Phosphorylation of alpha-synuclein (alpha-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 alpha-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 alpha-syn and beta-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate alpha-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate beta-syn at Ser-118, whereas no phosphorylation of gamma-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 alpha-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of alpha-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) alpha-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in alpha-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 alpha-syn and beta-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.
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"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). "
[Show abstract][Hide abstract] ABSTRACT: Alpha-Synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.
Full-text · Article · Jun 2014 · Neurobiology of Disease
"Comparative studies suggest that PLK2 and PLK3 directly phosphorylate α-synuclein at Ser129 in vitro with high stoichiometry, whilst PLK4 is unable to phosphorylate α-synuclein at this residue (Anderson et al., 2006; Inglis et al., 2009). The low kinase activity of PLK4 against α-synuclein, and other substrates, is partially explained by its unique structure, with only a single polo-box in the PBD, resulting in a much-reduced electropositive environment in its substrate-binding site (Mbefo et al., 2010). Human PLK5 lacks a functional kinase domain due to a premature stop codon in exon 6 and is therefore unable to phosphorylate α-synuclein. "
[Show abstract][Hide abstract] ABSTRACT: Substantial evidence implicates abnormal protein kinase function in various aspects of Parkinson's disease (PD) etiology. Elevated phosphorylation of the PD-defining pathological protein, α-synuclein, correlates with its aggregation and toxic accumulation in neurons, whilst genetic missense mutations in the kinases PTEN-induced putative kinase 1 and leucine-rich repeat kinase 2, increase susceptibility to PD. Experimental evidence also links kinases of the phosphoinositide 3-kinase and mitogen-activated protein kinase signaling pathways, amongst others, to PD. Understanding how the levels or activities of these enzymes or their substrates change in brain tissue in relation to pathological states can provide insight into disease pathogenesis. Moreover, understanding when and where kinase dysfunction occurs is important as modulation of some of these signaling pathways can potentially lead to PD therapeutics. This review will summarize what is currently known in regard to the expression of these PD-implicated kinases in pathological human postmortem brain tissue.
Full-text · Article · Jun 2014 · Frontiers in Molecular Neuroscience
"Syk also phosphorylates Y133 and Y136 (Negro et al., 2002), and c-Abl also phosphorylates Y39 aSyn (Mahul-Mellier et al., 2014). An emerging concept is that certain phosphorylation events might promote or prevent subsequent phosphorylation events in other residues (Negro et al., 2002; Mbefo et al., 2010). In fact, the double mutation of Y133 and Y136 to phenylalanines, designed to prevent phosphorylation in these residues, augments Y125 phosphorylation by Lyn (Negro et al., 2002). "
[Show abstract][Hide abstract] ABSTRACT: Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system.
Full-text · Article · May 2014 · Frontiers in Molecular Neuroscience