title: The role of Pak-interacting exchange factor-β phosphorylation at serines 340 and 583 by PKCγ in dopamine release.
abstract: Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-β (βPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates βPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated βPIX in vivo. Classical PKC inhibitors and βPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type βPIX, and not the βPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by βPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of βPIX at Ser340 and Ser583 has pivotal roles in Ca(2+)-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through β2PIX phosphorylation. The PKCγ-βPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Shirafuji
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Ueyama
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yoshino
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Adachi
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ago
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Koda
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tetsuaki
authors.initials: T
authors.lastname: Nashida
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Hiramatsu
authors.affiliation: Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan, and.
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Toda
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Sakai
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, naosaito@kobe-u.ac.jp.
authors.forename: Naoaki
authors.identifier: http://orcid.org/0000-0003-3389-1367
authors.initials: N
authors.lastname: Saito
mesh_terms: D000818:Animals; D001665:Binding Sites; D003342:Corpus Striatum / Q000378:metabolism*; D004298:Dopamine / Q000096:biosynthesis / Q000378:metabolism*; D059290:Dopaminergic Neurons / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D064067:Rho Guanine Nucleotide Exchange Factors; D012694:Serine; C087857:protein kinase C gamma; D011493:Protein Kinase C; D004298:Dopamine
keywords: Cdc42; PKC; Parkinson's disease; dopamine; phosphoproteome; βPIX