| Up-regulation of G-protein-coupled receptor kinase 5 in the a-Synuclein over-expression mouse model and cell model of Parkinson's disease Regulates Gene Transcription in NucleusParkinson's disease is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies (LBs). a-Synuclein as the major component of LBs was identified to be phosphorylated at Ser-129 (pS129-a-synuclein) in LBs. GRKs as members of serine/threonine protein kinases family phosphorylate activated GPCRs and thereby result in these receptors' desensitization. They play the critical role in cellular signaling. But the function of GRKs is not limited to the mediation of GPCR desensitization. Recently, some nonreceptor substrates of GRKs were found, including tubulin, synucleins and the ribosomal protein P2. Recently GRK5 has been correlated to the pathogenesis of Parkinson's disease, a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies (LBs).α-Synuclein as the major component of LBs was identified to be phosphorylated at Ser-129 (pS129-a-synuclein) in LBs. Recent event showed that phosphorylation at Ser-129 ofα-synuclein is essential for its toxicity to dopaminergic neurons.GRK5 was reported to phosphorylate a-synuclein at Ser-129 and in the following study GRK5 was detected to accumulate in Lewy bodies and colocalize withα-synuclein in the pathological structures of the brains of sPD patients. These evidents proved that GRK5 was involved in the pathogenesis of Parkinson's disease for the modification ofα-synuclein in LBs. In addition to the function of phosphorylation at Ser-129 ofα-synuclein, GRK5 has been found as a histone deacetylase kinase in the nucleus of cardiomyocytes. The DNA-binding nuclear localization sequence(NLS) contained in GRK5 and the activity of HDAC kinase detected in GRK5 make us explore the more potential function of GRK5 in parkinson's disease.We developed the human a-synuclein transgenic mice and the stable transfected humana-synuclein SHSY5Y cell lines to investigate the alters of GRK5 expression level and then determined whether it effects the activity of HDAC in Parkinson's disease. If it does, we want to quest the goal genes regulated by GRK5. Two parts were included in the research: 1 The expression level of GRK5 protein in the alpha-synuclein over-expression mouse model and cell model of Parkinson's disease and the distribution of the increased GRK5 protein:The mRNA and protein expression levels of GRK5 in various Parkinson's disease a-synuclein transgenic mice groups and 3m,6m and 9m hα-synuclein transgenic mice were measured by western blotting and real time PCR. At the same time, we used western blotting and Immunocytofluorescence to observe the distribution of the increased GRK5 protein. The results show to us, the expression level of GRK5 was elevated in Parkinson's diseaseα-synuclein transgenic mice and in the stable transfectedα-synuclein cell lines. And GRK5 protein accumulates not only in the cytosol but also in the nucleus.2 The function of GRK5 in cytosol and in nucleus:Western blotting, Immunohistoche-mistry, HDAC assay, ChIP and RNAi technique were used to detect the function of GRK5 in cytosol and in nucleus. The results suggest potential cytosolic GRK5 function, rather than alpha synuclein phosphorylative events, appears as the underlying mechanism of Parkinson's disease. Nuclear accumulation of GRK5 inhibits blc2 transcription and expression by enhancing the activity of HDAC and the opposite results are obtained by GRK5 repressed through shRNA.To sum up, we report in this study that overexpression of alpha synuclein in vivo or in vitro induces increase of cytosolic and nuclear GRK5 and we reveal an unexpected role for GRK5 in the regulation of apoptosis-or cell cycle-related genes at the first time. These results suggest that GRK5 has more extensive effects in the Parkinson's disease.
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