Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein.
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.
Okochi
M
M
Adolf-Butenandt Institute, Department of Biochemistry, Ludwig-Maximilians University, 80336 Munich, Germany.
Walter
J
J
Koyama
A
A
Nakajo
S
S
Baba
M
M
Iwatsubo
T
T
Meijer
L
L
Kahle
P J
PJ
Haass
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001923:Brain Chemistry; D047390:Casein Kinase II; D047388:Casein Kinases; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016716:PC12 Cells; D010300:Parkinson Disease / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000235:genetics; D051843:Synucleins; D051844:alpha-Synuclein
D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C497604:SNCA protein, human; C497606:Snca protein, rat; D051843:Synucleins; D051844:alpha-Synuclein; D012694:Serine; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases