Novel linear motif filtering protocol reveals the role of the LC8 dynein light chain in the Hippo pathway

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

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Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0103 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3107-3116 ◽

Cited By ~ 29

Author(s):

Anindya Ghosh-Roy ◽

Bela S. Desai ◽

Krishanu Ray

Keyword(s):

Light Chain ◽

Cytoplasmic Dynein ◽

Actin Dynamics ◽

Dynein Light Chain ◽

Actin Assembly ◽

Cellular Functions ◽

Directed Movement ◽

Cellular Processes ◽

Dynactin Complex

Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila.

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P04.20 The role of YAP in Glioblastoma cell lines

Neuro-Oncology ◽

10.1093/neuonc/noab180.074 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii22-ii23

Author(s):

G Casati ◽

L Giunti ◽

A Iorio ◽

A Marturano ◽

I Sardi

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Western Blotting ◽

Cytotoxic Effect ◽

Target Genes ◽

Hippo Pathway ◽

Combined Treatment ◽

Set Up ◽

The Hippo Pathway

Abstract BACKGROUND Glioblastoma (GBM) is a primary human malignant brain tumor, the most common in adults. Several studies have highlighted the Hippo-pathway as a cancer signalling network. The Hippo pathway is an evolutionarily conserved signal cascade, which is involved in the control of organ growth. Dysregulations among this pathway have been found in lung, ovarian, liver and colorectal cancer. The key downstream effector of the Hippo-pathway is the Yes-associated protein (YAP); in the nucleus, its function as transcription co-activator is to interact with transcription factors, resulting in the expression of target genes involved in pro-proliferating and anti-apoptotic programs. MATERIAL AND METHODS Using western blotting analysis, we determined the nuclear expression of YAP on three GBM cell lines (U87MG, T98G and A172). To investigate which inhibitors against the Hippo-pathway were the most efficient, we performed a cytotoxic assay: we treated all the three cell lines with different inhibitors such as Verteporfin (VP), Cytochalasin D (CIT), Latrunculin A (LAT), Dobutamine (DOB) and Y27632. Afterwards, we performed a treatment using Doxorubicin (DOX) combined with the inhibitors, evaluating its cytotoxic effect on our cell lines, through cell viability experiments. More western blotting experiments were performed to investigate the oncogenic role of YAP at nucleus level. Furthermore, preliminary experiments have been conducted in order to investigate the apoptosis, senescence and autophagy modulation due to the Hippo-pathway. RESULTS We showed our cell lines express nuclear YAP. We assessed the efficiency of the main inhibitors against Hippo-pathway, proving that VP, LAT A and CIT show a strong cytostatic effect, linked to time increase; plus we saw a cytotoxic effect on T98G. The association of DOX with selected inhibitors is able to reduce cell viability and nuclear YAP expression rate in all three GBM lines. Finally, preliminary experiments were set up to assess how and if the mechanisms of apoptosis, autophagy and senescence were affected by the Hippo-pathway. The combination of DOX with inhibitors promotes resistance to apoptosis. CONCLUSION Our results show that nuclear YAP is present in all tumor lines, thus confirming that this molecular pathway is functioning in GBM lines. Nuclear YAP is more highly expressed after DOX administration. Moreover, the combined treatment (DOX with Hippo-pathway inhibitors) reduces both cell proliferation and viability, and increases the rate of apoptosis. Preliminary experiments on senescence and autophagy were used to determine the best Hippo-pathway inhibitor. These data demonstrate that the Hippo-pathway plays a crucial role in GBM proliferation and resistance to apoptosis. Inhibiting this pathway and in particular the transcription factor YAP, in association with DOX, might be an excellent therapeutic target.

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1183Beta-Adrenoceptor activation increases cardiac galectin-3 levels via the hippo signaling pathway

European Heart Journal ◽

10.1093/eurheartj/ehz748.0025 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

X.-J Du ◽

W B Zhao ◽

Q Lu ◽

M N Nguyen ◽

M Ziemann ◽

...

Keyword(s):

Hippo Pathway ◽

Fold Increase ◽

Signalling Pathway ◽

Mutant Form ◽

Wild Type ◽

Galectin 3 ◽

Β Blockers ◽

The Hippo Pathway ◽

Hippo Signalling

Abstract Background Galectin-3 (Gal-3) is a clinical biomarker for risk of cardiovascular disease and a disease mediator forming a therapeutic target. However, the mechanism(s) that regulate cardiac expression of Gal-3 remains unknown. Activation of the sympatho-β-adrenergic system is a hallmark of heart disease, but the relationship of βAR activation and cardiac content of Gal-3 remains unknown. Purpose To determine the role of βAR activation in regulating cardiac Gal-3 level and the responsible mechanism focusing on the Hippo signalling pathway. Methods Wild-type and Gal-3 gene deleted (Gal3-KO) mice were used. To test the role of the Hippo pathway, we used transgenic (TG) mouse strains with cardiac overexpression of mammalian-20-like sterile kinase 1 (Mst1, mammalian orthology of Drosophila Hippo kinase) either in wild-type form (TG-Mst1) or dominative-negative kinase dead mutant form (TG-dnMst1). Effects of β-antagonist (isoprenaline, ISO) and antagonists were determined. We measured phosphorylation (Ser127) of YAP as a transcription co-regulator acting as the main signal output of the Hippo pathway. Results In wild-type mice, treatment with ISO led to a time- and dose-dependent increase in cardiac expression of Gal-3 (Fig. A) accompanied by elevated circulating Gal-3 levels (Fig. B). ISO treatment stimulated cardiac expression of Mst1 and YAP hyper-phosphorylation (i.e. inactivation, Fig. C), indicating activation of the Hippo signalling. These effects of ISO were inhibited by β-blockers (propranolol, Prop; carvedilol, Carv; Fig. D,E). Relative to non-TG controls, ISO-induced expression of Gal-3 was inhibited by 75% in TG-dnMst1 mice (inactivated Mst1), but exaggerated by 7-fold in TG-Mst1 mice (activated Mst1). Mst1-TG mice had a 45-fold increase in Gal-3 content, YAP hyper-phosphorylation and enhanced pro-fibrotic signaling. In Mst1-TG mice, whilst blood Gal-3 level was unchanged, treatment with ISO (6 mg, 2 days) evoked a marked increase in cardiac and blood Gal-3 levels. Using rat cardiomyoblasts, we showed that ISO-mediated Mst1 expression and YAP phosphorylation were PKA-dependent and that siRNA-mediated YAP knockdown led to Gal-3 upregulation. The role of Gal-3 in mediating ISO-induced cardiomyopathy was examined by treating wild-type and Gal3-KO mice with ISO (30 mg/kg, 7 days). ISO-treated wild-type mice had 8-fold increase in cardiac Gal-3, ventricular dysfunction, fibrosis, hypertrophy and activated inflammatory or fibrotic signalling. All these changes, except hypertrophy, were abolished by Gal3-KO. beta-AR regulates galectin-3 Conclusion βAR stimulation increases cardiac expression of Gal-3 through activation of the Hippo signalling pathway. This is accompanied by elevated circulating Gal-3 level. βAR antagonists inhibited βAR-Mst1 (Hippo) signalling and cardiac Gal-3 expression, actions likely contributing to the overall efficacy of β-blockers. Acknowledgement/Funding NHMRC of Australia; Nature Science Fund of China

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The Drosophila tctex-1 Light Chain Is Dispensable for Essential Cytoplasmic Dynein Functions but Is Required during Spermatid Differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-01-0013 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3005-3014 ◽

Cited By ~ 41

Author(s):

Min-gang Li ◽

Madeline Serr ◽

Eric A. Newman ◽

Thomas S. Hays

Keyword(s):

Light Chain ◽

Cytoplasmic Dynein ◽

Sperm Nucleus ◽

P Element ◽

Dynein Light Chain ◽

Null Mutant ◽

Multiple Functions ◽

Bona Fide ◽

Adult Organism

Variations in subunit composition and modification have been proposed to regulate the multiple functions of cytoplasmic dynein. Here, we examine the role of the Drosophila ortholog of tctex-1, the 14-kDa dynein light chain. We show that the 14-kDa light chain is a bona fide component of Drosophila cytoplasmic dynein and use P element excision to generate flies that completely lack this dynein subunit. Remarkably, the null mutant is viable and the only observed defect is complete male sterility. During spermatid differentiation, the 14-kDa light chain is required for the localization of a nuclear “cap” of cytoplasmic dynein and for proper attachment between the sperm nucleus and flagellar basal body. Our results provide evidence that the function of the 14-kDa light chain in Drosophila is distinct from other dynein subunits and is not required for any essential functions in early development or in the adult organism.

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The Hippo Pathway Effector YAP1 Regulates Intestinal Epithelial Cell Differentiation

Cells ◽

10.3390/cells9081895 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1895 ◽

Cited By ~ 1

Author(s):

Sepideh Fallah ◽

Jean-François Beaulieu

Keyword(s):

Stem Cells ◽

Cell Differentiation ◽

Epithelial Cells ◽

Cell Line ◽

Intestinal Epithelial Cells ◽

Hippo Pathway ◽

Intestinal Cell ◽

Intestinal Epithelial ◽

The Hippo Pathway

The human intestine is covered by epithelium, which is continuously replaced by new cells provided by stem cells located at the bottom of the glands. The maintenance of intestinal stem cells is supported by a niche which is composed of several signaling proteins including the Hippo pathway effectors YAP1/TAZ. The role of YAP1/TAZ in cell proliferation and regeneration is well documented but their involvement on the differentiation of intestinal epithelial cells is unclear. In the present study, the role of YAP1/TAZ on the differentiation of intestinal epithelial cells was investigated using the HT29 cell line, the only multipotent intestinal cell line available, with a combination of knockdown approaches. The expression of intestinal differentiation cell markers was tested by qPCR, Western blot, indirect immunofluorescence and electron microscopy analyses. The results show that TAZ is not expressed while the abolition of YAP1 expression led to a sharp increase in goblet and absorptive cell differentiation and reduction of some stem cell markers. Further studies using double knockdown experiments revealed that most of these effects resulting from YAP1 abolition are mediated by CDX2, a key intestinal cell transcription factor. In conclusion, our results indicate that YAP1/TAZ negatively regulate the differentiation of intestinal epithelial cells through the inhibition of CDX2 expression.

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Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain

Journal of Virology ◽

10.1128/jvi.74.21.10212-10216.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10212-10216 ◽

Cited By ~ 214

Author(s):

Hélène Raux ◽

Anne Flamand ◽

Danielle Blondel

Keyword(s):

Rabies Virus ◽

Light Chain ◽

Cytoplasmic Dynein ◽

Dynein Light Chain ◽

Directed Movement ◽

Virus Particles ◽

P Protein ◽

Infected Cells ◽

Transcription And Replication

ABSTRACT The rabies virus P protein is involved in viral transcription and replication but its precise function is not clear. We investigated the role of P (CVS strain) by searching for cellular partners by using a two-hybrid screening of a PC12 cDNA library. We isolated a cDNA encoding a 10-kDa dynein light chain (LC8). LC8 is a component of cytoplasmic dynein involved in the minus end-directed movement of organelles along microtubules. We confirmed that this molecule interacts with P by coimmunoprecipitation in infected cells and in cells transfected with a plasmid encoding P protein. LC8 was also detected in virus particles. Series of deletions from the N- and C-terminal ends of P protein were used to map the LC8-binding domain to the central part of P (residues 138 to 172). These results are relevant to speculate that dynein may be involved in the axonal transport of rabies virus along microtubules through neuron cells.

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Linking Extracellular Matrix Agrin to the Hippo Pathway in Liver Cancer and Beyond

Cancers ◽

10.3390/cancers10020045 ◽

2018 ◽

Vol 10 (2) ◽

pp. 45 ◽

Cited By ~ 21

Author(s):

Sayan Chakraborty ◽

Wanjin Hong

Keyword(s):

Extracellular Matrix ◽

Neuromuscular Junctions ◽

Hippo Pathway ◽

Cardiac Regeneration ◽

Matrix Rigidity ◽

Macroscopic Scale ◽

Cellular Processes ◽

Scale Matrix ◽

The Hippo Pathway

In addition to the structural and scaffolding role, the extracellular matrix (ECM) is emerging as a hub for biomechanical signal transduction that is frequently relayed to intracellular sensors to regulate diverse cellular processes. At a macroscopic scale, matrix rigidity confers long-ranging effects contributing towards tissue fibrosis and cancer. The transcriptional co-activators YAP/TAZ, better known as the converging effectors of the Hippo pathway, are widely recognized for their new role as nuclear mechanosensors during organ homeostasis and cancer. Still, how YAP/TAZ senses these “stiffness cues” from the ECM remains enigmatic. Here, we highlight the recent perspectives on the role of agrin in mechanosignaling from the ECM via antagonizing the Hippo pathway to activate YAP/TAZ in the contexts of cancer, neuromuscular junctions, and cardiac regeneration.

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

Biochemical Society Transactions ◽

10.1042/bst20140030 ◽

2014 ◽

Vol 42 (4) ◽

pp. 816-821 ◽

Cited By ~ 8

Author(s):

David Romano ◽

David Matallanas ◽

Dennie T. Frederick ◽

Keith T. Flaherty ◽

Walter Kolch

Keyword(s):

Protein Interactions ◽

Hippo Pathway ◽

Promoter Hypermethylation ◽

Protein Protein Interactions ◽

Evolutionarily Conserved ◽

Or Gene ◽

Phosphoinositide 3 Kinase ◽

Ras Isoforms ◽

The Hippo Pathway

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.

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