Regulatory mechanisms of multi-functional sheddase

我们已经获得了许多证据表明chat diacylglycerol kinase（DGK）6参与了多功能脱落酶--肿瘤坏死α转化酶（tumor necrosis α-converning enzyme，TACE）的调节。作为下一步，我们通过在刺激的细胞中修饰DGK 6及其密切相关的异构体（DGKs η和κ）。在佛波醇肉豆蔻酸酯乙酸酯（PMA）处理的细胞中，DGKδ1或其普列克底物蛋白同源（PH）结构域单独已显示从细胞质易位至质膜。在目前的工作中，我们确定了PH结构域中的Ser-22和Ser-26作为DGKδ1的PMA和表皮生长因子依赖性磷酸化位点。体外实验和完整细胞实验表明，传统的蛋白激酶C（cPKC）直接磷酸化这些丝氨酸残基。有趣的是，在Ser-22或Ser-26处模拟磷酸丝氨酸的Asp突变显著抑制全长DGKδ1和PH结构域的易位，表明磷酸化负调控酶的易位。我们鉴定了另一种DGKη亚型（η2，135 kDa），其与DGKη1共享相同的序列，除了在C末端添加了一个无菌a基序（SAM）结构域。显示DGKη2通过其SAM结构域形成同源寡聚体以及与其它含SAM的DGK形成异源寡聚体（81和62）。有趣的是，DGKη1和DGKη2响应于应激刺激而从细胞质迅速移位到内体。在这种情况下，DGKη1 1在去除应激刺激后迅速重新定位回细胞质，而DGKη2表现出持续的内体缔合。新的DGK同工酶在N-末端增加了33个Glu-Pro-Ala-Pro串联重复序列。有趣的是，在H_2O_2处理的细胞中，DGKκ而不是其他II型DGK被Src家族激酶特异性地酪氨酸磷酸化。

We had ahrady obtained several lit s of evidence suggesting chat diacylglycerol kinase (DGK) 6 participates m the regulation of a multi-functional sheddase, tumor necrosis α-converning enzyme (TACE). As a next step, we by modification of DGK6 and its closely related isofoms (DGKs η and κ) in stimulated cells. DGKδ1 or its pleckstrin homology (PH) domain alone has been shown to be translocated to the plasma membranes from the cytoplasm in phorbol myristate acetate (PMA)-treated cells. In the present work we identified Ser-22 and Ser-26 within the PH domain as the PMA-and epidermal growth factor-dependent phosphorylation sites of DGKδ1. Experiments in vitro and with intact cells suggested that the conventional protein kinase C (cPKC) directly phosphorylated these Ser residues. Interestingly, the Asp-mutation, which mimics phosphoserine, at Ser-22 or Ser-26, markedly inhibited the translocation of full-length DGKδ1 and the PH domain, suggesting that the phosphorylation regulates negatively the enzyme translocation.We identified another DGKη isoform (η2, 135 kDa) that shared the same sequence with DGKη1 except for a sterile a motif (SAM) domain added at the C-terminus. DGKη2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (81 and 62). Interestingly, DGKη1 and DGKη2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGKη1 l was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGKη2 exhibited sustained endosomal association.Recently, we identified a tenth member of the DGK family designated DGKκ. The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N-terminus. Interestingly, DGKκ, but not other type II DGKs, was specifically tyrosine-phosphorylated by Src-family kinase in H_2O_2-treated cells.