Phosphorylation of synucleins by members of the Polo-like kinase family.
ABSTRACT 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.
- SourceAvailable from: Longhou Fang[Show abstract] [Hide abstract]
ABSTRACT: Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates. Copyright © 2015. Published by Elsevier Inc.Developmental Biology 05/2015; 8. DOI:10.1016/j.ydbio.2015.05.011 · 3.64 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson's disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effect of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues S87 and S129. Whereas the A30P and A53T mutants slightly affected pS129 levels compared to WT α-syn, the E46K mutation significantly enhanced S129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate, nor due to alterations in endogenous kinase levels, but mostly linked with enhanced nuclear and ER accumulation. Importantly, lentiviral mediated overexpression in mice also showed enhanced pS129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 02/2015; 290(15). DOI:10.1074/jbc.M114.610774 · 4.60 Impact Factor
- [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.Neurobiology of Disease 06/2014; 70. DOI:10.1016/j.nbd.2014.06.018 · 5.20 Impact Factor