Tyrosine hydroxylase (TH) is a rate-limiting
enzyme for the biosynthesis of catecholamines including
dopamine. The relationship between proteasomal dysfunction
and the etiology of Parkinson’s disease has been
suggested, but it is unknown if TH protein is affected by
proteasomal dysfunctions. Here, we examined the effect of
inhibition of ubiquitin–proteasomal pathway on biochemical
characteristics of TH protein in the neuronal cells.
Inhibition of 20S or 26S proteasome by proteasome
inhibitor I, or MG-132 in NGF-differentiated PC12D cells
induced dot-like immunoreactivities with the anti-40Serphosphorylated
TH (p40-TH) antibody. These dots were
tightly co-localized with ubiquitin and positive to Thioflavine-
S staining. These dot-like immunoreactivities were
not obvious when immunostaining was performed against
total-TH or choline acetyltransferase. Western blotting
analysis showed time-dependent increase of p40-TH in the
Triton-insoluble fractions. We also examined the effect of
okadaic acid, an inhibitor of protein phosphatase 2A, which
is a phosphatase acting on p40-TH. Okadaic acid increased
the amount of insoluble p40-TH. These data suggest that
p40-TH is prone to be insolubilized and aggregated by
dysfunction of an ubiquitin–proteasome system in PC12D
cells.
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