The Study Of Mechanism In MST1 Promotion Of Apoptosis

Background and ObjectivesThe protein kinase mammalian Sterile 20-like kinase 1(MST1) contains a Ste20-related kinase catalytic domain in the amino-terminal segment followed by a regulatory domain at the COOH-terminus. It has been implicated in the diverse biological functions including cell proliferation, differentiation, morphogenesis and cytoskeleton rearrangement. Previous studies have indicated that non-catalytic tail of MST1 is cleaved by caspase-3 upon a numbers of apoptotic stimuli for example death receptor triggering by CD95/FasL and treatments with Staurosporine (STS), Ceramide, as well as heat shock and arsenite. The amino-terminal fragment of cleaved MST1 translocates into the nucleus, where it contributes to chromatin condensation and then apoptosis. Furthermore, under the condition of overexpressing MST1, the cleavage and herein induced apoptosis could also be observed. It has been reported that D326 and D349 are the two major cleavage sites. The kinase activation, nuclear translocation and ability to induce cell death of MST1 areapparently attenuated by mutating these cleavage sites. Recently it has been defined Threonine 183 as a dominating phosphoactivation site in subdomain VIII of MST1 and it is essential for kinase activation with the auto-phosphorylation of Threonine 183 within the MST1 kinase domain. Hippo, a mammalian homolog of MST1/2 in Drosophila, has been extensively shown to restrain cell from growth and proliferation through the inhibition of transcription and/or degradation of cyclin E and DIAPs or phosphorylation and inhibition of Yorkie. In mammals, it has been indicated that MST1 can activate c-Jun n-terminal kinase (JNK) and p38MAPK kinase signaling pathways through MKK4/MKK7 and MKK3/MKK6, respectively. Recently it is suggested that JNK is essential and sufficient for MST1 activation and MST1-mediated apoptosis via phosphorylating the serine 82 on MST1. Furthermore, MST1-induced caspase activation and apoptosis are inhibited by dominant-negative mutant of JNK, not dominant-negative p38 or the p38 inhibitor.MST1 induces apoptosis by phosphorylating Histone H2B on a relatively conserved site, Ser-14 in mammalian cells and Ser-10 in Saccharomyces cerevisiae, respectively. We reported that MST1 has also been implicated in the control of FOXO-dependent neuronal cell death via phosphorylating FOXO3a at Ser-207 and the corresponding site of FOXO1 at Ser-212. Recently we have shown that phosphorylation of Threonine 120 by phosphoinositide 3-kinase/Akt can inhibit MST1-mediated pro-apoptotic signaling pathway.Sirt1 is a NAD+-dependent deacetylase with numbers of substrates that participates in various cellular processes. Deacetylation of these target proteins may either inhibit or activate their activities, thus influences many aspects of organism physiology, such as transcriptional silencing, genetic control of aging, cell metabolism, energy homeostasis, DNA repair and cell survival. And the p53 protein functions as a key tumor suppressor, and plays a vital role in invoking cellular responses to numerous stress signals, including DNA damage, hypoxia and aberrant proliferation. The way of p53 in maintaining genome stability is mainly carried out by p53-dependent apoptosis, which is in mediating tumor suppression or tumor clearance. In general, p53 biological activity in responding to DNA damage is tightly regulated by its post-translational modification status, particularly by site-specific phosphorylation, acetylation and ubiquitination. Sirt1 has been reported to strongly bind to its substrate p53 and can deacetylate p53 at Lysine 382. The Sirt1-mediated deacetylation antagonizes p53-dependent transcriptional activation and specifically inhibits p53-dependent apoptosis in response to DNA damage as well as oxidative stress.Previous studies have shown that MST1 promotes cell death is p53-dependent, but the molecular mechanism underlying MST1-p53 signaling during apoptosis is still largely unknown. In this work, we aim to identify the role of MST1 in promotion of genotoxic agents-induced apoptosis dependent on p53. And we also expect to find out the regulator mechanism among MST1, Sirt1 and p53.Methods1.1 Effects of MST1 and Sirt1 on the p53 and p21 transcriptional activityA. H1299 cells were co-transfected with 14*p53 artificial-luciferase construct with p53, MST1, MST1 K59R or Sirt1. Lysates were assayed for the dual-luciferase activity. B. H1299 cells were co-transfected the p21-luciferase promoter construct with the plasmids encoding p53, MST1 or Sirt1. Lysates were assayed for the dual-luciferase activity as in A.1.2 Effects of MST1 and Sirt1 on the p53-mediated cell death A. U2OS cells were transfected with the plasmids encoding MST1 together with p53 shRNA or control vector, then treated with Etoposide (36h) before apoptosis analysis and the apoptosis was analyzed by Annexin-V staining followed by flow cytometry. B. Both HCT116 p53+/+ and p53-/- cells stably transfected with MST1 or control vectors were treated with Cisplatin (CDDP) and the cell death analysis was performed as in A. C. U2OS cells were transfected with the plasmids encoding MST1 together with Sirt1 or control empty vector and the cell death analysis was performed as in A.2.1 Effects of MST1 on Sirt1-mediated deacetylation of p53A. Lysates of HCT116 p53+/+ cells stably transfected with MST1 or control vector were immunoblotted with anti-p53-K382Ac or anti-p53 antibody and the loading was normalized by using the protein 14-3-3β. B. FLAG-Sirt1 immunoprecipitates from cells transfected with HA-p53 or GFP-MST1 were immunoblotted with anti-HA or anti-FLAG antibody. C. Lysates of 293T cells transfected with the plasmids encoding p53, p300, Sirt1, MST1 were immunoblotted with anti-p53-K382Ac antibody as well as the other antibodies.2.2 How MST1 enhances p53 acetylation through its interaction with Sirt1A. In vitro MST1 kinase assay was performed by incubating the recombinant active MST1 with GST-Sirt1 as substrate in the presence of 32P-ATP. The reaction was analyzed by SDS-PAGE followed by autoradiography. B. In vitro MST1 kinase assay was performed by incubating the recombinant active MST1 with different Sirt1 fragments (P1, P2, and P3) in the presence of 32P-ATP. The reaction was analyzed by SDS-PAGE followed by autoradiography. C. In vitro phosphorylation was performed by incubating active MST1 and Sirt1 in the presence of cold ATP. Then the deacetylation reaction was performed by incubating the products of in vitro phosphorylation reaction with the acetylated p53. The reaction was analyzed by SDS-PAGE followed by immunoblotting with anti-p53-K382Ac, anti-GST or Sirt1 antibody.Results and Conclusions1.1 MST1 promotes p53 transcriptional activities by inhibiting Sirt1 activity.Sirt1 dramatically inhibits p53-mediated expression of the 14*p53 reporter. Wild type MST1, significantly rescues Sirt1-induced p53 repression, but the kinase dead MST1 in which the ATP binding site was mutated (MST1 K59R) failed to do so. Similarly, Sirt1 inhibits p53-dependent p21 luciferase's activity and MST1 could reverse Sirt1-induced p21 repression. Taken together, MST1 promotes p53 biological activities by inhibiting Sirt1.1.2 MST1 promotes cell death is p53-dependent and Sirt1 can inhibition of MST1-induced apoptosis.P53 knockdown in U2OS cells reduces MST1 overexpression-induced cell death upon Etoposide treatment. We also found that MST1 cannot induce Cisplatin-triggered cell death in HCT116 p53-/- cells. However, MST1 increases Cisplatin-induced cell death in HCT116 p53+/+ cells. These experiments together strikingly support our conclusion that MST1 induces cell death under DNA damage is p53-dependent. And Sirt1 overexpression decreases MST1 mediated apoptosis.2.1 MST1 inhibits Sirt1-mediated deacetylation of p53.In vitro MST1 kinase assay using recombinant p53 or Histone H2B as the substrate, we found MST1 failed to phosphorylate p53 in vitro. Western blot analysis by using p53 specific acetylation antibody revealed that the acetylation of endogenous p53 was upregulated in the MST1 overexpressing cells. Then first, we examined the physical interaction between MST1 and Sirt1, indicating they might interact functionally. Secondly, we observed that MST1 overexpression reduced the interaction between p53 and Sirt1. Consistently, the Sirt1 mediated deacetylation of p53 is inhibited in the presence of MST1 in cells. Collectively, MST1 inhibits Sirt1 mediated p53 deacetylation. We also found that MST1 reduces Sirt1-mediated FOXO3 deacetylation in vitro, indicating that MST1 might regulate the biological function of other Sirt1's substrates.2.2 MST1 enhances p53 acetylation through phosphorylating Sirt1.In vitro MST1 kinase assay using the recombinant Sirt1 as the substrate indicates Sirt1 could be phosphorylated by MST1. We then delineated the phosphorylated region within Sirt1 using recombinant GST fusion proteins encoding three non-overlapping Sirt1 domains (peptides P1-P3). In vitro kinase assay shows the P3 fragment containing C-terminal 489-747 amino acids is the major region of phosphorylation. And MST1 phosphorylation of Sirt1 indeed decreases Sirt1-induced p53 deacetylation by using in vitro phosphorylation followed by in vitro deacetylation reaction. Here we elucidate the mechanism of MST1 regulating p53 through negative regulation of Sirt1's deacetylation activity.