Analysis of PKCγ substrates in nigro-striatum system: The role of βPIX phosphorylation for dopamine release

Toshihiko Shirafuji1,2, Takehiko Ueyama1, Ken-ichi Yoshino1, Naoko Adachi1, Hideyuki Takahashi1, Naoki Hiramatsu3, Yukio Ago3, Toshio Matsuda3, Tatsushi Toda4, Norio Sakai2, Naoaki Saito1

1:Lab.Mol.Pharmacol.Biosig.Res.Ctr.,Kobe Univ
2:Dept.Mol.Pharmacol.Neurosci.Inst.Biomed Health Sci.,Hiroshima Univ
3:Lab.Medicinal Pharmacol.,Grad.Sch.of Pharmaceut.Sci.,Osaka Univ
4:Dept.Neurol/Mol Brain Sci.,Grad.Sch.Med.,Kobe Univ

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 Parkinsonian like symptoms (PS). Here, we found that PKCγ was abolished in AS/AGU rat and that PKCγ-KO mice showed PS. However, the PKCγ substrates responsible for the regulated exocytosis of DA have not been elucidated. To identify the PKCγ substrates involved in DA release, we employed a phospho-proteome analysis. In the striatum of PKCγ-KO mice, We found 10 candidate proteins with PKC motif that exhibited decreased phosphorylation levels. Among them, we focused on βPIX, which is a Cdc42/Rac1 guanine nucleotide exchange factor and found that PKCγ directly phosphorylates βPIX at Serine (Ser)583 and indirectly at Ser 340 in cell culture. We also found that PKC phosphorylated βPIX in vivo. Classical PKC inhibitors and βPIX knockdown (KD) suppressed the DA release in PC12 cells. The expression of WT βPIX, but not βPIX mutants, whose ser 340 or 583 was substituted by alanine (S340A or S583A), fully rescued the decreased DA release of βPIX KD cells. Double KD of Cdc42/Rac1 decreased DA release from PC12 cells. Phospho-mimicking mutants of βPIX (S340E or S583E) interacted more strongly with Cdc42/Rac1 than WT βPIX. These findings indicate that the phosphorylation of βPIX at Ser 340 and 583 has pivotal roles in DA release through interaction with Cdc42/Rac1 in the striatum.