title: DGKζ is degraded through the cytoplasmic ubiquitin-proteasome system under excitotoxic conditions, which causes neuronal apoptosis because of aberrant cell cycle reentry.
abstract: Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin-proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.
authors.affiliation: Department of Anatomy & Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Okada
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Hozumi
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Tanaka
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Yanagida
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Araki
authors.forename: Camilla
authors.initials: C
authors.lastname: Evangelisti
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Yagisawa
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Topham
authors.forename: Alberto M
authors.initials: AM
authors.lastname: Martelli
authors.forename: Kaoru
authors.initials: K
authors.lastname: Goto
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism*; D019852:Diacylglycerol Kinase / Q000172:deficiency / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D018698:Glutamic Acid; D019852:Diacylglycerol Kinase; C512480:diacylglycerol kinase zeta, mouse; D046988:Proteasome Endopeptidase Complex