Regulatory mechanisms of multi-functional sheddase

我们已经获得了大量证据表明二酰基甘油激酶（DGK） 6参与了一种多功能脱落酶——肿瘤坏死α-转化酶（TACE）的调控。下一步，我们在受刺激的细胞中对DGK6及其密切相关的异构体（DGKs η和κ）进行修饰。DGKδ1或其pleckstrin同源结构域（PH）已被证明在phorbol肉豆蔻酸酯（PMA）处理的细胞中从细胞质转移到质膜上。在目前的工作中，我们确定了PH域中Ser-22和Ser-26是DGKδ1的pma和表皮生长因子依赖性磷酸化位点。体外和完整细胞实验表明，常规蛋白激酶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，模仿磷酸丝氨酸的asp突变显著抑制全长DGKδ1和PH结构域的易位，表明磷酸化负性调节酶易位。我们发现了另一个DGKη亚型（η 2,135 kDa），除了在c端增加了一个不育的a基序（SAM）结构域外，与DGKη1具有相同的序列。DGKη2通过其SAM结构域的同聚物和异聚物与其他含有SAM的DGKs形成（81和62）。有趣的是，DGKη1和DGKη2在应激刺激下迅速从细胞质转移到核内体。在这种情况下，DGKη1在去除应激刺激后迅速重新定位到细胞质中，而DGKη2则表现出持续的内体关联。最近，我们确定了DGK家族的第十个成员DGKκ。新的DGK同工酶在n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DGKs，被src家族激酶特异性酪氨酸磷酸化。

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.