Mst1 and Mst2 kinases: regulations and diseases

The Hippo signaling pathway has emerged as a critical regulator for organ size control. The serine/threonine protein kinases Mst1 and Mst2, mammalian homologs of the Hippo kinase from Drosophila, play the central roles in the Hippo pathway controlling the cell proliferation, differentiation, and apoptosis during development. Mst1/2 can be activated by cellular stressors and the activation of Mst1/2 might enforce a feedback stimulation system to regulate oxidant levels through several mechanisms, in which regulation of cellular redox state might represent a tumor suppressor function of Mst1/2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in multiple organs, although considerable diversification in the pathway composition and regulation is observed in some of them. Generally, loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by the reduction or elimination of YAP. The Hippo pathway integrates with other signaling pathways e.g. Wnt and Notch pathways and coordinates with them to impact on the tumor pathogenesis and development. Furthermore, Mst1/2 kinases also act as an important regulator in immune cell activation, adhesion, migration, growth, and apoptosis. This review will focus on the recent updates on those aspects for the roles of Mst1/2 kinases.     

哺乳动物不育系20样激酶MST1

哺乳动物不育系20样激酶1(mammalian sterile 20-like kinase 1,Mst1)基因是果蝇Hippo基因在哺乳动物中的同源基因,编码丝氨酸/苏氨酸激酶,主要参与细胞生长、增殖、凋亡以及器官大小等的调控。最近的研究表明,Mst1基因具有抑癌作用,其功能的缺失与肿瘤的发生密切相关。本文就MST1的结构与功能、促凋亡作用机制及其在医学研究中的应用作一简要综述。     

Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation

Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C-terminal coiled-coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled-coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N-terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.     

Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely perturb Hippo signaling. We found that Mst-/- ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although Mst-/- ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, Mst-/- ES cells are unable to form teratoma. Mst-/- ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in Mst-/- EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.     

Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) 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 the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.     

Mst1-FoxO Signaling Protects Na?ve T Lymphocytes from Cellular Oxidative Stress in Mice

Background

The Ste-20 family kinase Hippo restricts cell proliferation and promotes apoptosis for proper organ development in Drosophila. In C. elegans, Hippo homolog also regulates longevity. The mammalian Ste20-like protein kinase, Mst1, plays a role in apoptosis induced by various types of apoptotic stress. Mst1 also regulates peripheral naïve T cell trafficking and proliferation in mice. However, its functions in mammals are not fully understood.

Methodology/Principal Findings

Here, we report that the Mst1-FoxO signaling pathway plays a crucial role in survival, but not apoptosis, of naïve T cells. In Mst1^−/− mice, peripheral T cells showed impaired FoxO1/3 activation and decreased FoxO protein levels. Consistently, the FoxO targets, Sod2 and catalase, were significantly down-regulated in Mst1^−/− T cells, thereby resulting in elevated levels of intracellular reactive oxygen species (ROS) and induction of apoptosis. Expression of constitutively active FoxO3a restored Mst1^−/− T cell survival. Crossing Mst1 transgenic mice (Mst1 Tg) with Mst1^−/− mice reduced ROS levels and restored normal numbers of peripheral naïve T cells in Mst1 Tg;Mst1^−/− progeny. Interestingly, peripheral T cells from Mst1^−/− mice were hypersensitive to γ-irradiation and paraquat-induced oxidative stresses, whereas those from Mst1 Tg mice were resistant.

Conclusions/Significance

These data support the hypothesis that tolerance to increased levels of intracellular ROS provided by the Mst1-FoxOs signaling pathway is crucial for the maintenance of naïve T cell homeostasis in the periphery.     

Proinflammation effect of Mst1 promotes BV-2 cell death via augmenting Drp1-mediated mitochondrial fragmentation and activating the JNK pathway

Inflammation has been increasingly studied as part of the pathophysiology of neurodegenerative diseases. Mammalian Ste20-like kinase 1 (Mst1), a key factor of the Hippo pathway, is connected to cell death. Unfortunately, little study has been performed to detect the impact of Mst1 in neuroninflammation. The results indicated that Mst1 expression was upregulated because of LPS treatment. However, the loss of Mst1 sustained BV-2 cell viability and promoted cell survival in the presence of LPS treatment. Molecular investigation assay demonstrated that Mst1 deletion was followed by a drop in the levels of mitochondrial fission via repressing Drp1 expression. However, Drp1 adenovirus transfection reduced the protective impacts of Mst1 knockdown on mitochondrial stress and neuronal dysfunction. Finally, our results illuminated that Mst1 affected Drp1 content and mitochondrial fission in a JNK-dependent mechanism. Reactivation of the JNK axis inhibited Mst1 knockdown-mediated neuronal protection and mitochondrial homeostasis. Altogether, our results indicated that Mst1 upregulation and the activation of JNK-Drp1-mitochondrial fission pathway could be considered as the novel mechanism regulating the progression of neuroninflammation. This finding would pave a new road for the treatment of neurodegenerative diseases via modulating the Mst1-JNK-Drp1-mitochondrial fission axis.     

The Mammalian Ste20-like Kinase 2 (Mst2) Modulates Stress-induced Cardiac Hypertrophy

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2^−/− mice showed no alteration in cardiomyocyte proliferation. However, Mst2^−/− mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart.     

Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway

The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4^DCAF1. Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin''s auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin''s binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin''s auto-inhibitory tail does not obviously alter Merlin''s conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.     

Atypical protein kinase C induces cell transformation by disrupting Hippo/Yap signaling

Epithelial cells are major sites of malignant transformation. Atypical protein kinase C (aPKC) isoforms are overexpressed and activated in many cancer types. Using normal, highly polarized epithelial cells (MDCK and NMuMG), we report that aPKC gain of function overcomes contact inhibited growth and is sufficient for a transformed epithelial phenotype. In 2D cultures, aPKC induced cells to grow as stratified epithelia, whereas cells grew as solid spheres of nonpolarized cells in 3D culture. aPKC associated with Mst1/2, which uncoupled Mst1/2 from Lats1/2 and promoted nuclear accumulation of Yap1. Of importance, Yap1 was necessary for aPKC-mediated overgrowth but did not restore cell polarity defects, indicating that the two are separable events. In MDCK cells, Yap1 was sequestered to cell–cell junctions by Amot, and aPKC overexpression resulted in loss of Amot expression and a spindle-like cell phenotype. Reexpression of Amot was sufficient to restore an epithelial cobblestone appearance, Yap1 localization, and growth control. In contrast, the effect of aPKC on Hippo/Yap signaling and overgrowth in NMuMG cells was independent of Amot. Finally, increased expression of aPKC in human cancers strongly correlated with increased nuclear accumulation of Yap1, indicating that the effect of aPKC on transformed growth by deregulating Hippo/Yap1 signaling may be clinically relevant.     

Scutellarin exerts protective effects against atherosclerosis in rats by regulating the Hippo–FOXO3A and PI3K/AKT signaling pathways

Atherosclerosis (AS), a progressive disorder, is one of the tough challenges in the clinic. Scutellarin, an extract from Herba Erigerontis, is found to have oxygen-free radicals scavenging effects and antioxidant effects. In this study, we aimed to investigate the anti-AS effects of scutellarin is related to controlling the Hippo–FOXO3A and PI3K/AKT signal pathway. To establish an AS model, the rats in the scutellarin and model groups were intraperitoneally injected with vitamin D ₃ and then fed a high-fat diet for 12 weeks. In addition, in vitro angiotensin II-induced apoptosis of human aortic endothelial cells (HAECs) were used to establish models. Scutellarin significantly reduced blood lipid levels and increased antioxidase levels in both models. Additionally, scutellarin inhibited reactive oxygen species generation and apoptosis in HAECs. The impaired vascular barrier function was restored by using scutellarin in AS rats and in HAECs cells characterized by inhibiting mammalian sterile-20-like kinases 1 (Mst1) phosphorylation, Yes-associated protein (YAP) phosphorylation, forkhead box O3A (FOXO3A) phosphorylation at serine 207, nuclear translocation of FOXO3A, and upregulating protein expression of AKT and FOXO3A phosphorylation at serine 253. Scutellarin significantly reduced Bcl-2 interacting mediator of cell death (Bim), caspase-3, APO-1, CD95 (Fas), and Bax: Bcl-2-associated X (Bax) levels and activated Bcl-2: B-cell lymphoma-2 (Bcl-2). Scutellarin also significantly inhibited the expression of Mst1, YAP, FOXO3A at the messenger RNA level. When Mst1 was overexpressed or phosphoinositide 3-kinases suppressed, the effects of scutellarin were significantly blocked. In conclusion, the results of the present study suggest that scutellarin exerts protective effects against AS by inhibiting endothelial cell injury and apoptosis by regulating the Hippo–FOXO3A and PI3K/AKT signal pathways.     

CD44 acts through RhoA to regulate YAP signaling

The Hippo pathway plays an important role in both physical and pathogenesis processes. As crucial downstream effectors of Hippo pathway, YAP is inhibited by Lats1/2 through phosphorylation. However, upstream signals that regulate the Hippo pathway have been still poorly understood. Here, we found that knockdown of CD44 reduced YAP expression and nuclear localization, but nearly had no effect on its upstream effectors, Mst1 and Lats1. Downregulated CD44 expression also significantly decreased the expression of YAP downstream effectors CTGF, Cyr61 and EDN1 at mRNA level. Our next study showed that knockdown of CD44 inhibited RhoA expression, which was consistent with RhoA knockdown mediated YAP downregulation. Furthermore, we demonstrated that over expression of the constitutively active RhoA (RhoA-V14) could block the YAP expression decrease mediated by CD44 knockdown. Moreover, downregulation of CD44 significantly promoted cell apoptosis and inhibited cell proliferation, cell cycle progression and migration, which were consistent with the effects of RNAi-mediated YAP knockdown in both A549 and HepG2 cells. Overall, data are presented showing that CD44 could act through RhoA signaling to regulate YAP expression and this study also provide new insights into the regulatory mechanisms of the Hippo–YAP pathway.     

Hippo signaling: growth control and beyond

The Hippo pathway has emerged as a conserved signaling pathway that is essential for the proper regulation of organ growth in Drosophila and vertebrates. Although the mechanisms of signal transduction of the core kinases Hippo/Mst and Warts/Lats are relatively well understood, less is known about the upstream inputs of the pathway and about the downstream cellular and developmental outputs. Here, we review recently discovered mechanisms that contribute to the dynamic regulation of Hippo signaling during Drosophila and vertebrate development. We also discuss the expanding diversity of Hippo signaling functions during development, discoveries that shed light on a complex regulatory system and provide exciting new insights into the elusive mechanisms that regulate organ growth and regeneration.