Regulatory mechanisms of multi-functional sheddase

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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.