The ESCRT-0 subcomplex component Hrs/Hgs is a master regulator of myogenesis via modulation of signaling and degradation pathways

Abstract Background Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis. Results We uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited. Conclusions We show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.

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AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e12-03-0251 ◽

2012 ◽

Vol 23 (18) ◽

pp. 3612-3623 ◽

Cited By ~ 35

Author(s):

Michael R. Dores ◽

May M. Paing ◽

Huilan Lin ◽

William A. Montagne ◽

Adriano Marchese ◽

...

Keyword(s):

Adaptor Protein ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Early Endosomes ◽

Lysosomal Sorting ◽

Protease Activated Receptor 1 ◽

Signaling Receptors ◽

G Protein Coupled ◽

Hepatocyte Growth

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

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Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012303 ◽

2020 ◽

Vol 295 (23) ◽

pp. 7941-7957 ◽

Cited By ~ 1

Author(s):

Shiho Torii ◽

Yasuko Orba ◽

Michihito Sasaki ◽

Koshiro Tabata ◽

Yuji Wada ◽

...

Keyword(s):

Chikungunya Virus ◽

Host Factors ◽

Genome Replication ◽

Sirna Screen ◽

Chikv Infection ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Viral Genome Replication ◽

Human Host Factors ◽

Hepatocyte Growth

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor–regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

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Arrestin-2 Interacts with the Endosomal Sorting Complex Required for Transport Machinery to Modulate Endosomal Sorting of CXCR4

Molecular Biology of the Cell ◽

10.1091/mbc.e10-02-0169 ◽

2010 ◽

Vol 21 (14) ◽

pp. 2529-2541 ◽

Cited By ~ 67

Author(s):

Rohit Malik ◽

Adriano Marchese

Keyword(s):

Molecular Mechanisms ◽

Lysosomal Degradation ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Adaptor Molecule ◽

Escrt Machinery ◽

Early Endosomes ◽

G Protein Coupled ◽

Hepatocyte Growth ◽

Dependent Mechanism

The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.

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ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

The Journal of Cell Biology ◽

10.1083/jcb.201110031 ◽

2012 ◽

Vol 197 (3) ◽

pp. 407-419 ◽

Cited By ~ 98

Author(s):

Michael R. Dores ◽

Buxin Chen ◽

Huilan Lin ◽

Unice J.K. Soh ◽

May M. Paing ◽

...

Keyword(s):

Mammalian Cells ◽

Interacting Protein ◽

Kinase Substrate ◽

Endosomal Sorting ◽

Ubiquitin Binding ◽

Lysosomal Sorting ◽

Protease Activated Receptor 1 ◽

Signaling Receptors ◽

G Protein Coupled ◽

Hepatocyte Growth

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX3L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPXnL motifs.

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SNX-1 and RME-8 oppose the assembly of HGRS-1/ESCRT-0 degradative microdomains on endosomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612730114 ◽

2017 ◽

Vol 114 (3) ◽

pp. E307-E316 ◽

Cited By ~ 33

Author(s):

Anne Norris ◽

Prasad Tammineni ◽

Simon Wang ◽

Julianne Gerdes ◽

Alexandra Murr ◽

...

Keyword(s):

Kinase Substrate ◽

Endosomal Sorting ◽

Sorting Nexin ◽

Binding Partner ◽

Regulatory Interactions ◽

C Elegans ◽

J Domain ◽

Vacuolar Protein ◽

Hepatocyte Growth

After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1–labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8 microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenon we observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.

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Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and guanylate kinase 1 (GUK1) are differentially expressed in GH-secreting adenomas

Pituitary ◽

10.1007/s11102-006-9277-1 ◽

2006 ◽

Vol 9 (2) ◽

pp. 83-92 ◽

Cited By ~ 3

Author(s):

Anderson Alves da Rocha ◽

Ricardo Rodrigues Giorgi ◽

Sandra Valeria de Sa ◽

Maria Lucia Correa-Giannella ◽

Maria Angela Fortes ◽

...

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Kinase ◽

Differentially Expressed ◽

Guanylate Kinase ◽

Kinase Substrate ◽

Hepatocyte Growth ◽

Tyrosine Kinase Substrate

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EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.281.4.g890 ◽

2001 ◽

Vol 281 (4) ◽

pp. G890-G898 ◽

Cited By ~ 56

Author(s):

Suzana D. Savkovic ◽

Akila Ramaswamy ◽

Athanasia Koutsouris ◽

Gail Hecht

Keyword(s):

Tight Junction ◽

Signaling Cascades ◽

Extracellular Signal Regulated Kinase ◽

Intestinal Epithelia ◽

Extracellular Signal ◽

Tnf Α ◽

Functional Consequences ◽

First Time ◽

Necrosis Factor ◽

Iκbα Degradation

Enteropathogenic Escherichia coli (EPEC) alters many functions of the host intestinal epithelia. Inflammation is initiated by activation of nuclear factor (NF)-κB, and paracellular permeability is enhanced via a Ca2+- and myosin light-chain kinase (MLCK)-dependent pathway. The aims of this study were to identify signaling pathways by which EPEC triggers inflammation and to determine whether these pathways parallel or diverge from those that alter permeability. EPEC-induced phosphorylation and degradation of the primary inhibitor of NF-κB (IκBα) were tumor necrosis factor (TNF)-α and interleukin (IL)-1β independent. In contrast to Salmonella typhimurium, EPEC-stimulated IκBα degradation and IL-8 expression did not require Ca2+. Instead, extracellular signal-regulated kinase (ERK)-1/2 was significantly and rapidly activated. ERK1/2 inhibitors attenuated IκBα degradation and IL-8 expression. Although ERK1/2 can activate MLCK, its inhibition had no impact on EPEC disruption of the tight junction barrier. In conclusion, EPEC-induced inflammation 1) is TNF-α and IL-1β receptor independent, 2) utilizes pathways differently from S. typhimurium, 3) requires ERK1/2, and 4) employs signals that are distinct from those that alter permeability. This is the first time that EPEC-activated signaling cascades have been linked to independent functional consequences.

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Identification of MEF2-regulated genes during muscle differentiation

Physiological Genomics ◽

10.1152/physiolgenomics.00149.2004 ◽

2004 ◽

Vol 20 (1) ◽

pp. 143-151 ◽

Cited By ~ 19

Author(s):

James Paris ◽

Carl Virtanen ◽

Zhibin Lu ◽

Mark Takahashi

Keyword(s):

Gene Regulation ◽

Transcription Factor ◽

Transcription Factors ◽

Neuromuscular Junctions ◽

Muscle Differentiation ◽

Signaling Cascades ◽

Specific Gene ◽

Genetic Mechanisms ◽

Selection Parameters ◽

First Time

Although a great deal has been elucidated concerning the mechanisms regulating muscle differentiation, little is known about transcription factor-specific gene regulation. Our understanding of the genetic mechanisms regulating cell differentiation is quite limited. Much of what has been defined centers on regulatory signaling cascades and transcription factors. Surprisingly few studies have investigated the association of genes with specific transcription factors. To address these issues, we have utilized a method coupling chromatin immunoprecipitation and CpG microarrays to characterize the genes associated with MEF2 in differentiating C2C12 cells. Results demonstrated a defined binding pattern over the course of differentiation. Filtered data demonstrated 9 clones to be elevated at 0 h, 792 at 6 h, 163 by 1 day, and 316 at 3 days. Using unbiased selection parameters, we selected a subset of 291 prospective candidates. Clones were sequenced and filtered for removal of redundancy between clones and for the presence of repetitive elements. We were able to place 50 of these on the mouse genome, and 20 were found to be located near well-annotated genes. From this list, previously undefined associations with MEF2 were discovered. Many of these genes represent proteins involved in neurogenesis, neuromuscular junctions, signaling and metabolism. The remaining clones include many full-length cDNA and represent novel gene targets. The results of this study provides for the first time, a unique look at gene regulation at the level of transcription factor binding in differentiating muscle.

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Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7685-7692.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7685-7692 ◽

Cited By ~ 86

Author(s):

Sylvie Urbé ◽

Ian G. Mills ◽

Harald Stenmark ◽

Naomi Kitamura ◽

Michael J. Clague

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Membrane Trafficking ◽

Signal Transduction Pathway ◽

Dominant Negative ◽

Kinase Substrate ◽

Hepatocyte Growth ◽

Tyrosine Kinase Substrate

ABSTRACT Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

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A Functional SNP in the Promoter of LBX1 Is Associated With the Development of Adolescent Idiopathic Scoliosis Through Involvement in the Myogenesis of Paraspinal Muscles

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.777890 ◽

2021 ◽

Vol 9 ◽

Author(s):

Leilei Xu ◽

Zhenhua Feng ◽

Zhicheng Dai ◽

Wayne Y. W. Lee ◽

Zhichong Wu ◽

...

Keyword(s):

Adolescent Idiopathic Scoliosis ◽

Idiopathic Scoliosis ◽

Muscle Development ◽

Luciferase Assay ◽

Paraspinal Muscles ◽

Mobility Shift ◽

Putative Promoter Region ◽

Mobility Shift Assay ◽

First Time ◽

Functional Snp

Previous studies have shown that LBX1 is associated with adolescent idiopathic scoliosis (AIS) in multiple populations. For the first time, rs1322330 located in the putative promoter region of LBX1 was found significantly associated with AIS in the Chinese population [p = 6.08 × 10–14, odds ratio (OR) = 1.42, 95% confidence interval of 1.03–1.55]. Moreover, the luciferase assay and electrophoretic mobility shift assay supported that the allele A of rs1322330 could down-regulate the expression of LBX1 in the paraspinal muscles of AIS. In addition, silencing LBX1 in the myosatellite cells resulted in significantly inhibited cell viability and myotube formation, which supported an essential role of LBX1 in muscle development of AIS. To summarize, rs1322330 may be a novel functional SNP regulating the expression of LBX1, which was involved in the etiology of AIS possibly via regulation of myogenesis in the paraspinal muscles.

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