The role of Pak-interacting exchange factor-β phosphorylation at serines 340 and 583 by PKCγ in dopamine release.

Protein kinase C has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is (KO) and that mice showed parkinsonian syndrome. However, the substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the duanyu1531γ substrates involved in dopamine release, we used duanyu1531γ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of duanyu1531γ-KO mice. We focused on Pak-interacting exchange factor-β (βPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that duanyu1531γ directly phosphorylates βPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that phosphorylated βPIX in vivo. Classical duanyu1531 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 duanyu1531γ positively modulates dopamine release through β2PIX phosphorylation. The phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

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