title: Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells.
abstract: Abnormal protein accumulation and cell death with cytoplasmic vacuoles are hallmarks of several neurodegenerative disorders. We previously identified p97/valosin-containing protein (VCP), an AAA ATPase with two conserved ATPase domains (D1 and D2), as an interacting partner of the Machado-Joseph disease (MJD) protein with expanded polyglutamines that causes Machado-Joseph disease. To reveal its pathophysiological roles in neuronal cells, we focused on its ATPase activity. We constructed and characterized PC12 cells expressing wild-type p97/VCP and p97(K524A), a D2 domain mutant. The expression level, localization, and complex formation of both proteins were indistinguishable, but the ATPase activity of p97(K524A) was much lower than that of the wild type. p97(K524A) induced cytoplasmic vacuoles that stained with an endoplasmic reticulum (ER) marker, and accumulation of polyubiquitinated proteins in the nuclear and membrane but not cytoplasmic fractions was observed, together with the elevation of ER stress markers. These results show that p97/VCP is essential for degrading membrane-associated ubiquitinated proteins and that profound deficits in its ATPase activity severely affect ER quality control, leading to abnormal ER expansion and cell death. Excessive accumulation of misfolded proteins may inactivate p97/VCP in several neurodegenerative disorders, eventually leading to the neurodegenerations.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000502:physiology*; D016923:Cell Death; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008858:Microscopy, Phase-Contrast; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D016716:PC12 Cells; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism; D000074405:Valosin Containing Protein
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C497249:DDIT3 protein, human; D018076:DNA, Complementary; C497250:Ddit3 protein, mouse; C497251:Ddit3 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D014157:Transcription Factors; D051742:Transcription Factor CHOP; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse; C000614180:Vcp protein, rat