alpha-Synuclein has been implicated in the pathogenesis of
Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and
alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed
alpha-synuclein expression in transfected cell lines. In pulse-chase experiments
alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed.
alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat
pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to
phosphatases, since
okadaic acid markedly stabilized
phosphate incorporation. Phosphoamino
acid analysis revealed that phosphorylation occurred predominantly on
serine. Using site-directed mutagenesis we have identified a major phosphorylation site at
serine 129 within the C-terminal domain of
alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to
serine 87. The major phosphorylation site was located within a consensus recognition sequence of
casein kinase 1 (CK-1). In vitro experiments and two-dimensional
phosphopeptide mapping provided further evidence that
serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of
serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that
alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of
alpha-synuclein is regulated by phosphorylation/dephosphorylation.