Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain–mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

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Dynamic patterns of YAP1 expression and cellular localization in the developing and injured utricle

10.1101/2020.05.06.081323 ◽

2020 ◽

Cited By ~ 1

Author(s):

Vikrant Borse ◽

Matthew Barton ◽

Harry Arndt ◽

Tejbeer Kaur ◽

Mark E. Warchol

Keyword(s):

Hair Cell ◽

Cellular Localization ◽

Cell Injury ◽

Nuclear Translocation ◽

Cell Damage ◽

Hippo Pathway ◽

Hippo Signaling ◽

Supporting Cells ◽

The Hippo Pathway

AbstractThe Hippo pathway is an evolutionarily conserved signaling pathway involved in regulating organ size, development, homeostasis and regeneration1–4. YAP1 is a transcriptional coactivator and the primary effector of Hippo signaling. Upstream activation of the Hippo pathway leads to nuclear translocation of YAP1, which then evokes changes in gene expression and cell cycle entry5. A prior study has demonstrated nuclear translocation of YAP1 in the supporting cells of the developing utricle6, but the possible role of YAP1 in hair cell regeneration is unclear. The present study characterizes the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after hair cell injury. During neonatal development of the mouse utricle, YAP1 expression was observed in the cytoplasm of supporting cells, and was also transiently expressed in the cytoplasm of hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle at short time periods after placement in organotypic culture. However, little or no nuclear translocation of YAP1 was observed after injury to the utricles of neonatal or mature mice. In contrast, a significant degree of YAP1 nuclear translocation was observed in the chicken utricle after streptomycin-induced hair cell damage in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may be partly responsible for the distinct regenerative abilities of the avian vs. mammalian inner ear.

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Spontaneous recovery from sunitinib-induced disruption of sarcomere in human iPSC-cardiomyocytes and possible involvement of the Hippo pathway

BMC Pharmacology and Toxicology ◽

10.1186/s40360-021-00527-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Toshikatsu Matsui ◽

Tadahiro Shinozawa

Keyword(s):

Nuclear Localization ◽

Spontaneous Recovery ◽

Hippo Pathway ◽

Clinical Settings ◽

Hippo Signaling ◽

Cardiac Events ◽

Drug Induced ◽

Adverse Cardiac Events ◽

The Hippo Pathway

Abstract Background Sunitinib is known to cause cardiotoxicity in clinical settings. However, among sunitinib-treated patients experiencing adverse cardiac events, decreased cardiac function was reportedly reversible in > 50% of the patients. We previously showed that anti-cancer drugs such as sunitinib cause marked sarcomere disruption in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and the extent of sarcomere disruption can be used to predict drug-induced cardiotoxicity in humans. The aim of this study is to investigate whether the reversibility of sunitinib-induced cardiac events in clinical settings can be mimicked in vitro, and to examine the molecular mechanism responsible for sunitinib-induced cardiotoxicity focusing on the Hippo pathway. Methods iPSC-CMs were stimulated with sunitinib for 72 h and the morphology of sarcomere structures were analyzed by high-content analysis before and after sunitinib washout. To examine the involvement of the Hippo pathway in the sunitinib-induced sarcomere disruption, the extent of nuclear localization of YAP1 (yes-associated protein 1, a Hippo signaling target) was determined. iPSC-CMs were also stimulated with sunitinib and a small molecule inhibitor of the Hippo pathway, XMU-MP-1 and sarcomere structures were analyzed. Results We observed a spontaneous recovery in cardiac sarcomeres in iPSC-CMs that were significantly disrupted by sunitinib treatment after a 72 h or 144 h washout of sunitinib. The extent of nuclear localization of YAP1 was significantly reduced after sunitinib stimulation and tended to return to normal levels after drug washout. Simultaneous stimulation of iPSC-CM with sunitinib and XMU-MP-1 suppressed the sunitinib-induced disruption of sarcomeres. Conclusions These results indicate that iPSC-CMs have the ability to recover from sunitinib-induced sarcomere disruption, and the Hippo pathway plays a role in the process of sunitinib-induced disruption of sarcomere and its recovery. Inhibition of the Hippo pathway may help to develop a co-medication strategy for mitigating the risk of sunitinib-induced adverse cardiac events.

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Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro†

Biology of Reproduction ◽

10.1093/biolre/ioz139 ◽

2019 ◽

Vol 101 (5) ◽

pp. 1001-1017 ◽

Cited By ~ 7

Author(s):

Michele R Plewes ◽

Xiaoying Hou ◽

Pan Zhang ◽

Aixin Liang ◽

Guohua Hua ◽

...

Keyword(s):

Signaling Pathway ◽

Nuclear Localization ◽

Granulosa Cells ◽

Hippo Pathway ◽

Critical Role ◽

Small Cells ◽

Hippo Signaling ◽

Hippo Signaling Pathway ◽

The Hippo Pathway

Abstract Yes-associated protein 1 (YAP1) is a major component of the Hippo signaling pathway. Although the exact extracellular signals that control the Hippo pathway are currently unknown, increasing evidence supports a critical role for the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. Granulosa cells (GCs) within the ovarian follicle proliferate and produce steroids and growth factors, which facilitate the growth of follicle and maturation of the oocyte. We hypothesize that YAP1 plays a role in proliferation and estrogen secretion of GCs. In the current study, we examined the expression of the Hippo signaling pathway in bovine ovaries and determined whether it was important for GC proliferation and estrogen production. Mammalian STE20-like protein kinase 1 (MST1) and large tumor suppressor kinase 2 (LATS2) were identified as prominent upstream components of the Hippo pathway expressed in granulosa and theca cells of the follicle and large and small cells of the corpus luteum. Immunohistochemistry revealed that YAP1 was localized to the nucleus of growing follicles. In vitro, nuclear localization of the downstream Hippo signaling effector proteins YAP1 and transcriptional co-activator with PDZ-binding motif (TAZ) was inversely correlated with GC density, with greater nuclear localization under conditions of low cell density. Treatment with verteporfin and siRNA targeting YAP1 or TAZ revealed a critical role for these transcriptional co-activators in GC proliferation. Furthermore, knockdown of YAP1 in GCs inhibited follicle-stimulating hormone (FSH)-induced estradiol biosynthesis. The data indicate that Hippo pathway transcription co-activators YAP1/TAZ play an important role in GC proliferation and estradiol synthesis, two processes necessary for maintaining normal follicle development.

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Dynamic patterns of YAP1 expression and cellular localization in the developing and injured utricle

Scientific Reports ◽

10.1038/s41598-020-77775-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Vikrant Borse ◽

Matthew Barton ◽

Harry Arndt ◽

Tejbeer Kaur ◽

Mark E. Warchol

Keyword(s):

Inner Ear ◽

Cellular Localization ◽

Nuclear Translocation ◽

Hippo Pathway ◽

Hippo Signaling ◽

Supporting Cells ◽

The Hippo Pathway

AbstractThe Hippo signaling pathway is a key regulator of tissue development and regeneration. Activation of the Hippo pathway leads to nuclear translocation of the YAP1 transcriptional coactivator, resulting in changes in gene expression and cell cycle entry. Recent studies have demonstrated the nuclear translocation of YAP1 during the development of the sensory organs of the inner ear, but the possible role of YAP1 in sensory regeneration of the inner ear is unclear. The present study characterized the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after HC injury. During neonatal development, YAP1 expression was observed in the cytoplasm of supporting cells, and was transiently expressed in the cytoplasm of some differentiating hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle after short periods in organotypic culture. However, little or no nuclear translocation of YAP1 was observed in the utricles of neonatal or mature mice after ototoxic injury. In contrast, substantial YAP1 nuclear translocation was observed in the chicken utricle after streptomycin treatment in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may partially account for the differing regenerative abilities of the avian vs. mammalian inner ear.

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Using Biosensors to Study Protein–Protein Interaction in the Hippo Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.660137 ◽

2021 ◽

Vol 9 ◽

Author(s):

Alexander Pipchuk ◽

Xiaolong Yang

Keyword(s):

Protein Interactions ◽

High Throughput Screening ◽

Cellular Proliferation ◽

Affinity Purification ◽

Hippo Pathway ◽

High Sensitivity ◽

Live Cells ◽

Hippo Signaling ◽

The Hippo Pathway

The Hippo signaling network is dependent on protein–protein interactions (PPIs) as a mechanism of signal transduction to regulate organ size, cellular proliferation and differentiation, tumorigenesis, and other cellular processes. Current efforts aim to resolve the complex regulation of upstream Hippo components or focus on identifying targeted drugs for use in cancer therapy. Despite extensive characterization of the Hippo pathway interactome by affinity purification mass spectrometry (AP-MS) and other methodologies, previous research methods have not been sufficient to achieve these aims. In this review, we describe several recent studies that make use of luciferase-based biosensors as a new approach to study the Hippo Pathway. These biosensors serve as powerful tools with which to study PPIs both in vitro using purified biosensor proteins, and in real time in live cells. Notably, luciferase biosensors have excellent sensitivity and have been used to screen for upstream kinase regulators of the Hippo pathway. Furthermore, the high sensitivity and stability of these biosensors enables their application in high throughput screening for Hippo-targeted chemotherapeutics. Finally, we describe the strengths and weaknesses of this method relative to AP-MS and discuss potential future directions for using biosensors to study Hippo signaling.

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PMEPA1 isoform a drives progression of glioblastoma by promoting protein degradation of the Hippo pathway kinase LATS1

Oncogene ◽

10.1038/s41388-019-1050-9 ◽

2019 ◽

Vol 39 (5) ◽

pp. 1125-1139 ◽

Cited By ~ 2

Author(s):

Jianxiong Ji ◽

Kaikai Ding ◽

Tao Luo ◽

Ran Xu ◽

Xin Zhang ◽

...

Keyword(s):

Tumor Suppressor ◽

Signaling Pathway ◽

Transmembrane Protein ◽

Hippo Pathway ◽

Ectopic Expression ◽

Hippo Signaling ◽

Human Gliomas ◽

Hippo Signaling Pathway ◽

The Hippo Pathway

Abstract The Hippo signaling pathway controls organ development and is also known, in cancer, to have a tumor suppressing role. Within the Hippo pathway, we here demonstrate, in human gliomas, a functional interaction of a transmembrane protein, prostate transmembrane protein, androgen induced 1 (PMEPA1) with large tumor suppressor kinase 1 (LATS1). We show that PMEPA1 is upregulated in primary human gliomas. The PMEPA1 isoform PMEPA1a was predominantly expressed in glioma specimens and cell lines, and ectopic expression of the protein promoted glioma growth and invasion in vitro and in an orthotopic xenograft model in nude mice. In co-immunoprecipitation experiments, PMEPA1a associated with the Hippo tumor suppressor kinase LATS1. This interaction led to a proteasomal degradation of LATS1 through recruitment of the ubiquitin ligase, neural precursor cell expressed, developmentally downregulated 4 (NEDD4), which led to silencing of Hippo signaling. Alanine substitution in PMEPA1a at PY motifs resulted in failed LATS1 degradation. Targeting of a downstream component in the Hippo signaling pathway, YAP, with shRNA, interfered with the growth promoting activities of PMEPA1a in vitro and in vivo. In conclusion, the presented work shows that PMEPA1a contributes to glioma progression by a dysregulation of the Hippo signaling pathway and thus represents a promising target for the treatment of gliomas.

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Angiomotins stimulate LATS kinase autophosphorylation and act as scaffolds that promote Hippo signaling

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004187 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18230-18241 ◽

Cited By ~ 17

Author(s):

Sebastian Mana-Capelli ◽

Dannel McCollum

Keyword(s):

Kinase Activity ◽

Hippo Pathway ◽

Kinase Domain ◽

Activation Loop ◽

Hippo Signaling ◽

Transcriptional Coactivator ◽

Mechanical Environment ◽

Unknown Mechanism ◽

The Hippo Pathway ◽

Kinase A

The Hippo pathway controls cell proliferation, differentiation, and survival by regulating the Yes-associated protein (YAP) transcriptional coactivator in response to various stimuli, including the mechanical environment. The major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP. LATS1/2 are activated by phosphorylation on a hydrophobic motif (HM) outside of the kinase domain by MST1/2 and other kinases. Phosphorylation of the HM motif then triggers autophosphorylation of the kinase in the activation loop to fully activate the kinase, a process facilitated by MOB1. The angiomotin family of proteins (AMOT, AMOTL1, and AMOTL2) bind LATS1/2 and promote its kinase activity and YAP phosphorylation through an unknown mechanism. Here we show that angiomotins increase Hippo signaling through multiple mechanisms. We found that, by binding LATS1/2, SAV1, and YAP, angiomotins function as a scaffold that connects LATS1/2 to both its activator SAV1–MST1 and its target YAP. Deletion of all three angiomotins reduced the association of LATS1 with SAV1–MST1 and decreased MST1/2-mediated LATS1/2-HM phosphorylation. Angiomotin deletion also reduced LATS1/2's ability to associate with and phosphorylate YAP. In addition, we found that angiomotins have an unexpected function along with MOB1 to promote autophosphorylation of LATS1/2 on the activation loop motif independent of HM phosphorylation. These results indicate that angiomotins enhance Hippo signaling by stimulating LATS1/2 autophosphorylation and by connecting LATS1/2 with both its activator SAV1–MST1/2 and its substrate YAP.

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Hippo-YAP/MCP-1 mediated tubular maladaptive repair promote inflammation in renal failed recovery after ischemic AKI

Cell Death and Disease ◽

10.1038/s41419-021-04041-8 ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Zhihuang Zheng ◽

Chuanlei Li ◽

Guangze Shao ◽

Jinqing Li ◽

Kexin Xu ◽

...

Keyword(s):

Chronic Inflammation ◽

Ischemia Reperfusion ◽

Hippo Pathway ◽

Glucose Deprivation ◽

Macrophage Infiltration ◽

Renal Inflammation ◽

Monocyte Chemoattractant Protein 1 ◽

The Hippo Pathway

AbstractAcute kidney injury (AKI) is associated with significant morbidity and its chronic inflammation contributes to subsequent chronic kidney disease (CKD) development. Yes-associated protein (YAP), the major transcriptional coactivator of the Hippo pathway, has been shown associated with chronic inflammation, but its role and mechanism in AKI-CKD transition remain unclear. Here we aimed to investigate the role of YAP in AKI-induced chronic inflammation. Renal ischemia/reperfusion (I/R) was used to induce a mouse model of AKI-CKD transition. We used verteporfin (VP), a pharmacological inhibitor of YAP, to treat post-IRI mice for a period, and evaluated the influence of YAP inhibition on long-term outcomes of AKI. In our results, severe IRI led to maladaptive tubular repair, macrophages infiltration, and progressive fibrosis. Following AKI, the Hippo pathway was found significantly altered with YAP persistent activation. Besides, tubular YAP activation was associated with the maladaptive repair, also correlated with interstitial macrophage infiltration. Monocyte chemoattractant protein 1 (MCP-1) was found notably upregulated with YAP activation. Of note, pharmacological inhibition of YAP in vivo attenuated renal inflammation, including macrophage infiltration and MCP-1 overexpression. Consistently, in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) induced YAP activation and MCP-1 overproduction whereas these could be inhibited by VP. In addition, we modulated YAP activity by RNA interference, which further confirmed YAP activation enhances MCP-1 expression. Together, we concluded tubular YAP activation with maladaptive repair exacerbates renal inflammation probably via promoting MCP-1 production, which contributes to AKI-CKD transition.

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NPHP4, a cilia-associated protein, negatively regulates the Hippo pathway

The Journal of Cell Biology ◽

10.1083/jcb.201009069 ◽

2011 ◽

Vol 193 (4) ◽

pp. 633-642 ◽

Cited By ~ 89

Author(s):

Sandra Habbig ◽

Malte P. Bartram ◽

Roman U. Müller ◽

Ricarda Schwarz ◽

Nikolaos Andriopoulos ◽

...

Keyword(s):

Cell Proliferation ◽

Cellular Proliferation ◽

Nuclear Translocation ◽

Hippo Pathway ◽

Negative Regulator ◽

Hippo Signaling ◽

Transcriptional Coactivator ◽

Hippo Signaling Pathway ◽

The Hippo Pathway ◽

Potent Negative Regulator

The conserved Hippo signaling pathway regulates organ size in Drosophila melanogaster and mammals and has an essential role in tumor suppression and the control of cell proliferation. Recent studies identified activators of Hippo signaling, but antagonists of the pathway have remained largely elusive. In this paper, we show that NPHP4, a known cilia-associated protein that is mutated in the severe degenerative renal disease nephronophthisis, acts as a potent negative regulator of mammalian Hippo signaling. NPHP4 directly interacted with the kinase Lats1 and inhibited Lats1-mediated phosphorylation of the Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding domain), leading to derepression of these protooncogenic transcriptional regulators. Moreover, NPHP4 induced release from 14-3-3 binding and nuclear translocation of YAP and TAZ, promoting TEA domain (TEAD)/TAZ/YAP-dependent transcriptional activity. Consistent with these data, knockdown of NPHP4 negatively affected cellular proliferation and TEAD/TAZ activity, essentially phenocopying loss of TAZ function. These data identify NPHP4 as a negative regulator of the Hippo pathway and suggest that NPHP4 regulates cell proliferation through its effects on Hippo signaling.

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The Hippo Pathway: A Master Regulatory Network Important in Cancer

Cells ◽

10.3390/cells10061416 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1416

Author(s):

Qiuping Liu ◽

Xiaomeng Liu ◽

Guanbin Song

Keyword(s):

Metabolic Regulation ◽

Human Cancer ◽

Hippo Pathway ◽

Tumor Development ◽

Biological Significance ◽

Cancer Development ◽

Hippo Signaling ◽

Complex Regulation ◽

And Function ◽

The Hippo Pathway

The Hippo pathway is pervasively activated and has been well recognized to play critical roles in human cancer. The deregulation of Hippo signaling involved in cancer development, progression, and resistance to cancer treatment have been confirmed in several human cancers. Its biological significance and deregulation in cancer have drawn increasing interest in the past few years. A fundamental understanding of the complexity of the Hippo pathway in cancer is crucial for improving future clinical interventions and therapy for cancers. In this review, we try to clarify the complex regulation and function of the Hippo signaling network in cancer development, including its role in signal transduction, metabolic regulation, and tumor development, as well as tumor therapies targeting the Hippo pathway.

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