Phosphorylation of Synucleins by Members of the Polo-like Kinase Family

Abstract

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...

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner

“…Samples used for NMR spectroscopy contained 15 N-labeled αS in 50 mM HEPES buffer, 100 mM NaCl at pH 7.4. αS was phosphorylated at S129 by polo-like kinase 3 (PLK3) at 30°C and reached full phosphorylation after 2 h in agreement with previous studies (Mbefo et al, 2010). Phosphorylated αS (pS129-αS) was purified by semipreparative reverse-phase HPLC.…”

“…S129 can be phosphorylated by a variety of kinases in vitro and in vivo (Waxman and Giasson, 2010). In particular, polo-like kinases quantitatively and selectively phosphorylate αS at S129 and levels of polo-like kinase 2 are increased in the brains of patients with Lewy body disease (Inglis et al, 2009;Mbefo et al, 2010). However, the relevance of αS phosphorylation at S129 for neurotoxicity and disease remains controversial (Basso et al, 2013).…”

α-Synuclein disrupts stress signaling by inhibiting polo-like kinase Cdc5/Plk2

“…Specifically, αS(103-140) fails to be phosphorylated by all four Plks (Plk1-4) tested, whereas the intact protein is efficiently phosphorylated (28). Our results show that deletion of only 9 N-terminal residues abolishes the ability of Cdc5/Plk2 to phosphorylate αS in vitro and in vivo (Figs.…”

Serum inducible kinase is a positive regulator of cortical dendrite development and is required for BDNF-promoted dendritic arborization

“…In addition, aberrant BDNF signaling is involved in an array of neurological disorders, including mental retardation, autism, epilepsy, and neurodegenerative diseases [47]. Interestingly, SNK is regulated by LTP and epileptic stimuli [14], and is implicated in Parkinson disease and Alzheimer disease via α-synuclein phosphorylation [50][51][52]. Our work is the first to establish a relationship between BDNF and SNK in vitro; however, in this work we only show the effects of exogenous BDNF on SNK.…”