| Co-Investigator(Kenkyū-buntansha) |
KANOH Hideo Sapporo Medical University, Department of Biochemistry, School of Medicine, Honorary Professor (70045475)
IMAI Shin-ichi Sapporo Medical University, Department of Biochemistry, School of Medicine, Assistant Professor (20213209)
KAI Masahiro Sapporo Medical University, Department of Biochemistry, School of Medicine, Assistant Professor (80260777)
安田 智 札幌医科大学, 医学部, 助教 (20381262)
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| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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
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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
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