title: Protein kinase C stabilizes X-linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser(87) to suppress apoptotic cell death.
abstract: BACKGROUND
  Multiple protein kinases have been shown to be involved in the apoptotic neuronal loss of Alzheimer's disease (AD). Although some studies support the role of protein kinase C (PKC) in amyloid precursor protein processing as well as in tau phosphorylation, a direct role for PKC in apoptotic neuronal death remains to be clarified. In the present study, we report on the possible role of PKC in cell survival during conditions of stress through phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP).
  
  
  METHODS
  Phosphorylation of XIAP at Ser87 was confirmed by western blot analysis employing phosphorylation dependent anti-XIAP antibody after incubation of recombinant XIAP with active PKC in vitro. And increased phosphorylation of XIAP at the site was also confirmed in SH-SY5Y cells treated with PKC activator, phorbol 12-myristate 13-acetate (PMA). A mutant XIAP construct in which Ser87 was substituted by Ala, was prepared, and transfected to cells. After the transfection of wild or mutant XIAP, cells viability was evaluated by counting living and dead cells treated with PMA during etoposide-induced apoptosis.
  
  
  RESULTS
  Recombinant XIAP was phosphorylated at Ser(87) by PKC in vitro and treatment of XIAP-transfected SH-SY5Y cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of XIAP at Ser(87) . Pulse chase experiments revealed that, when phosphorylated at Ser(87) , wild-type XIAP is more stable than XIAP with a Ser87Ala substitution, which is degraded faster. Importantly, the phosphorylation of XIAP at the site by PKC significantly increased cell survival up to approximately 2.5 times under the condition of apoptosis induced by 25 µg/ml etoposide.
  
  
  CONCLUSIONS
  The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser(87) and its stabilization, in cell survival under conditions of stress and lend strength to the idea that PKC is crucial in regulating neuronal homeostasis, which may be impaired in AD.
authors.affiliation: Department of Psychiatry, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Kato
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Tanaka
authors.forename: Golam
authors.initials: G
authors.lastname: Sadik
authors.forename: Miyako
authors.initials: M
authors.lastname: Baba
authors.forename: Daisuke
authors.initials: D
authors.lastname: Maruyama
authors.forename: Kanta
authors.initials: K
authors.lastname: Yanagida
authors.forename: Takashi
authors.initials: T
authors.lastname: Kodama
authors.forename: Takashi
authors.initials: T
authors.lastname: Morihara
authors.forename: Shinji
authors.initials: S
authors.lastname: Tagami
authors.forename: Masayasu
authors.initials: M
authors.lastname: Okochi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kudo
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Takeda
mesh_terms: D000544:Alzheimer Disease / Q000503:physiopathology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D001921:Brain / Q000503:physiopathology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D012694:Serine; D005047:Etoposide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate