坂根 郁夫

Phosphatidylcholine- and phosphatidylethanolamine-specific phospholipase C (PC-PLC and PE-PLC) activities, which generate diacylglycerol (DG) and are tricyclodecan-9-yl-xanthogenate (D609)-sensitive, have been detected in both the membrane and cytosolic fractions. We have previously demonstrated that sphingomyelin synthase isozymes, which are transmembrane proteins, exhibit PC-/PE-PLC activities. However, mammalian cytosolic PC-PLC and PE-PLC remain unidentified. Here, we demonstrated that phosphatase orphan 1 (PHOSPHO1), a cytosolic protein, exhibits D609-sensitive PC-PLC and PE-PLC activities. Moreover, the overexpression of PHOSPHO1 in HEK293 cells significantly increased the levels of cellular saturated and/or monounsaturated fatty acid-containing DG. Furthermore, DGKδ cosedimented and colocalized with PHOSPHO1. Collectively, these in vitro findings provide, for the first time, a promising candidate for the long-sought cytosolic PC-/PE-PLC, which may act as DG supply enzyme upstream of DGKδ.

Diacylglycerol kinase δ (DGKδ) phosphorylates diacylglycerol and converts it into phosphatidic acid. DGKδ contributes to glucose uptake as one of its cellular functions. However, detail mechanisms underlying the regulation of DGKδ protein stability remain unelucidated. Herein, we identified ubiquitin-specific peptidase 11 (USP11) in the DGKδ protein complex by DGKδ-interactome analysis. By mapping analysis, we clarified that a wider region of USP11, including the catalytic domain 1 region, and both the C1 domains and catalytic subdomain-a of DGKδ mainly contributed to their association. Cellular dysfunction of USP11 by mitoxiantrone (a USP11-specific inhibitor) or siRNA knockdown markedly decreased DGKδ protein levels. Additionally, we found that DGKδ ubiquitination was increased by USP11 dysfunction, and cumulative ubiquitination was reduced by rescue manipulation. Functionally, USP11 dysfunction reduced cellular glucose uptake. Altogether, our findings provide the first evidence that USP11 deubiquitination-dependently stabilizes DGKδ to maintain cellular glucose uptake.

Abstract Diacylglycerol kinase δ (DGKδ) phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we demonstrated that down‐regulation of DGKδ suppresses the myogenic differentiation of C2C12 myoblasts. However, the myogenic roles of DGKδ in vivo remain unclear. In the present study, we generated DGKδ‐conditional knockout mice under the control of the myogenic factor 5 (Myf5) gene promoter, which regulates myogenesis and brown adipogenesis. The knockout mice showed a significant body weight reduction and apparent mass decrease in skeletal muscle, including the tibialis anterior (TA) muscle. Moreover, the thickness of a portion of the myofibers was reduced in DGKδ‐deficient TA muscles. However, DGKδ deficiency did not substantially affect brown adipogenesis, suggesting that Myf5‐driven DGKδ deficiency mainly affects muscle development. Notably, skeletal muscle injury induced by a cardiotoxin highly up‐regulated DGKδ protein expression, and the DGKδ deficiency significantly reduced the thickness of myofibers, the expression levels of myogenic differentiation markers such as embryonic myosin heavy chain and myogenin, and the number of newly formed myofibers containing multiple central nuclei during muscle regeneration. DGKδ was strongly expressed in myogenin‐positive satellite cells around the injured myofibers and centronucleated myofibers. These results indicate that DGKδ has important roles in muscle regeneration in activated satellite cells. Moreover, the conditional knockout mice fed with a high‐fat diet showed increased fat mass and glucose intolerance. Taken together, these results demonstrate that DGKδ plays crucial roles in skeletal muscle development, regeneration, and function.