The Phosphorylation Of SIRT2 By GSK3β

With the continuous improvement in medicine, people pay more and more attention to therapies of nervous system diseases. However, due to the particularity of the nervous system, we have known less about how the nervous system works and pathogenesis of neurological diseases so far. Moreover, it is still lack of effective treatment for diseases of the nervous system in clinical practice.The basic unit of the structure and function in nervous system is the neuron, also known as a "nerve cell". One of the pathological characteristics of many nervous system diseases is the death of neurons. After the cell is damaged, it can activate or inhibit the transcription of a series of genes, some of which probably involved in maintaining the cellular homeostasis and repairing. Eventually, the cells will be destined to die or live due to the transcription of genes.Cell apoptosis plays a very important role in the process of organism growth and responding to external stimuli. In normal physiological conditions, various kinds of neurons keep balance. If some kinds of neurons lose, it likely leads to pathological changes, such as Parkinson’s disease. As the pathogenesis of the diseases is still unclear, the treatment of them is symptomatic. Although we have known that Parkinson’s disease has a relationship with genetic mutations and environmental toxins, the molecular mechanism of neuron death is still unclear. Clarifying the mechanism may do a favor for seeking the effective treatment of neurological diseases. When the neuron responds to the external and internal stimuli, it will activate the cell survival pathways. The pathways that rely on gene transcription are of more importance.Gene transcription is mainly regulated by the transcription factors and under the epigenetic control. Histone deactylases are a class of enzymes that remove acetyl groups from a ε-N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly and keeping histones balance in acetylating. Histone deacetylases have a tight relationship with the inhibition of gene transcription, involving in the process of gene silencing. At the chromatin level, deactylating histones can regulate a series of biological activities including chromatin reorganization, transcriptional activation or inhibition, cell cycle, cell differentiation, cell survival and apoptosis.Sirtuin proteins are a class of enzymes that need NAD+(nicotinamide adenine dinucleotide) to deactylate histones. Sir2, the first member of the sirtuin family, was identified in yeast cells. And, in mammal animal, there are seven homologs of Sir2, SIRT1-SIRT7.They all contain a conserved catalytic core domain and unique additional N-terminal and/or C-terminal sequences of variable length. Moreover, their subcellular localizations are not the same and they exert the different biological functions.SIRT2 is the only member of Sirtuin family, which resides most prominently in the cytoplasm. But it also participates in regulating the transcription activities happening in the nucleus. Some researches show that SIRT2 also interacts with and deacetylates Lys16 on histone H4. What’s more, some researches show that SIRT2 exhibits a protective effect on Parkinson’s disease model.So far, the researches have shown that histone deacetylases activities can be regulated by protein kinases, influencing chromatin reorganization, transcription activation or inhibition, cell cycle, cell differentiation and apoptosis, etc.Protein kinase is a kind of enzyme that modifies other proteins by chemically adding phosphate groups to them. And, protein kinases are distributed in nuclei, mitochondria, microsome and cytoplasm of cells. They are capable of transferring a phosphate group from a nucleoside triphosphate to specific amino acids with a free hydroxyl group. Most of them usually act on serine, threonine or tyrosine. In addition, protein kinases are able to phosphorylate amino acids in other proteins.At present, kinases CDK5 and GSK3 beta have been widely studied in neurodegenerative diseases.It has been reported that the abnormal accumulation of CDK5 regulatory subunits p35 induced tau protein phosphorylation in Alzheimer’s disease. And, the tau protein can be directly phosphorylated by CDK5. CDK5 may also indirectly exert its functions by interacting with other kinases and phosphatases of the tau protein. The abnormal activity and distribution of CDK5 are toxic to neurons and may cause some neurodegenerative diseases. Studies show that Cdk5 could phosphorylate SIRT2 in neurons, affecting cell adhesion, migration, neurite outgrowth, and growth cone collapse.Another important protein kinase is Glycogen synthase kinase 3 beta (GSK3 beta). GSK3 beta is a key enzyme involved in glycogen metabolism. GSK3 beta also can affect mitochondrial permeability and neuronal survival factor to get involved in regulation of cell apoptosis.GSK3 beta can regulate cancer cell differentiation, proliferation, apoptosis and even cytoskeleton dynamics by regulating glycogen synthase, protooncogene or participating in the intracellular classic signal pathways, such as nuclear factor kappa B signal pathway.Some research shows that, in Parkinson’s disease mouse models, which are mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the activation of GSK3 beta can induce cell death in DA neurons. And, the inhibition of GSK3 beta can not only protect DA neurons to reduce MPTP mediated apoptosis, but also restore the loss of DA neurons to mitigate behaviour disorders.It has been reported in the Alzheimer’s disease that GSK3 beta cooperates with CDK5 to regulate Tau proteins. Does another substrate regulated by GSK3 beta and CDK5 exist? Therefore, maybe some substrates regulated by both GSK3 beta and CDK5 exists in PD. In patients with PD, one of pathologic characteristics is forming Lewy body. PD is caused by loss of dopaminergic neurons and development of Lewy bodies containing a-synuclein in the substantia nigra. Sirtuin 2 inhibitors rescue alpha-synuclein-mediated toxicity in models of Parkinson’s disease. Cdk5 could phosphorylate SIRT2 and we found that potential phosphorylation sites, which are targeted by GSK3 beta.Does GSK3 beta cooperate with CDK5 or other enzymes to interact with SIRT2?After analysing the candidates that probably participates in regulating activities of SIRT2, we chose GSK3 beta and CDK5 as our research objects. As it had been reported that CDK5 could interact with SIRT2, we wanted to determine whether the GSK3 beta associate with SIRT2 or not. Firstly, we constructed the SIRT2 plasmid vector and transformed it to the bacteria. Then, after inducing the expression of SIRT2, we extracted and purified the SIRT2 protein from the bacteria. Subsequently, we used the protein to perform the kinase assay in vitro. Then we used the method of Pro-Q diamond to measure SIRT2 phosphorylation level and the results show that GSK3 beta has a relationship with SIRT2.However, we found that the Pro-Q diamond isn’t sensitive to determine the phosphorylation of SIRT2 protein in vitro. We made use of the radioautography method, which is more sensitive and targeted to test the level of protein phosphorylation. In the kinase assay, the kinases can transfer a phosphate group containing radio-label γ [32P] from γ [32P]-ATP to SIRT2. The SIRT2 phosphorylation level in vitro is measured by assessing phosphate groups containing radio. We found that, the kinase GSK3 beta and CDK5 could phosphorylate SIRT2 respectively. Interestingly, when the kinase GSK3 beta and CDK5 were added simultaneously to react with SIRT2 in test tubes, the level of its phosphorylation increased and was higher than it was when GSK3 beta or CDK5 was added alone. The results indicated that, GSK3 beta and CDK5 could interact with SIRT2 simultaneously to enhance its phosphorylation.To detect the SIRT2 phosphorylation sites, we retrieved the phosphorylated SIRT2 in kinase assay to run the SDS-PAGE gel and then performed phosphorylation mass spectrometry. We get some sites that might be phosphorylated by GSK3 beta. Then, we mutated the potential phosphorylation sites of SIRT2 and did the kinase assay. The results showed a decrease in the level of SIRT2 phosphorylation, in which GSK3 beta get involved. And, this finding further proved GSK3 beta might phosphorylate sites.The results of kinase assay in vitro had shown that GSK3beta interacted with SIRT2. Does it happen in the cells too? This problem prompted us to further study in the cells. We transfected the plasmids expressing HA-GSK3 beta or SIRT2-FLAG into HEK293 cells. Then, we tried to pull down the compounds containing either GSK3 beta or SIRT2 from cell lysate by co-immunoprecipitation. Next, we used the FLAG-tag antibody targeting SIRT2-FLAG protein to perform co-immunoprecipitation assay. The result showed that HA-GSK3 beta protein could be co-immunoprecipitated with SIRT2-FLAG In addiction, HA-tag antibody attaching to HA-GSK3 beta was used to perform co-immunoprecipitation assay. We found that HA-GSK3 beta was able to co-immunoprecipitate with SIRT2-FLAG. Together, these experiments demonstrated that HA-GSK3 beta could interact with SIRT2 in the cells.In neurons, what a role SIRT2 is playing? And, could the amount of SIRT2 have an effect on neurons? Whether over expression of SIRT2 affects neuronal survival or not? We transfected the plasmids expressing phMGFP or phMGFP-SIRT2 into neurons respectively and measured the cell survival. We found that the mortality of phMGFP-SIRT2 group was higher than phMGFP group. Cell survival rate was calculated and P= 0.024< 0.05, which indicates over expression of SIRT2 leads to neuron death.And, whether the neuron death caused by over expression of SIRT2 is related with GSK3 beta or not? The problem will be answered in our further study.Our study identified the phosphorylation sites recognized by the GSK3 beta and CDK5. And, we demonstrated that GSK3 beta and CDK5 could interact with SIRT2 simultaneously to increase the level of its phosphorylationon. These findings provide novel insights to explain the pathogenesis of neuron death, doing a favor for seeking the molecular targets and providing novel methods in treating neurodegenerative diseases.