Phosphorylation Of MST1 Research Articles

OSM mitigates post-infarction cardiac remodeling and dysfunction by up-regulating autophagy through Mst1 suppression

The incidence and prevalence of heart failure (HF) in the world are rapidly rising possibly attributed to the worsened HF following myocardial infarction (MI) in recent years. Here we examined the effects of oncostatin M (OSM) on postinfarction cardiac remodeling and the underlying mechanisms involved. MI model was induced using left anterior descending coronary artery (LAD) ligation. In addition, cultured neonatal mouse cardiomyocytes were subjected to simulated MI. Our results revealed that OSM alleviated left ventricular remodeling, promoted cardiac function, restored mitochondrial cristae density and architecture disorders after 4weeks of MI. Enhanced autophagic flux was indicated in cardiomyocytes transduced with Ad-GFP -LC3 in the OSM treated group as compared with the MI group. OSM receptor Oβ knockout blocked the beneficial effects of OSM in postinfarction cardiac remodeling and cardiomyocytes autophagy. OSM pretreatment significantly alleviated left ventricular remodeling and dysfunction in Mst1 transgenic mice, while it failed to reverse further the postinfarction left ventricular dilatation and cardiac function in the Mst1 knockout mice. Our data revealed that OSM alleviated postinfarction cardiac remodeling and dysfunction by enhancing cardiomyocyte autophagy. OSM holds promise as a therapeutic target in treating HF after MI through Oβ receptor by inhibiting Mst1 phosphorylation.

Melatonin alleviates postinfarction cardiac remodeling and dysfunction by inhibiting Mst1.

Melatonin reportedly protects against several cardiovascular diseases including ischemia/reperfusion (I/R), atherosclerosis, and hypertension. The present study investigated the effects and mechanisms of melatonin on cardiomyocyte autophagy, apoptosis, and mitochondrial injury in the context of myocardial infarction (MI). We demonstrated that melatonin significantly alleviated cardiac dysfunction after MI. Four weeks after MI, echocardiography and Masson staining indicated that melatonin notably mitigated adverse left ventricle remodeling. The mechanism may be associated with increased autophagy, reduced apoptosis, and alleviated mitochondrial dysfunction. Furthermore, melatonin significantly inhibited Mst1 phosphorylation while promoting Sirt1 expression after MI, which indicates that Mst1/Sirt1 signaling may serve as the downstream target of melatonin. We thus constructed a MI model using Mst1 transgenic (Mst1 Tg) and Mst1 knockout (Mst1-/- ) mice. The absence of Mst1 abolished the favorable effects of melatonin on cardiac injury after MI. Consistently, melatonin administration did not further increase autophagy, decrease apoptosis, or alleviate mitochondrial integrity and biogenesis in Mst1 knockout mice subjected to MI injury. These results suggest that melatonin alleviates postinfarction cardiac remodeling and dysfunction by upregulating autophagy, decreasing apoptosis, and modulating mitochondrial integrity and biogenesis. The attributed mechanism involved, at least in part, Mst1/Sirt1 signaling.

Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration

Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration.

Malibatol A enhances alternative activation of microglia by inhibiting phosphorylation of Mammalian Ste20-like kinase1 in OGD-BV-2 cells

Objective: To investigate the polarization effect of Malibatol A on oxygen-glucose deprivation (OGD)-BV-2 cells, and the possible molecular mechanism involved in c-Abl-MST signaling pathway.Method: The OGD BV-2 cell model was established. BV-2 cells were exposed to OGD for 8 h followed by reperfusion for 15 h with Malibatol A at different concentration of 0.5, 1, 2, 4, 8, 16 μM or without it. And then cells, mRNA and protein were harvested respectively. The cell viability and apoptosis were measured by MTT assay and flow cytometry. The mRNA of classical activated microglia (M1) markers (MCP-1, IL-1 and TNF-α) and alternatively activated microglia (M2) markers (Ym-1, CD206, IL-10, TGF-β) in BV-2 cells were measured by RT-PCR. Meanwhile, the proteins of Ym-1 and CD206 was assayed by flow cytometry. Furthermore, the expression of c-Abl and MST was measured by Western blot.Result: Malibatol A significantly decreased apoptosis and increased viability of OGD BV-2 cells in a dose-dependent manner. In the presence of Malibatol A, the mRNA levels of Ym-1, CD206, IL-10 and TGF-β mRNA was significantly increased in OGD-BV-2 cells, while the mRNA levels of MCP-1, IL-1 and TNF-α was obviously down-regulated. Meanwhile, the proteins of Ym-1 and CD206 was raised in OGD BV-2 cells with Malibatol A. Besides, Malibatol A also inhibited OGD-induced p-MST1(Y433) in BV-2 cells.Conclusion: Malibatol A could attenuate OGD-induced BV-2 cell injury and promote M2 microglia polarization. The mechanism may be related to inhibition of MST1 phosphorylation at Y433.

Abstract 3291: Geranylgeranylation signals to the hippo pathway for breast cancer cell proliferation and migration

Abstract Protein geranylgeranylation (GGylation) is an important biochemical process for cellular localization of many signaling molecules including Rho and Rab small GTPases and heterotrimeric GTPase gamma subunits. Previous studies have shown that GGylation is essential for cancer cell survival. However, the molecular mechanism mediating the cell survival effect remains elusive. Here we show that the hippo pathway is the downstream pathway of GGylation in breast cancer cells. Inhibition of GGylation enhanced phosphorylation of Mst1, Mst2 and Lats1 kinases, and eliminated LPA-induced activation of Yap and Taz and the hippo pathway-dependent transcription of Cyr61 and CTGF. We have demonstrated that the inhibitory effects of atorvastatin, an inhibitor of the biosynthesis of geranylgeranyl pyrophosphate (GGPP), and GGTI-298, an inhibitor of geranylgeranyltransferase I (GGTase I), on cell proliferation and migration are dependent on the hippo pathway in the breast cancer cells. In addition, knockdown of Yap and Taz produced the similar inhibitory effect on the cell proliferation and migration to that of atorvastatin and GGTI-298, suggesting the functional correlation between GGylation and the hippo signaling. Thus, our studies have demonstrated that the hippo pathway mediates the GGylation-dependent cancer cell survival and migration. Citation Format: Wenyi Mi, Qiong Lin, Chandra Childress, Wannian Yang. Geranylgeranylation signals to the hippo pathway for breast cancer cell proliferation and migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3291. doi:10.1158/1538-7445.AM2014-3291

Abstract 18363: Activation of the Hippo Pathway, Associated With Suppression the Canonical Wnt Signaling, Contributes to the Pathogenesis of Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an enigmatic disease characterized by excess fibro-adipocytes in the heart, cardiac arrhythmias and heart failure. Molecular genetic studies have identified mutations in several desmosome proteins as causes of ARVC. Our objective is to delineate the molecular links between the desmosomes and fibro-adiposis in ARVC. Expression levels and localization of plakophilin 2 (PKP2), plakoglobin (JUP), desmocollin 2 (DSC2), desmosome proteins and connexin 43 (GJA1), a member of Gap junction, in the human hearts with ARVC, were reduced. To test whether molecular remodeling of the junctions affected signaling pathways, we analyzed the Hippo kinase pathway, which is regulated by cell-cell contact and known to suppress the canonical Wnt signaling, the latter implicated in the pathogenesis of ARVC. Accordingly, Merlin, an upstream regulator of the Hippo kinase cascade, was activated and YAP, the downstream effector was inactivated by phosphorylation. To gain mechanistic insights, expression of Pkp2, the most common causal gene for ARVC, was stably knocked down using shRNA in the HL-1 cells. Transcriptome analysis showed reduced mRNA levels of the canonical Wnt and Hippo targets including Ctgf, an inhibitor of adipogenesis. KD of PKP2 was associated with a dramatic reduction in pPKC-α level, in accord with the known function of PKP2 as a platform for phosphorylation of PKC-α. Reduced pPKCα, an inhibitor of Merlin through phosphorylation, was associated with activation of Merlin and cascade phosphorylation of MST1, LATS1/2 and YAP, constituents of the Hippo pathway. Consequently, signaling through TEAD, the transcription factor of the Hippo pathway, was reduced and sequestration of pβ-catenin in the cytoplasm was increased, leading to suppression of the canonical Wnt signaling and enhanced adipogenesis. KD of pLAT1/2 using shRNAs partially rescued the molecular phenotype. Thus, human ARVC is characterized by molecular remodeling of the intercalated discs, which results in inactivation of PKCα, phosphorylation of the Hippo kinases, suppression of the canonical Wnt signaling and enhanced adipogenesis. The findings implicate the Hippo pathway in the pathogenesis of ARVC.

Okadaic Acid: A Tool to Study the Hippo Pathway

Mammalian Ste20-like kinases 1 and 2 (MST1 and MST2) are activated in NIH3T3 cells exposed to okadaic acid. The Hippo pathway is a newly emerging signaling that functions as a tumor suppressor. MST1 and MST2 work as core kinases of the Hippo pathway and their activities depend on the autophosphorylation, which is negatively regulated by protein phosphatase 2A (PP2A). Okadaic acid has been frequently used to enhance the phosphorylation of MST1 and MST2 and to trigger the activation of the Hippo pathway. However other components of the Hippo pathway could also be targets of okadaic acid. In this review we first briefly summarize the molecular architecture of the Hippo pathway for the reference of researchers outside the field. We explain how MST kinases are regulated by PP2A and how okadaic acid activates MST2. Thereafter we discuss which components of the Hippo pathway are candidate substrates of protein phosphatases and which points we need to consider in the usage of okadaic acid to study the Hippo pathway.

Abstract 17445: NF2 Regulates Mst1 Activation and Ischemia Reperfusion Injury

Neurofibromin 2 (NF2)/merlin is a tumor suppressor that can regulate activation of the hippo pathway in Drosophila and the mammalian liver, thereby inhibiting cell growth and promoting apoptosis. We have shown previously that the mammalian homologue of hippo, Mst1, has a similar function in the heart. Therefore, we sought to determine whether NF2 can mediate activation of Mst1 in the heart and modulate cardiac ischemia/reperfusion (I/R) injury. Both NF2 and Mst1 are activated by oxidative stress in isolated cardiomyocytes in vitro and in the heart following I/R. Increased expression of NF2 in cardiomyocytes increased Mst1 phosphorylation (2.9±0.2-fold vs LacZ, p<0.05) and elicited apoptosis (4.8±1.7-fold vs LacZ, p<0.05) that was prevented by co-expression of kinase inactive Mst1 (61±12% decrease vs. NF2, p<0.05). To examine the importance of endogenous NF2 in I/R injury, we employed two different genetic models - systemic heterozygous nf2 deletion ( nf2 +/- ) and cardiomyocyte-specific disruption using nf2 floxed mice crossed with αMHC-Cre transgenic mice ( nf2 +/flox Cre). We performed global I/R (30/60min) injury on isolated perfused hearts using the Langendorff method. Functional parameters and infarct size were determined following the reperfusion phase. While nf2 +/- hearts showed no difference in function and infarct size (45±5% vs 48±7%, NS) compared to WT, nf2 +/flox Cre hearts had significantly smaller infarcts (22±5% vs 53±8%, p<0.05) and improved function versus nf2 +/flox controls. nf2 +/- hearts also failed to protect against I/R injury in vivo as Infarct/AAR (34±3% vs 32±2%, NS) and myocyte apoptosis (1.5±0.2% vs 1.7±0.3%, NS) were unchanged despite significant attenuation of Mst1 activation in ventricular homogenates (71±8% reduced vs WT, p<0.05). Preliminary experiments to determine the effect of cardiomyocyte nf2 deletion show a trend toward protection (Infarct/AAR, 23±6% vs 33±6%, n=4) in nf2 +/flox Cre versus nf2 +/flox control mice. Based on these data we conclude that nf2 disruption in cardiomyocytes is protective against I/R injury through inhibition of Mst1 activity. However, NF2 signaling in residential non-myocytes appears beneficial for the heart and obscures the detrimental function of NF2 in cardiomyocytes during I/R.

Abstract 2928: Regulation of MST1 and MST2 by mTOR and androgen receptor signaling in prostate cancer

Abstract The serine/threonine kinases MST1 and MST2 (hippo in Drosophila) were originally identified as pro-apoptotic proteins. In addition to their pro-apoptotic functions, evidence suggests that deficiency in MST1 and MST2 and their downstream effectors has been linked to developmental defects, tissue overgrowth, and cancer, including prostate cancer (PCa). However, the molecular mechanism that may modulate MST1 or MST2 signaling in PCa cells remains unknown. Here we investigate the impact of androgen receptor (AR) and PI3K-AKT-mTOR signaling on MST1 or MST2. We used biochemical, mutagenesis, histochemical, and pharmacological approaches to carry out this investigation. First, we demonstrate that AR and MST1 co-localize and form protein complexes in cell nuclei. Androgen further stimulates the interaction between the two proteins and reduces MST1 kinase activity in LNCaP PCa cells, suggesting that AR may sequester MST1 in an inactive form. Second, we showed that MST1 at Threonine120 (T120) was exclusively phosphorylated in cell nuclei, whereas MST2 at Threonine117 (T117), corresponding to MST1-T120, was phosphorylated only in the cytoplasm, although MST1 and MST2 were found in both locations. Similar phosphorylation patterns were also observed in prostate cancer tissue samples. Third, we have found that the T120 is not a physiologic target of AKT kinase in vivo, because the inhibition of AKT activity by a specific PI3K inhibitor, LY294002, had no effect on the phosphorylation of MST1 at the T120 site, but it did inhibit MST1-T117 phosphorylation. Furthermore, attenuation of mTOR activity by Ku0063794, a specific mTOR1 and mTOR2 inhibitor, abolished MST2-T117 phosphorylation, but without having an effect on the levels of phospho-MST1-Thr120. Lastly, the use of combined LY294002 and Ku0063794 compounds synergistically inhibited PCa cell growth, relative to a single compound. Our data suggest that AR and mTOR pathway signaling may oppose MST1 and MST2 in discrete cell locations. Thus, understanding the functional relationship between these signal networks in the context of cell proliferation and cell death may have important therapeutic implications in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2928. doi:10.1158/1538-7445.AM2011-2928

Epigenetic and post-transcriptional mechanisms are responsible for disruption of the Hippo tumor suppressor pathway in human hepatocellular carcinoma

Mounting evidence indicates that disruption of the Hippo tumor suppressor pathway contributes to hepatocarcinogenesis. In this study, we investigated the expression levels of the members of the Hippo signaling in a collection of human hepatocellular carcinoma (HCC) as well as in in vivo and in vitro models. Thus, promoter and genomic status of Hippo pathway members were assessed in HCC by methylation and microsatellite analyses, while effects of Hippo modulation on HCC growth were evaluated in HCC cell lines. We found that suppression of FAT4, NF2, MST1, MST2, LATS1, LATS2 and SAV1 expression by promoter hypermethylation and/or loss of heterozygosity was rare in HCC. However, levels of activated YAP, the protooncogene negatively modulated by the Hippo cascade, were high in most HCC when compared with corresponding non-tumorous surrounding livers, implying the existence of post-transcriptional mechanisms responsible for suppression of the Hippo pathway in human HCC. Indeed, we found that MST1 and MST2 were inactivated via phosphorylation at serine 120 and 117 residues, respectively, by the AKT. In HCC cell lines, transfection of AKT led to increased proliferation and decreased apoptosis that were paralleled by phosphorylation of MST1 and MST2 and activation of YAP protooncogene. AKT-driven growth of HCC cell lines was significantly decreased when AKT transfection was associated with silencing of YAP via siRNA. Accordingly, a strong suppression of MST1 and MST2 activity, with resulting activation of YAP, followed the stable transfection of an activated form of AKT into the mouse liver by hydrodynamic gene delivery. In conclusion, we have defined the epigenetic and post-transcriptional mechanisms responsible for suppression of Hippo signaling pathway in liver cancer. Also, our data imply AKT as the major negative regulator of Hippo cascade in human HCC and support the use of AKT inhibitors as therapeutic modality for human HCC.

Mst1 Stimulates Cell Protective Mechanisms of FoxO1 Through Phosphorylation

FoxO transcription factors control both survival and death. Mst1, a proapoptotic kinase, phosphorylates FoxO1. We investigated the role of FoxO1 in regulating survival and death and the effect of Mst1 upon the function of FoxO1 in cardiomyocytes. Mst1 physically interacts with FoxO1 and induces nuclear translocation of FoxO1. Mst1 fails to induce nuclear translocation of a FoxO1 mutant, in which putative Mst1 phosphorylation sites are mutated (S209/216/231/232A). Overexpression of FoxO1 significantly inhibited whereas downregulation of FoxO1 exacerbated Mst1-induced apoptosis.

Structural Basis for DNA Recognition by FoxO1 and Its Regulation by Posttranslational Modification

FoxO transcription factors regulate the transcription of genes that control metabolism, cellular proliferation, stress tolerance, and possibly life span. A number of posttranslational modifications within the forkhead DNA-binding domain regulate FoxO-mediated transcription. We describe the crystal structures of FoxO1 bound to three different DNA elements and measure the change in FoxO1-DNA affinity with acetylation and phosphorylation. The structures reveal additional contacts and increased DNA distortion for the highest affinity DNA site. The flexible wing 2 region of the forkhead domain was not observed in the structures but is necessary for DNA binding, and we show that p300 acetylation in wing 2 reduces DNA affinity. We also show that MST1 phosphorylation of FoxO1 prevents high-affinity DNA binding. The observation that FoxO-DNA affinity varies between response elements and with posttranslational modifications suggests that modulation of FoxO-DNA affinity is an important component of FoxO regulation in health and misregulation in disease.

Mapping of MST1 Kinase Sites of Phosphorylation: ACTIVATION AND AUTOPHOSPHORYLATION

MST1 is a member of the Sterile-20 family of cytoskeletal, stress, and apoptotic kinases. MST1 is activated by phosphorylation at previously unidentified sites. This study examines the role of phosphorylation at several sites and effects on kinase activation. We define Thr(183) in subdomain VIII as a primary site of phosphoactivation. Thr(187) is also critical for kinase activity. Phosphorylation of MST1 in subdomain VIII was catalyzed by active MST1 via intermolecular autophosphorylation, enhanced by homodimerization. Active MST1 (wild-type or T183E), but not inactive Thr(183)/Thr(187) mutants, was also highly autophosphorylated at the newly identified Thr(177) and Thr(387) residues. Cells expressing active MST1 were mostly detached, whereas with inactive MST1, adhesion was normal. Active MKK4, JNK, caspase-3, and caspase-9 were detected in the detached cells. These cells also contained all autophosphorylated and essentially all caspase-cleaved MST1. Similar phenotypes were elicited by a caspase-insensitive D326N mutant, suggesting that kinase activity, but not cleavage of MST1, is required. Interestingly, an S327E mutant mimicking Ser(327) autophosphorylation was also caspase-insensitive, but only when expressed in caspase-3-deficient cells. Together, these data suggest a model whereby MST1 activation is induced by existing, active MST kinase, which phosphorylates Thr(183) and possibly Thr(187). Dimerization promotes greater phosphorylation. This leads to induction of the JNK signaling pathway, caspase activation, and apoptosis. Further activation of MST1 by caspase cleavage is best promoted by caspase-3, although this appears to be unnecessary for signaling and morphological responses.