Mammalian Ste20-like Kinase 1 Research Articles

Intermittent Leucine Pulses during Continuous Feeding Alters Novel Components Involved in Skeletal Muscle Growth of Neonatal Pigs

Extra‐uterine growth restriction in low‐birth‐weight (LBW) infants is associated with the development of chronic metabolic derangements, and emphasizes the need for improved nutritional protocols to optimize growth. Supplementation with intermittent pulses of leucine in continuously fed neonatal pigs enhanced skeletal muscle protein synthesis through the upregulation of mammalian target of rapamycin signaling. Piglets (n = 23, 7‐d‐old) were continuously fed a milk formula via orogastric tube for 21 d, with an additional parenteral infusion of either leucine (800 μmol·kg−1·h−1, LEU) or alanine (CON) for 1 h every 4 h. To identify novel genes by which LEU may promote neonatal muscle growth, we performed RNA‐Seq analysis of longissimus dorsi muscle from 4 pigs per group, followed by gene ontology (GO) analysis of differentially expressed genes (DEG). In addition, protein abundance/activation of DEG products involved in both muscle protein anabolic or catabolic pathways was assessed by immunoblots. RNA‐Seq was performed on a HiSeq2000 sequencer analyzer. Reads were analyzed using CLCBio Genomics workbench software, assembled against the porcine reference genome, and normalized by a threshold of ‘reads per kilo base per million mapped reads’ (RPKM) ≥ 0.2. A total of 465 genes were differentially expressed between LEU and CON groups (RPKM P < 0.05). GO analyses showed developmental processes, cell junction assembly, regulation of cellular process and organ development as the terms most significantly enriched among the 465 genes. Applying more stringent parameters (RPKM P < 0.01 + >2 fold change), 18 differential expressed genes were identified between CON and LEU pigs, of which tumor necrosis factor receptor‐associated factor 6 (TRAF6) and mammalian Ste20‐like kinase 1 (MST1), both negative regulators of muscle growth, were decreased by > 2‐fold (P < 0.01) in LEU compared to CON. Western blot analysis also showed a downregulation of MST1 and TRAF6 abundance in LEU animals (P < 0.01) compared to CON. Together, these findings suggest that intermittent leucine pulses in continuously fed neonate pigs lead to downregulation in the gene and protein expression of signaling components that limit muscle growth.Support or Funding InformationNIH AR444474, NIHHD072891, USDA NIFA 2013‐67015‐20438, and USDA/ARS 6250‐51000‐055

TRAF2 functions as an activator switch in the reactive oxygen species-induced stimulation of MST1

Reactive oxygen species (ROS) have many physiological and pathological effects on diverse cellular events. In particular, excessive ROS causes oxidative stress that leads to cell death. The mammalian STE20-like kinase-1 (MST1), a multifunctional serine–threonine kinase, plays a pivotal role in oxidative stress-induced cellular signaling events. Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is also known to be essential for oxidative stress-induced cell death. Here, we showed that H2O2 induced the physical interaction between TRAF2 and MST1, and that this interaction promoted the homodimerization as well as the activation of MST1. Furthermore, TRAF2 was required for MST1 to mediate the H2O2-induced stimulation of c-Jun N-terminal kinase and p38 kinase as well as apoptosis. Taken together, our results suggest that TRAF2 functions as a key activator of MST1 in oxidative stress-induced intracellular signaling processes.

Deacetylation of tumor-suppressor MST1 in Hippo pathway induces its degradation through HBXIP-elevated HDAC6 in promotion of breast cancer growth.

Reduction or loss of tumor-suppressor mammalian STE20-like kinase 1 (MST1) in Hippo pathway contributes to the tumorigenesis. However, the mechanism leading to reduction of MST1 in cancers remains poorly understood. In this study, we explored the hypothesis that the oncoprotein hepatitis B X-interacting protein (HBXIP) is involved in the reduction of MST1 in breast cancer. Immunohistochemical analysis of tissue microarrays revealed that the expression of HBXIP was negatively associated with that of MST1 in 98 clinical breast tissue samples. Then we found that HBXIP could posttranslationally downregulate MST1 in breast cancer cells. Mechanistically, we identified that MST1 could be acetylated on its lysine 35 residue in the cells. Strikingly, the treatment with trichostatin A, an inhibitor of histone deacetylases (HDACs), markedly increased the levels of MST1 acetylation and protein in the cells. Interestingly, the oncoprotein HBXIP could significantly inhibit acetylation of MST1, resulting in the reduction of MST1 protein. Notably, we revealed that the HDAC6 could reduce the protein levels of MST1 through deacetylation modification of MST1 in the cells. Moreover, our data revealed that HBXIP upregulated HDAC6 at the levels of mRNA and protein by activating transcription factor nuclear factor-κB. Deacetylation of MST1 promoted the interaction of MST1 with HSC70 in the cells, resulting in a lysosome-dependent degradation of MST1 via chaperone-mediated autophagy (CMA). Functionally, the reduction of tumor-suppressor MST1 mediated by HBXIP promoted the growth of breast cancer cells in vitro and in vivo. Thus we conclude that the deacetylation of MST1 mediated by HBXIP-enhanced HDAC6 results in MST1 degradation in a CMA manner in promotion of breast cancer growth. Our finding provides new insights into the mechanism of tumor-suppressor MST1 reduction in breast cancer.

Mst-1 switches between cardiac cell life and death

Autophagy and apoptosis are ancient processes that regulate cell fate under normal and disease conditions. A new study in mice identifies mammalian Ste20-like kinase-1 (Mst-1) as a missing link that interfaces between these pathways for cell survival and death during cardiac stress (pages 1478–1488).

Thioredoxin-1 functions as a molecular switch regulating the oxidative stress-induced activation of MST1

The mammalian STE20-like kinase-1 (MST1), a multifunctional serine–threonine kinase in mammalian cells, has been recently implicated in the mediation of oxidative stress-induced signaling processes that lead to cell death. However, the molecular mechanism by which oxidative stress induces the stimulation of MST1 remains unclear. In this study, we found that thioredoxin-1 was physically associated with MST1 in intact cells and that this interaction was abolished by H2O2. Thioredoxin-1, by binding to the SARAH domain of MST1, inhibited the homodimerization and autophosphorylation of MST1, thereby preventing its activation. Furthermore, TNF-α prevented the physical interaction between thioredoxin-1 and MST1 and promoted the homodimerization and activation of MST1. The effect of TNF-α on MST1 activation was reversed by the reducing agent N-acetyl-l-cysteine. Taken together, our results suggest that thioredoxin-1 functions as a molecular switch to turn off the oxidative stress-induced activation of MST1.

Protein Glutathionylation in the Regulation of Peroxiredoxins: A Family of Thiol-Specific Peroxidases That Function As Antioxidants, Molecular Chaperones, and Signal Modulators

Reversible protein glutathionylation plays an important role in cellular regulation, signaling transduction, and antioxidant defense. This redox-sensitive mechanism is involved in regulating the functions of peroxiredoxins (Prxs), a family of ubiquitously expressed thiol-specific peroxidase enzymes. Glutathionylation of certain Prxs at their active-site cysteines not only provides reducing equivalents to support their peroxidase activity but also protects Prxs from irreversible hyperoxidation. Typical 2-Cys Prx also functions as a molecular chaperone when it exists as a decamer and/or higher molecular weight complexes. The hyperoxidized sulfinic derivative of 2-Cys Prx is reactivated by sulfiredoxin (Srx). In this review, the roles of glutathionylation in the regulation of Prxs are discussed with respect to their molecular structure and functions as antioxidants, molecular chaperones, and signal modulators. Recent findings reveal that glutathionylation regulates the quaternary structure of Prx. Glutathionylation of Prx I at Cys(83) converts the decameric Prx to its dimers with the loss of chaperone activity. The findings that dimer/oligomer structure specific Prx I binding proteins, e.g., phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST1), regulate cell cycle and apoptosis, respectively, suggest a possible link between glutathionylation and those signaling pathways. Knowing how glutathionylation affects the interaction between Prx I and its nearly 20 known interacting proteins, e.g., PTEN and MST1 kinase, would reveal new insights on the physiological functions of Prx. In vitro studies reveal that Prx oligomerization is linked to its functional changes. However, in vivo dynamics, including the effect by glutathionylation, and its physiological significance remain to be investigated.

The c-Abl-MST1 Signaling Pathway Mediates Oxidative Stress-Induced Neuronal Cell Death

Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. The protein kinase MST1 (mammalian Ste20-like kinase 1) plays a major role in oxidative stress-induced cell death in primary mammalian neurons. However, the mechanisms that regulate MST1 in oxidative stress responses remain largely unknown. In the present study, we demonstrate that the protein kinase c-Abl phosphorylates MST1 at Y433, which triggers the stabilization and activation of MST1. Inhibition of c-Abl promotes the degradation of MST1 through C terminus of Hsc70-interacting protein (CHIP)-mediated ubiquitination, and thereby attenuates cell death. Oxidative stress induces the c-Abl-dependent tyrosine phosphorylation of MST1 and increases the interaction between MST1 and FOXO3 (Forkhead box O3), thereby activating the MST1-FOXO signaling pathway, leading to cell death in both primary culture neurons and rat hippocampal neurons. The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST1 suggests that the c-Abl-MST1 signaling cascade plays an important role in cellular responses to oxidative stress.

Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling

Microglia, the resident macrophages of the mammalian central nervous system, migrate to sites of tissue damage or infection and become activated. Although the persistent secretion of inflammatory mediators by the activated cells contributes to the pathogenesis of various neurological disorders, most activated microglia eventually undergo apoptosis through the process of activation-induced cell death (AICD). The molecular mechanism of AICD, however, has remained unclear. Here, we show that Daxx and mammalian Ste20-like kinase-1 (MST1) mediate apoptosis elicited by interferon-γ (IFN-γ) in microglia. IFN-γ upregulated the expression of Daxx, which in turn mediated the homodimerization, activation, and nuclear translocation of MST1 and apoptosis in microglial cells. Depletion of Daxx or MST1 by RNA interference also attenuated IFN-γ-induced cell death in primary rat microglia. Furthermore, the extent of IFN-γ-induced death of microglia in the brain of MST1-null mice was significantly reduced compared with that apparent in wild-type mice. Our results thus highlight new functions of Daxx and MST1 that they are the key mediators of microglial cell death initiated by the proinflammatory cytokine IFN-γ.

Oligomeric peroxiredoxin-I is an essential intermediate for p53 to activate MST1 kinase and apoptosis

Mammalian Ste20-like kinase-1 (MST1) kinase mediates H₂O₂-induced cell death by anticancer drugs such as cisplatin in a p53-dependent manner. However, the mechanism underlying MST1 activation by H₂O₂ remains unknown. Here we show that peroxiredoxin-I (PRX-I) is an essential intermediate in H₂O₂-induced MST1 activation and cisplatin-induced cell death through p53. Cell stimulation with H₂O₂ resulted in PRX-I oxidation to form homo-oligomers and interaction with MST1, leading to MST1 autophosphorylation and augmentation of kinase activity. In addition, RNA interference knockdown experiments indicated that endogenous PRX-I is required for H₂O₂-induced MST1 activation. Live-cell imaging showed H₂O₂ generation by cisplatin treatment, which likewise caused PRX-I oligomer formation, MST1 activation and cell death. Cisplatin-induced PRX-I oligomer formation was not observed in embryonic fibroblasts obtained from p53-knockout mice, confirming the importance of p53. Indeed, ectopic expression of p53 induced PRX-I oligomer formation and cell death, both of which were cancelled by the antioxidant NAC. Moreover, we succeeded in reconstituting H₂O₂-induced MST1 activation in vitro, using purified PRX-I and MST1 proteins. Collectively, our results show a novel PRX-I function to cause cell death in response to high levels of oxidative stress by activating MST1, which underlies the p53-dependent cytotoxicity caused by anticancer agents.

MST1 Promotes Apoptosis through Phosphorylation of Histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphorylates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phosphorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphorylation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.

NDR Kinase Is Activated by RASSF1A/MST1 in Response to Fas Receptor Stimulation and Promotes Apoptosis

Human NDR1 and 2 (NDR1/2) are serine-threonine protein kinases in a subgroup of the AGC kinase family. The mechanisms of physiological NDR1/2 activation and their function remain largely unknown. Here we report that Fas and TNF-alpha receptor stimulation activates human NDR1/2 by promoting phosphorylation at the hydrophobic motif (Thr444/442). Moreover, NDR1/2 are essential for Fas receptor-induced apoptosis as shown by the fact that NDR knockdown significantly reduced cell death whereas overexpression of the NDR1 kinase further potentiated apoptosis. Activation of NDR1/2 by death receptor stimulation is mediated by the tumor suppressor RASSF1A. Furthermore, RASSF1A-induced apoptosis largely depends on the presence of NDR1/2. Fas receptor stimulation promoted direct phosphorylation and activation of NDR1/2 by the mammalian STE20-like kinase 1 (MST1), a downstream effector of RASSF1A. Concurrently, the NDR1/2 coactivator MOB1 induced MST1-NDR-MOB1 complex formation, which is crucial for MST1-induced NDR1/2 phosphorylation upon induction of apoptosis. Our findings identify NDR1/2 as novel proapoptotic kinases and key members of the RASSF1A/MST1 signaling cascade.

Nuclear Trip After Caspase Clip

Mammalian STE20-like kinase 1 (MST1) (so-named for its similarity to the yeast mitogen-activated protein kinase kinase kinase kinase STE20) is activated during apoptosis. In fact, it appears to be a direct target of caspases. Cleavage of the enzyme by caspases, along with phosphorylation, contributes to activation of the enzyme. Ura et al. now show that proteolytic removal of the COOH-terminus of the enzyme not only relieves inhibition of the enzyme, but also removes two nuclear export signals and thus causes localization of MST1 to the nucleus. Studies of mutants directed into or out of the nucleus showed that accumulation of MST1 in the nucleus is associated with increased chromatin condensation. Expression of a dominant-negative catalytically inactive mutant of MST1 inhibited chromatin condensation in 293T cells treated with staurosporin (an apoptotic stimulus). Also, a mutant of MST1 that cannot be cleaved was less effective in causing chromatin condensation when overexpressed in cultured cells. The results reveal a new mechanism for regulating nuclear localization of an enzyme and support a role for MST1 in mediating nuclear signals that lead to apoptosis. S. Ura, N. Masuyama, J. D. Graves, Y. Gotoh, Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation. Proc. Natl. Acad. Sci. U.S.A. 98 , 10148-10153 (2001) [Abstract] [Full Text]