P38Regulates G2/M Arrest Of U2OS Cell Through Phosphorylation Of HPC2

ObjectivesCheckpoints are control mechanisms that ensure the proper timing of cell cycle events by enforcing the dependency of late events on the completion of early events. Entry into mitosis is blocked by the G2checkpoint mechanism when DNA is damaged. The G2/M checkpoints is consisted of complicated signal molecules in which cdc2is essential for entry into mitosis. Activated Cdc2binds to cyclin B initiate mitosis and its activation is controlled by multiple proteins such as p53, p21, cdc25and which could phosphorylate different sites of cdc2and regulate its location and expression. Recent research suggest that Rb protein repress the activity of cdc2promoter in coordination with HPC2. HPC2is a member of Polycomb complex group and known as the function of gene expression repression and SUMO E3ligase activity. Our group used to find that p38MAP kinase bind to HPC2in vitro which give us a hypothesis that p38may take part in the regulation of cell cyle through HPC2. In order to investigate the relationship between p38and HPC2, we predicted the potential p38regulated phosphorylation sites in HPC2and mutated them to detect their roles in manipulation of cell cycle. Moreover, HPC2carries out transcriptional repression of targeted genes through formation of PRC1complex and binding to the pormoter of genes to interfere the initiation and elongation of transcription. We also assumed that p38may regulate formation of PRC1through phosphorylation dependent manner. By means of over-expressing wild type HPC2, HPC2T497A, HPC2T494R and RING1into U2OS cells, we explored the mechanism of PRC1stability and formation.Methods1. Human U2OS cells were stimulated by arsenic to induce G2/M arrest;2. Over-express HPC2-V5in U2OS cells, and stimulate cells with arsenic at different timing, then immuno-precipitate HPC2-V5by V5-beads. Detect p38by western blot if any binds to HPC2-V5.3. Overexpress HPC2-V5in U2OS cells and observe the subcellular location of HPC2-V5and endogenous p38with immunofluorescence.4. In order to investigate which domain is responsible for the interaction between p38and HPC2, we construct the deletional plasmids of HP2-V5, and express them in U2OS cells. Then precipitate HPC2-V5by I.P. and compare the difference of p38precipitated by those HPC2mutants with western blot.5. Predict the potential phosphorylation sites modified by p38in HPC2, mutate and express them in U2OS cells and observe the their roles in arsenic induced G2/M arrest.6. Over-express the phosphoryaltion sites mutated HPC2-V5and Ringl in U2OS cells and precipitate HPC2using I.P. in order to detect any RING1binds to HPC2which may help to investigate the mechanism of PRC1formation and stability. Results1. Most of the normal U2OS cells are in G1phase, but arsenic could increase the amount of cells in G2/M phase significantly through decreasing the expression of cdc2.2. Inhibition of p38by SB203580alleviated G2/M arrest induced by arsenic, and the interferencence of HPC2by siRNA had a similar effect. Activation of p38by adeno-virus MKK6b E mimiced G2/M arrest cells induced by arsenic successfully.3. Arsenic induced interaction of endogenous p38and over-expressed HPC2in U2OS cells through a time-dependent manner.4. Arsenic induced co-localization of endogenous p38and over-expressed HPC2in nucleus of U2OS cells, then HPC2migrated into cytosol with p38.5. Deletion of the first374amino acids of HPC2abolished the interaction between p38and HPC2in U2OS cells.6. HPC2T497A mutant reversed the arsenic induced G2/M arrest in U2OS cells by protection of cdc2expression.7. Arsenic induced interaction of HPC2, Ringl and Rb, but mutation of threonine497site and lysine495site in HPC2damaged the interaction of HPC2and Ringl.Conclusions1. Arsenic induced U2OS G2/M arrest through p38and HPC2pathway, which repress the expression of cdc2after activated by arsenic.2. Arsenic could induce participation of p38in the complex which HPC2belong to during G2/M arrest, and the first373amino acids are responsible for the interaction between them.3. Phosphorylation and sumoylation of HPC2at threonine497and lysine495 sites respectively are vital for the formation and stability of PRC1complex.4. Phosphorylation of HPC2threonine497site is crucial for the repression of cdc2expression induced by aresenic.