Phosphorylation of ␣-synuclein (␣-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating ␣-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate ␣-syn and ␤-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate ␣-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate ␤-syn at Ser-118, whereas no phosphorylation of ␥-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103-140) of ␣-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of ␣-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) ␣-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in ␣-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of ␣-syn and ␤-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.Increasing evidence suggests that phosphorylation may play an important role in the oligomerization and fibrillogenesis (1), Lewy body formation (1, 2) and neurotoxicity of ␣-synuclein (␣-syn) 5 in vivo (3). The majority of ␣-syn within Lewy bodies (LBs) in diseased human brains and animal models of Parkinson disease (PD) and related synucleinopathies is phosphorylated at Ser-129 (Ser(P)-129) (1, 2, 4 -7). Although recent studies support the notion that phosphorylation at Ser-129 is related to pathology and blocks ␣-syn fibrillization in vitro (8, 9), the exact mechanisms by which phosphorylation at Ser-129 modulates ␣-syn aggregation and toxicity in vivo remain elusive. Unraveling the role of phosphorylation in modulating the physiological and pathogenic activities of ␣-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo.Several kinases that phosphorylate ␣-syn at serine and tyrosine residues, primarily in its C-terminal region, have been identified using in vitro kinase assays and co-transfection studies. Casein kinase I and II, G-protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6), and calmodulin-dependent kinase II (10 -12) phosphorylate ␣-syn at Ser-129. Ser-87 is the only residue outside the C-terminal region report...

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The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.