| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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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 DGKα, 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 DGKα 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 DGKα 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 and hydrogen peroxide/phorbol 12-myristate 13-acetate (PMA). PMA was found to be mainly required for a conversion of 82-chimaerin to an active form. On the other hand, H_2O_2 was suggested to induce a release of Zn^<2+> from the Cl domain of β2-chimaerin. By stepwise deletion analysis, we demonstrated that the Src homology 2 (SH2) and Cl domains of β2-chimaerin interacted with the N-terminal half of catalytic region of DGKγ. Taken together, these results suggest that the functional link between DGKγ and β2-chimaerin (and Racl) has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in cooperation with the Cl domain.
Lipid phosphate phosphatases (LPPs, type 2 phosphatidic acid phosphatases), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we found that LPP1 is also associated with lipid rafts (Triton X-100-soluble but CHAPS-insoluble) distinct from those harboring LPP3. LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions. Less
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