Mammalian Sterile 20-like Kinase Research Articles

MiR-155-dependent regulation of mammalian sterile 20-like kinase 2 (MST2) coordinates inflammation, oxidative stress and proliferation in vascular smooth muscle cells

In response to vascular injury, inflammation, oxidative stress, and cell proliferation often occur simultaneously in vascular tissues. We previously observed that microRNA-155 (miR-155), which is implicated in proliferation and inflammation is involved in neointimal hyperplasia; however, the molecular mechanisms by which it regulates these processes remain largely unknown. In this study, we observed that vascular smooth muscle cell (VSMC) proliferation and neointimal formation in wire-injured femoral arteries were reduced by the loss of miR-155 and increased by the gain of miR-155. The proliferative effect of miR-155 was also observed in cultured VSMCs. Notably, expression of the miR-155-target protein mammalian sterile 20-like kinase 2 (MST2) was increased in the injured arteries of miR-155−/− mice. miR-155 directly repressed MST2 and thus activated the extracellular signal-regulated kinase (ERK) pathway by promoting an interaction between RAF proto-oncogene serine/threonine-protein kinase (Raf-1) and mitogen-activated protein kinase kinase (MEK) and stimulating inflammatory and oxidative stress responses; together, these effects lead to VSMC proliferation and vascular remodeling. Our data reveal that MST2 mediates miR-155-promoted inflammatory and oxidative stress responses by altering the interaction of MEK with Raf-1 and MST2 in response to vascular injury. Therefore, suppression of endogenous miR-155 might be a novel therapeutic strategy for vascular injury and remodeling.

Mst2 Controls Bone Homeostasis by Regulating Osteoclast and Osteoblast Differentiation.

Mammalian sterile 20-like kinase 2 (Mst2) plays a central role in the Hippo pathway, controlling cell proliferation, differentiation, and apoptosis during development. However, the roles of Mst2 in osteoclast and osteoblast development are largely unknown. Here, we demonstrate that mice deficient in Mst2 exhibit osteoporotic phenotypes with increased numbers of osteoclasts and decreased numbers of osteoblasts as shown by micro-computed tomography (µCT) and histomorphometric analyses. Osteoclast precursors lacking Mst2 exhibit increased osteoclastogenesis and Nfatc1, Acp5, and Oscar expression in response to receptor activator of NF-κB ligand (RANKL) exposure. Conversely, Mst2 overexpression in osteoclast precursors leads to the inhibition of RANKL-induced osteoclast differentiation. Osteoblast precursors deficient in Mst2 exhibit attenuated osteoblast differentiation and function by downregulating the expression of Runx2, Alpl, Ibsp, and Bglap. Conversely, ectopic expression of Mst2 in osteoblast precursors increases osteoblastogenesis. Finally, we demonstrate that the NF-κB pathway is activated by Mst2 deficiency during osteoclast and osteoblast development. Our findings suggest that Mst2 is involved in bone homeostasis, functioning as a reciprocal regulator of osteoclast and osteoblast differentiation through the NF-κB pathway.

Mst1 inhibition rescues β1-adrenergic cardiomyopathy by reducing myocyte necrosis and non-myocyte apoptosis rather than myocyte apoptosis

It is generally held that inhibition of mammalian sterile 20-like kinase 1 (Mst1) protects the heart through reducing myocyte apoptosis. We determined whether inhibition with a dominant-negative Mst1 (DN-Mst1) would protect against the cardiomyopathy induced by chronic β1-adrenergic receptor (β1-AR) stimulation by preventing myocyte apoptosis. DN-Mst1 mice were mated with β1-AR transgenic (Tg) mice and followed for 20 months. β1-AR Tg mice developed cardiomyopathy as they aged, as reflected by premature mortality and depressed cardiac function, which were rescued in β1-AR × DN-Mst1 bigenic mice. Surprisingly, myocyte apoptosis did not significantly decrease with Mst1 inhibition. Instead, Mst1 inhibition predominantly reduced non-myocyte apoptosis, e.g., fibroblasts, macrophages, neutrophils and endothelial cells. Fibrosis in the hearts with cardiomyopathy increased fivefold and this increase was nearly abolished in the bigenic mice with Mst1 inhibition. Regression analysis showed no correlation between myocyte apoptosis and cardiac function or myocyte number, whereas the latter two correlated significantly, p < 0.05, with fibrosis, which generally results from necrosis. To examine the role of myocyte necrosis, chronic β-AR stimulation with isoproterenol was induced for 24 h and myocyte necrosis was assessed by 1% Evans blue dye. Compared to WT, DN-Mst1 mice showed significant inhibition, p < 0.05, of myocyte necrosis. We confirmed this result in Mst1-knockout mice, which also showed significant protection, p < 0.05, against myocyte necrosis compared to WT. These data indicate that Mst1 inhibition rescued cardiac fibrosis and myocardial dysfunction in β1-AR cardiomyopathy. However, this did not occur through Mst1 inhibition of myocyte apoptosis but rather by inhibition of cardiomyocyte necrosis and non-myocyte apoptosis, features of Mst1 not considered previously.

Role of Mammalian Sterile20-like Kinase 1 and 2 in Oxidative Stress

Mammalian sterile 20 like kinase (Mst) 1 and Mst2 are stress-activated kinases, activated by various stress stimuli including oxidative stress. Mst1/2 act as tumor suppressor proteins and their deregulation is associated with the development of cancers, such as liver cancer and prostate cancer. Cellular oxidative stress is known to upregulate Mst1/2 activity; activation of Mst1/2 induces apoptosis in oxidative stress conditions. Until recently, the exact mechanism/s of Mst1/2 activation and Mst1/2induced apoptosis under oxidative stress conditions was not clear. But some recent reports have begun to elucidate the mechanisms of Mst1/2 activation by oxidative stress. Not only Mst1/2 activity is regulated by oxidative stress, but Mst1/2 are also involved in the regulation of redox levels in cells. In this short report we will discuss various mechanisms of Mst1/2 activation and Mst1/2-dependent apoptosis in response to oxidative stress.

RASSF5 inhibits growth and invasion and induces apoptosis in osteosarcoma cells through activation of MST1/LATS1 signaling.

Ras association (RalGDS/AF-6) domain family member RASSF5 has been implicated in a variety of key biological processes, including cell proliferation, cell cycle regulation and apoptosis. It is believed to play an important role in tumorigenesis as a tumor suppressor in a number of malignancies. Yet, little is known concerning the function and underlying mechanisms of RASSF5 in human osteosarcoma (OS). The expression of RASSF5 was examined by immunohistochemical assay using a tissue microarray in 45cases of OS tissues. A gain-of-function approach was used to observe the effects of lentiviral vector-mediated overexpression of RASSF5 (Lv-RASSF5) on cell growth, invasion and apoptosis, respectively, as indicated by MTT, Transwell and flow cytometry assays, and the expression levels of mammalian sterile 20-like (MST1) kinase, large tumor suppressor1 (LATS1), proliferating cell nuclear antigen (PCNA), matrix metallopeptidase-9 (MMP-9) and p53 were detected by real-time PCR and western blot assays in OS cells (MG-63 and U-2 OS). The results indicated that the expression of RASSF5 protein was significantly downregulated in OS tissues compared to that in adjacent non-cancerous tissues (ANCT) (40.0 vs. 73.3%, P=0.002), and had a negative correlation with distant metastasis of the tumor (P=0.01). Overexpression of RASSF5 markedly suppressed cell proliferation and invasion, and induced cell apoptosis in the OS cell lines with increased expression of MST1, LATS1 and p53 and decreased expression of PCNA and MMP-9. Taken together, our findings demonstrate that RASSF5 expression is negatively correlated with distant metastasis of OS, and RASSF5 may function as a tumor suppressor in OS cells through activation of the MST1/LATS1 pathway.

One Hippo and many masters: differential regulation of the Hippo pathway in cancer.

The Hippo/MST2 (mammalian sterile 20-like kinase 2) pathway is a signalling cascade evolutionarily conserved in its structure. Originally described in Drosophila melanogaster as a regulator of organ size, this pathway has greater functions in mammals. Disturbance of mammalian MST2 pathway is associated with tumorigenesis by affecting apoptosis, cell cycle and polarity. In addition, this pathway has been shown to cross-talk with mitogenic pathways at multiple levels. In the present mini-review, we discuss our contribution highlighting the regulation of MST2 signalling by frequently observed oncogenic perturbations affecting mitogenic pathways. In particular, we review the role of RAS isoforms and PI3K (phosphoinositide 3-kinase)/Akt in the regulation of MST2 activity by phosphorylation. We also put the emphasis on RAF-induced control of MST2 signalling by protein-protein interactions. Finally, we recapitulate some of the direct mechanisms, such as ubiquitin-dependent degradation or gene silencing by promoter hypermethylation, involved in MST2 pathway component down-regulation in cancers.

Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

Gemcitabine-induced pancreatic cancer cell death is associated with MST1/cyclophilin D mitochondrial complexation.

The pancreatic adenocarcinoma remains the most aggressive human malignancy with an extremely low 5-year overall survival. Postoperative gemcitabine could significantly delay recurrence after complete resection of pancreatic cancer. However, the underlying mechanisms are not fully understood. The chemo-resistance factors against gemcitabine still need further characterizations. Here we studied the mechanism of gemcitabine-induced pancreatic cancer cell death by focusing on mammalian sterile 20-like kinase 1 (MST1) and cyclophilin D (Cyp-D). We found that MST1 and Cyp-D expressions were significantly lower in gemcitabine-resistant pancreatic cancer tissues and cell lines. In vitro, gemcitabine activated MST1 through reactive oxygen species (ROS) production, which was prevented by antioxidant n-acetyl-cysteine (NAC). We found that gemcitabine-activated MST1 translocated to mitochondria and formed a complex with the local protein Cyp-D. Gemcitabine-induced cell death was alleviated by MST1 or Cyp-D shRNA silencing, but was aggravated by MST1 or Cyp-D over-expression. Further, cyclosporin A (CsA), the Cyp-D inhibitor, prevented gemcitabine-induced MST1/Cyp-D mitochondrial complexation and cancer cell death. We suggest that gemcitabine-induced death of pancreatic cancer cells requires MST1/Cyp-D mitochondrial complexation.

Inhibition of Mammalian Sterile 20‐like Kinase 1 Rescues Cardiomyopathy through Protection from Myocyte Necrosis as well as Apoptosis (LB700)

Inhibition of Mammalian Sterile 20‐like Kinase 1 (MST1) is known to have cardioprotective effects by preventing myocyte apoptosis. We examined the effects of MST1 inhibition in β1‐adrenergic receptor (β1‐AR) cardiomyopathy by mating dominant negative (DN)‐MST1 mice with β1‐AR mice. As the mice aged, the bigenic mice had higher left ventricular ejection fraction (67+12% vs 43+5%) and decreased cardiac fibrosis (8.8+1.5% vs 19.5+2.7%). It is generally believed that fibrosis in cardiomyopathy is secondary to necrosis, more than apoptosis. Accordingly, we hypothesized that MST1 may also mediate necrosis. We examined necrosis through in‐vivo labeling by Evans Blue Dye in DN‐MST1 mice after continuous isoproterenol (ISO) challenge. At 1 day after ISO the wild type hearts showed massive myocyte necrotic death (0.90+0.37%), which was significantly protected in DN‐MST1 (0.32+0.14% p&lt;0.05), while having no discernible differences in myocyte apoptosis (0.10+0.03% vs 0.08+0.01%). These results suggest that MST1 may also have an important role in myocyte necrosis as well as apoptosis.

MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes

Apoptotic cell death is a hallmark of the loss of insulin producing beta-cells in all forms of diabetes mellitus. Current treatment fails to halt the decline in functional beta-cell mass. Strategies to prevent beta-cell apoptosis and dysfunction are urgently needed. Here, we identified Mammalian Sterile 20-like kinase 1 (MST1) as a critical regulator of apoptotic beta-cell death and function. MST1 was strongly activated in beta-cells under diabetogenic conditions and correlated with beta-cell apoptosis. MST1 specifically induced the mitochondrial-dependent pathway of apoptosis in beta-cells through up-regulation of the BH3-only protein Bim. MST1 directly phosphorylated PDX1 at Thr11, resulting in its ubiquitination, degradation and impaired insulin secretion. Mst1 deficiency completely restored normoglycemia, beta-cell function and survival in vitro and in vivo. We show MST1 as novel pro-apoptotic kinase and key mediator of apoptotic signaling and beta-cell dysfunction, which may serve as target for the development of novel therapies for diabetes.

The role of reciprocal activation of cAbl and Mst1 in the Oxidative death of cultured astrocytes

The protein kinase Mst1 (mammalian Sterile 20-like kinase 1) likely plays a role in oxidative neuronal cell death as a target of its activator, cAbl. We previously found that H2O2-induced death of astrocytes is mediated by cAbl in a metallothionein-3 (Mt3)-dependent manner. In the present study, we examined a possible role for Mst1 in the oxidative death of astrocytes. Treatment of cortical astrocytes with 170 µM H2O2 activated Mst1. Knockdown of Mst1 reduced H2O2-induced cell death, indicating that Mst1 activation contributes to astrocytic cell death. STI571, an inhibitor of cAbl, blocked induction/activation of Mst1 and H2O2-induced cell death. However, Mst1 silencing also inhibited induction/activation of cAbl, suggesting that the two kinases are regulated by a reciprocal activating mechanism. The zinc chelator TPEN blocked induction/activation of cAbl and Mst1, indicating that these phenomena are dependent on the rise of intracellular zinc. Moreover, H2O2 exposure did not increase free zinc levels in Mt3-null astrocytes, suggesting that the increased levels of free zinc were largely from Mt3. Consistent with the involvement of FoxO1/3, which may play a role in the Mst1-cell death cascade, we found an increase in the level of phosphorylated FoxO1/3 in H2O2-treated astrocytes. Moreover, inhibition of cAbl or Mst1 reversed this effect. The present results suggest the interesting possibility that cAbl and Mst1 are reciprocally activated under oxidative stress conditions in astrocytes. Both kinases appear to be regulated by changes in the levels of free zinc originating from Mt3 and contribute to oxidative cell death through a FoxO-dependent mechanism.

Mammalian sterile 20-like kinase 1/2 inhibits the Wnt/β-catenin signalling pathway by directly binding casein kinase 1ε

The Hippo signalling pathway can suppress the Wnt/β-catenin signalling pathway through the last downstream effectors YAP (Yes-associated protein)/TAZ (tafazzin). MST (mammalian sterile 20-like kinase) 1 functions as the upstream kinase of the Hippo pathway, and CK1ε (casein kinase 1ε) plays roles in the up-stream signal transduction of the Wnt/β-catenin pathway. In the present study, using tandem affinity purification and MS analysis, CK1ε was identified as a novel partner of MST1. Further analysis showed that the interaction between MST1 and CK1ε was mediated by their kinase domains and enhanced by the activation of MST1. To exclude the interference of the phosphorylated YAP/TAZ, the transduction from MST1 to YAP/TAZ was blocked using anti-WW45 shRNA. In the sh-WW45 cells, MST1 still inhibited the Wnt3A-induced phosphorylation of DVL2 (dishevelled 2) and Wnt/β-catenin signalling by disturbing the interaction of DVL2 and CK1ε. The growth-suppressive effect of MST1 in the presence of Wnt3A was effectively relieved by the downstream activation of the Wnt/β-catenin pathway. Moreover, MST2, the close homologue of MST1, also displayed the similar function in suppressing the Wnt/β-catenin pathway. Therefore the results of the present study revealed that, in addition to the phosphorylated YAP/TAZ, the Hippo pathway can suppress the Wnt/β-catenin pathway directly through MST1/2.

MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

A DAF‐16/FoxO3a‐dependent longevity signal is initiated by antioxidants

The precise mechanisms of antioxidant-mediated longevity are poorly understood. We show that an antioxidant treatment can extend the lifespan of Caenorhabditis elegans (C. elegans) through the nuclear translocation of the forkhead box O transcription factor (FoxO) homolog DAF-16. This pathway was found to involve 3-phosphoinositide-dependent kinase-1 (PDK-1) and serum- and glucocorticoid-regulated kinase-1 (SGK-1), distinct from the known oxidative stress-mediated mechanism in which FoxO3a translocation is regulated by c-Jun N-terminal kinase (JNK) and mammalian sterile 20-like kinase-1 (MST-1). The differences in the mechanisms of FoxO activation by antioxidants and oxidants result in differences in FoxO phosphorylation and target gene expression. Based on these results, we found that a combination of early antioxidant treatment and late oxidant treatment is most effective for lifespan extension in C. elegans.

Abstract 148: Mst1 Mediates Cell-protective Mechanisms In The Heart Through Phosphorylation Of FoxO1 And C/EBP-beta

Forkhead box O1 (FoxO1) regulates both cell survival and death in the heart. The function of FoxO1 is modulated by posttranslational modifications, including phosphorylation. Mst1 (mammalian sterile 20-like kinase 1), a stress-activated pro-apoptotic kinase, phosphorylates FoxO1 at Ser209/216/231/232. We investigated the molecular mechanism by which FoxO1 regulates both cell survival and death in the heart. Overexpression of FoxO1 significantly suppressed, whereas downregulation of endogenous FoxO1 exacerbated, Mst1 activity and Mst1-induced apoptosis. In the presence of Mst1, FoxO1 downregulated pro-apoptotic genes, including FasL, but upregulated antioxidant genes, including catalase. Chromatin immunoprecipitation assays revealed that Mst1 attenuated FoxO1 binding to the FasL promoter, but it enhanced binding to the catalase promoter. Reporter gene assays showed that C/EBP-β binding elements, but not the FoxO binding ones, in the catalase promoter, are critical for Mst1-mediated upregulation of the catalase gene. FoxO1 interacted with C/EBP-β and their interaction was enhanced in the presence of Mst1. Mass spectrometry analyses revealed that Mst1 phosphorylates C/EBP-β at Thr 299 in its leucine zipper domain. Downregulation of endogenous C/EBP-β reversed the cell-protective effects of FoxO1 expression against Mst1-induced apoptosis. We evaluated the role of Mst1-mediated phosphorylation of FoxO1 and C/EBP-β in mediating survival and death of cardiomyocytes in response to ischemia/reperfusion (I/R). The size of myocardial infarction (MI) in left ventricle (LV) after I/R was significantly greater in cardiac-specific FoxO1 knockout ( c-FoxO1 -/- ) mice than in wild-type mice (MI/area at risk: 59±2, 41±1%, p &lt;0.05, n=6). Injection of a C/EBP-β phospho-mimetic mutant adenovirus into the LV of c-FoxO1 -/- significantly reduced the I/R-induced LV infarct size observed in c-FoxO1 -/- (50±2%, p &lt;0.05, n=6). In summary, Mst1-mediated phosphorylation of FoxO1 inhibits the DNA binding of FoxO1 to the cell death promoting genes whereas it enhances FoxO1-C/EBP-β interaction, Mst1-mediated phosphorylation of C/EBP-β, and C/EBP-β-mediated transcription, which activates cell-protective mechanisms in the heart.

MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species-dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.

Prognostic significance of mammalian sterile 20-like kinase 1 in breast cancer

Mammalian sterile 20-like kinase 1 (Mst1) has been proved in the process of apoptosis and tumor suppression. The aim of the study was to investigate the expression of Mst1 in breast cancer and to evaluate its prognostic significance. The expression of Mst1 was examined in 110 breast cancer patients by immunohistochemistry, in which 80 (72.7 %) were defined as positive for Mst1 expression. Patients with negative expression of Mst1 had poor overall survival, comparing with those with positive expression using Kaplan-Meier survival analysis (P = 0.009). Multivariate analysis using Cox proportional hazards model showed that Mst1 expression was a significant independent prognostic factor in breast cancer (P = 0.030). Our results presented the tumor suppressive role of Mst1, and confirmed Mst1 was a prognostic factor in human breast cancer.

OP0195 Genetic and Pharmacologic Inhibition of MST1 Blocks Lymphocyte Function and Protects Against Inflammation and Autoimmunity

BackgroundThe mammalian sterile 20-like kinase 1 (MST1) is a MAP4 kinase which acts upstream of a wide range of signal transduction pathways. It can modify signaling events mediated by Jnk,...

Ras and Rheb Signaling in Survival and Cell Death

One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase protects cardiomyocytes against hypoxia induced apoptosis through inhibiting proapoptotic kinase Mst1

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known. To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis. These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.