The Role Of Pak-Interacting Exchange Factor-Beta Phosphorylation At Serines 340 And 583 By Pkc Gamma In Dopamine Release

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKC gamma, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKC gamma-knock-out (KO) and that PKC gamma-KO mice showed parkinsonian syndrome. However, the PKC gamma substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKC gamma substrates involved in dopamine release, we used PKC gamma-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKC gamma-KO mice. We focused on Pak-interacting exchange factor-beta(beta PIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKC gamma directly phosphorylates beta PIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated beta PIX in vivo. Classical PKC inhibitors and beta PIX knock-down (KD) significantly suppressed Ca2+ evoked dopamine release in PC12 cells. Wild-type beta PIX, and not the beta PIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by beta PIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of beta PIX at Ser340 and Ser583 has pivotal roles in Ca2+-evoked dopamine release in the striatum. Therefore, we propose that PKC gamma positively modulates dopamine release through beta 2PIX phosphorylation. The PKC gamma-beta PIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.