，显著抑制了肿瘤坏死因子a诱导的AKI人黑色素瘤细胞凋亡。在反向实验中，小干扰rna介导的DGKα敲低显著增强了细胞凋亡，从而令人信服地表明该亚型具有细胞保护功能。此外，我们获得了几条证据表明DGKa通过激活更多的核因子κB离子来抑制肿瘤坏死因子a诱导的黑色素瘤细胞凋亡。因此，该转录因子可能是DGKa在细胞增殖和存活过程中下游的关键因子之一。我们已经证明DGKγ通过其催化作用发挥作用，作为Racl的上游抑制因子，因此，板状基底/膜皱褶的形成。最近，我们进一步发现DGKγ特异性地与β2-嵌合蛋白（一种rac特异性的gtpase激活蛋白）相互作用并激活β2-嵌合蛋白，以响应表皮生长因子（EGF）和过氧化氢/酚酯对细胞的刺激。这些结果表明β2-嵌合蛋白是连接DGKγ与Racl功能的效应分子。我们生成并分析了dgko基因敲除小鼠。DGKδ缺乏降低了EGF受体蛋白的表达和活性。DGK8缺乏引起的DAG积累激活了PKC，使EGF受体Thr-654位点磷酸化。磷酸化因此使受体失活并增强其降解。在这种情况下，DGKδ与常规蛋白激酶C （PKCα）和新型PKC （PKC_s, δ， a和η）相关。因此，DGKδ至少在一定程度上通过调节PKC信号来调节EGF受体。我们进一步报道DGKδ的反义沉默足以阻止高糖对PKCα活性、胰岛素受体信号传导和葡萄糖摄取的影响。因此，骨骼肌细胞短期暴露于葡萄糖会导致DGKδ的快速诱导，随后是PKCα活性的降低和胰岛素受体信号的反激活。此外，我们发现DGKδ表达降低，同时伴有2型糖尿病患者骨骼肌DGK活性降低。在糖尿病动物中，纠正血糖后，DGK8蛋白和激酶活性降低。DGKδ单倍体不足增加DAG含量，导致外周胰岛素抵抗，骨骼肌胰岛素信号和葡萄糖转运受损，以及年龄依赖性肥胖。这种损伤与胰岛素刺激下胰岛素受体底物-1的酪氨酸磷酸化减少以及Akt和RabGAP AS160的丝氨酸/苏氨酸磷酸化减少有关，后者是典型胰岛素信号级联的组成部分，对骨骼肌的葡萄糖摄取和代谢很重要。DGKδ蛋白表达的降低直接导致外周胰岛素抵抗和轻度肥胖的发生。通过调节DGKδ靶向DAG和PA代谢的治疗方法可能控制和预防代谢性疾病中的胰岛素抵抗。少

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways.Recently, we found that DGKa is expressed in several human melanoma cell lines but not in noncancerous melanocytes. Intriguingly, the overexpression of wild-type DGKa, but not of its kinase-dead mutant, markedly suppressed tumor necrosis factor-a-induced apoptosis of AKI human melanoma cells. In the reverse experiment, small interfering RNA-mediated knockdown of DGKα significantly enhanced the apoptosis, thus convincingly indicating that this isoform possesses a cytoprotective function. Moreover, we obtained several lines of evidence suggesting that DGKa suppresses tumor necrosis factor-a-induced melanoma cell apoptosis through activat … More ion of nuclear factor κB. Thus, this transcription factor is likely to be one of the key factors downstream of DGKa during cell proliferation and survival.We had already demonstrated that DGKγ functions through its catalytic action as an upstream suppressor of Racl, and consequently, lamellipodium/membrane ruffle formation. Recently, we further found that DGKγ specifically interacts with and activates β2-chimaerin, a Rac-specific GTPase-activating protein, in response to cell stimulation with epidermal growth factor (EGF) and hydrogen peroxide/phorbol ester. These results suggest that β2-chimaerin is an effector molecule linking DGKγ functionally with Racl.We generated and then analyzed DGKO-knockout mice. DGKδ deficiency reduced EGF receptor protein expression and activity. DAG accumulation caused by DGK8 deficiency activated PKC, which phosphorylates EGF receptor at Thr-654. The phosphorylation consequently inactivates the receptor and enhances its degradation. In this case, DGKδ was associated with conventional protein kinase C (PKC) (PKCα) and novel PKC (PKC_s, δ, a and η). Thus, DGKδ is concluded to regulate EGF receptor, at least in part, by modulating PKC signaling.We further reported that antisense silencing of DGKδ was sufficient to prevent the effect of high glucose on PKCα activity, insulin receptor signaling and glucose uptake. Thus, the short-term exposure of skeletal muscle cells to glucose caused a rapid induction of DGKδ, followed by a reduction of PKCα activity and transactivation of the insulin receptor signaling. Moreover, we identified decreased DGKδ expression, concomitant with reduced DGK activity in skeletal muscle from type II diabetic patients. In diabetic animals, reduced DGK8 protein and kinase activity were restored upon correction of glycemia. DGKδ haploinsufficiency increased DAG content and caused peripheral insulin resistance, impaired skeletal muscle insulin signaling and glucose transport, and age-dependent obesity. The impairment was associated with reduced insulin-stimulated turosine phosphorylation of insulin receptor substrate-1 and serine/threonine phosphorylation of Akt and the RabGAP AS160, components of the canonical insulin signaling cascade important for glucose uptake and metabolism in skeletal muscle. Reduced DGKδ protein expression directly contributes to the development of peripheral insulin resistance and mild obesity. Therapeutic approaches to target DAG and PA metabolism via regulation of DGKδ may control and prevent insulin resistance in metabolic disease. Less