Article

Contribution of endogenous G-protein-coupled receptor kinases to Ser129 phosphorylation of α-synuclein in HEK293 cells

Department of Neurology, Hematology, Metabolism, Endocrinology, and Diabetology, Faculty of Medicine, Yamagata University, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 06/2009; 384(3):378-82. DOI: 10.1016/j.bbrc.2009.04.130
Source: PubMed

ABSTRACT The majority of alpha-synuclein (alphaS) deposited in Lewy bodies, the pathological hallmark of Parkinson's disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of alphaS has been demonstrated to enhance the alphaS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of alphaS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of alphaS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of alphaS; however, knockdown of GRK2 or GRK5 did not decrease alphaS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of alphaS in HEK293 cells.

1 Follower
 · 
96 Views
 · 
0 Downloads
  • Source
    • "However, knockdown of either GRK5 or GRK2 failed to diminish the phosphorylation of α-synuclein in cell models (Sakamoto et al., 2009; Liu et al., 2010). In contrast, knockdown of GRK3 or GRK6 significantly decreased α-synuclein Ser129 phosphorylation levels (Sakamoto et al., 2009), suggesting further work is required to verify the role of GRK isoforms in phosphorylating α-synuclein. G protein coupled receptor kinase isoforms, 2, 3, 5, and 6, are highly expressed in the human brain. "
    [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.
    Frontiers in Molecular Neuroscience 06/2014; 7:57. DOI:10.3389/fnmol.2014.00057 · 4.08 Impact Factor
  • Source
    • "Overexpression of GRK2 or GRK5 in COS-1 cells showed that these kinases phosphorylate aSyn at S129 (Pronin et al., 2000). Phosphorylation of aSyn at S129 by endogenous GRKs was also demonstrated in HEK293 cells and it was observed that GRK3 and GRK6 play the main role in this modification (Sakamoto et al., 2009). GRK5 was found to colocalize with aSyn in the LBs of the substantia nigra of PD patients, but was not detected in cortical LBs of DLB, or in the glial cytoplasmic inclusions of MSA (Arawaka et al., 2006). "
    [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.
    Frontiers in Molecular Neuroscience 05/2014; 7. DOI:10.3389/fnmol.2014.00042 · 4.08 Impact Factor
  • Source
    • "In addition to their roles in regulating GPCR signaling, they can phosphorylate additional substrates including α -syn (Pronin et al. , 2000 ). The specifi c relevant isoforms are unclear as GRK2 and GRK5 phosphorylate α -syn in vitro and with overexpression in cells (Pronin et al. , 2000 ), while in knockdown experiments, GRK2 and GRK5 appear not to modulate α -syn phosphorylation, but GRK3 and GRK6 have signifi cant effects (Sakamoto et al. , 2009 ). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorylation is a key post-translational modification necessary for normal cellular signaling and, therefore, lies at the heart of cellular function. In neurodegenerative disorders, abnormal hyperphosphorylation of pathogenic proteins is a common phenomenon that contributes in important ways to the disease process. A prototypical protein that is hyperphosphorylated in the brain is α-synuclein (α-syn) - found in Lewy bodies and Lewy neurites - the pathological hallmarks of Parkinson's disease (PD) and other α-synucleinopathies. The genetic linkage of α-syn to PD as well as its pathological association in both genetic and sporadic cases have made it the primary protein of interest. In understanding how α-syn dysfunction occurs, increasing focus is being placed on its abnormal aggregation and the contribution of phosphorylation to this process. Studies of both the kinases and phosphatases that regulate α-syn phosphorylation are beginning to reveal the roles of this post-translational modification in disease pathogenesis. Modulation of α-syn phosphorylation may ultimately prove to be a viable strategy for disease-modifying therapeutic interventions. In this review, we explore mechanisms related to α-syn phosphorylation, its biophysical and functional consequences, and its role in neurodegeneration.
    Reviews in the neurosciences 04/2012; 23(2):191-8. DOI:10.1515/revneuro-2011-0067 · 3.31 Impact Factor
Show more