Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's dise ase, since rare autosomal dominant mutations are associated with early onse t of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected ce ll lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t(1/2) 54 h) and no endoproteolytic processing was obser ved. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines pho sphorylation was highly sensitive to phosphatases, since okadaic acid marke dly 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 pho sphorylation site was located within a consensus recognition sequence of ca sein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopepti de 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-synuclei n is constitutively phosphorylated within its C terminus and may indicate t hat the function of alpha-synuclein is regulated by phosphorylation/dephosp horylation.