The role of Pak-interacting exchange factor-ß 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-ß (ßPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates ßPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated ßPIX in vivo. Classical PKC inhibitors and ßPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type ßPIX, and not the ßPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by ßPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of ßPIX 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 ß2PIX phosphorylation. The PKCγ-ßPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.