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1.
PLoS Biol ; 17(10): e3000509, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613895

RESUMO

The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ or WW-domain-containing transcriptional activator [WWTR1]), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. Here, we show that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. We find similar results in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transativadores/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Fenômenos Biomecânicos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Polaridade Celular , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mecanotransdução Celular , Camundongos , Proteínas Nucleares/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transativadores/metabolismo , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Proteínas de Sinalização YAP
2.
J Cell Sci ; 132(8)2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-30872454

RESUMO

The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, regulating both known and unknown effectors. Here, we identify FARP2 as a 'RIPR' motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6-aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι-FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors.This article has an associated First Person interview with the first author of the paper.


Assuntos
Células Epiteliais/citologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína Quinase C/metabolismo , Junções Íntimas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Células CACO-2 , Polaridade Celular , Fatores de Troca do Nucleotídeo Guanina/genética , Células HCT116 , Humanos , Fosforilação
3.
J Cell Sci ; 131(22)2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30404826

RESUMO

Human cells can sense mechanical stress acting upon integrin adhesions and respond by sending the YAP (also known as YAP1) and TAZ (also known as WWTR1) transcriptional co-activators to the nucleus to drive TEAD-dependent transcription of target genes. How integrin signaling activates YAP remains unclear. Here, we show that integrin-mediated mechanotransduction requires the Enigma and Enigma-like proteins (PDLIM7 and PDLIM5, respectively; denoted for the family of PDZ and LIM domain-containing proteins). YAP binds to PDLIM5 and PDLIM7 (hereafter PDLIM5/7) via its C-terminal PDZ-binding motif (PBM), which is essential for full nuclear localization and activity of YAP. Accordingly, silencing of PDLIM5/7 expression reduces YAP nuclear localization, tyrosine phosphorylation and transcriptional activity. The PDLIM5/7 proteins are recruited from the cytoplasm to integrin adhesions and F-actin stress fibers in response to force by binding directly to the key stress fiber component α-actinin. Thus, forces acting on integrins recruit Enigma family proteins to trigger YAP activation during mechanotransduction.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas com Domínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CACO-2 , Fibroblastos/metabolismo , Células HEK293 , Humanos , Integrinas/metabolismo , Mecanotransdução Celular , Camundongos , Transdução de Sinais , Transativadores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP
4.
EMBO J ; 34(7): 940-54, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25712476

RESUMO

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.


Assuntos
Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mecanotransdução Celular/fisiologia , Folículo Ovariano/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espectrina/metabolismo , Animais , Linhagem Celular , Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Folículo Ovariano/citologia , Proteínas Serina-Treonina Quinases/genética , Espectrina/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas de Sinalização YAP
5.
Development ; 143(10): 1674-87, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26989177

RESUMO

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Homeostase , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Pele/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dasatinibe/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Neoplasias de Células Escamosas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Cicatrização/efeitos dos fármacos , Proteínas de Sinalização YAP , Quinases da Família src/metabolismo
6.
J Cell Sci ; 129(13): 2651-9, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27231092

RESUMO

In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli.


Assuntos
Proteínas de Drosophila/genética , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Microvilosidades/metabolismo , Miosina Tipo V/genética , Proteínas rab de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Caderinas/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Polaridade Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Humanos , Microtúbulos/genética , Microvilosidades/genética , Miosina Tipo V/metabolismo , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
Bioessays ; 38(7): 644-53, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27173018

RESUMO

The YAP/TAZ family of transcriptional co-activators drives cell proliferation in epithelial tissues and cancers. Yet, how YAP and TAZ are physiologically regulated remains unclear. Here we review recent reports that YAP and TAZ act primarily as sensors of epithelial cell polarity, being inhibited when cells differentiate an apical membrane domain, and being activated when cells contact the extracellular matrix via their basal membrane domain. Apical signalling occurs via the canonical Crumbs/CRB-Hippo/MST-Warts/LATS kinase cascade to phosphorylate and inhibit YAP/TAZ. Basal signalling occurs via Integrins and Src family kinases to phosphorylate and activate YAP/TAZ. Thus, YAP/TAZ is localised to the nucleus in basal stem/progenitor cells and cytoplasm in differentiated squamous cells or columnar cells. In addition, other signals such as mechanical forces, tissue damage and possibly receptor tyrosine kinases (RTKs) can influence MST-LATS or Src family kinase activity to modulate YAP/TAZ activity.


Assuntos
Polaridade Celular , Proteínas Nucleares/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/fisiologia , Animais , Fenômenos Biomecânicos , Proteínas de Ciclo Celular , Proteínas de Drosophila/fisiologia , Epitélio/metabolismo , Epitélio/fisiologia , Humanos , Proteínas de Ligação a RNA , Proteínas Repressoras , Células-Tronco/fisiologia , Transativadores/fisiologia , Proteínas de Sinalização YAP
9.
Biology (Basel) ; 13(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38927266

RESUMO

The repurposing of previously clinically approved drugs as an alternative therapeutic approach to treating disease has gained significant attention in recent years. A multitude of studies have demonstrated various and successful therapeutic interventions with these drugs in a wide range of neoplastic diseases, including multiple myeloma, leukaemia, glioblastoma, and colon cancer. Drug repurposing has been widely encouraged due to the known efficacy, safety, and convenience of already established drugs, allowing the bypass of the long and difficult road of lead optimization and drug development. Repurposing drugs in cancer therapy is an exciting prospect due to the ability of these drugs to successfully target cancer-associated genes, often dysregulated in oncogenic signalling pathways, amongst which are the classical cancer signalling pathways; WNT (wingless-related integration type) and Hippo signalling. These pathways play a fundamental role in controlling organ size, tissue homeostasis, cell proliferation, and apoptosis, all hallmarks of cancer initiation and progression. Prolonged dysregulation of these pathways has been found to promote uncontrolled cellular growth and malignant transformation, contributing to carcinogenesis and ultimately leading to malignancy. However, the translation of cancer signalling pathways and potential targeted therapies in cancer treatment faces ongoing challenges due to the pleiotropic nature of cancer cells, contributing to resistance and an increased rate of incomplete remission in patients. This review provides analyses of a range of potential anti-cancer compounds in drug repurposing. It unravels the current understanding of the molecular rationale for repurposing these drugs and their potential for targeting key oncogenic signalling pathways.

10.
Cells ; 10(11)2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34831369

RESUMO

The activation of microglia, the inflammatory cells of the central nervous system (CNS), has been linked to the pathogenesis of Alzheimer's disease and other neurodegenerative diseases. How microglia sense the changing brain environment, in order to respond appropriately, is still being elucidated. Microglia are able to sense and respond to the mechanical properties of their microenvironment, and the physical and molecular pathways underlying this mechanosensing/mechanotransduction in microglia have recently been investigated. The Hippo pathway functions through mechanosensing and subsequent protein kinase cascades, and is critical for neuronal development and many other cellular processes. In this review, we examine evidence for the potential involvement of Hippo pathway components specifically in microglia in the pathogenesis of Alzheimer's disease. We suggest that the Hippo pathway is worth investigating as a mechanosensing pathway in microglia, and could be one potential therapeutic target pathway for preventing microglial-induced neurodegeneration in AD.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Via de Sinalização Hippo , Mecanotransdução Celular , Microglia/metabolismo , Microglia/patologia , Animais , Humanos , Modelos Biológicos
11.
Cells ; 9(1)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936297

RESUMO

In order to ascertain their external environment, cells and tissues have the capability to sense and process a variety of stresses, including stretching and compression forces. These mechanical forces, as experienced by cells and tissues, are then converted into biochemical signals within the cell, leading to a number of cellular mechanisms being activated, including proliferation, differentiation and migration. If the conversion of mechanical cues into biochemical signals is perturbed in any way, then this can be potentially implicated in chronic disease development and processes such as neurological disorders, cancer and obesity. This review will focus on how the interplay between mechanotransduction, cellular structure, metabolism and signalling cascades led by the Hippo-YAP/TAZ axis can lead to a number of chronic diseases and suggest how we can target various pathways in order to design therapeutic targets for these debilitating diseases and conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Doença Crônica/epidemiologia , Mecanotransdução Celular , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Aciltransferases , Via de Sinalização Hippo , Humanos , Transdução de Sinais
12.
Elife ; 82019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31661072

RESUMO

Mask family proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. Here we show that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving formation of liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction.


Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , Sinais Direcionadores de Proteínas , Transativadores/genética , Proteínas de Sinalização YAP
13.
Curr Opin Cell Biol ; 51: 117-123, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29477107

RESUMO

Mechanical stretch forces can control the growth of epithelial tissues such as mammalian skin, whose surface area is precisely coordinated with body size. In skin keratinocytes cultured in vitro, mechanical forces acting via Integrin adhesions and the actin cytoskeleton have been shown to induce nuclear translocation of YAP/TAZ co-activators to induce cell proliferation. Furthermore, conditional knockouts of both YAP (also called YAP1) and TAZ (also called WWTR1) in mouse skin resemble the phenotype of skin-specific loss of Integrin beta1 (ITGB1), indicating that this signalling mechanism is important in vivo. Curiously, Integrins are dispensable in Drosophila to activate the sole YAP/TAZ homolog Yorkie (Yki), which has lost the C-terminal PDZ-binding motif needed to promote nuclear localization of YAP/TAZ in mammalian cells. Differences in the structure of the epidermis between deuterostomes (e.g.: stratified squamous skin of mammals) and protostomes (e.g.: monolayered columnar epidermis of Drosophila) may explain this evolutionary divergence. Monolayered columnar epithelia feature a well-differentiated apical membrane domain, where proteins such as Crumbs, Expanded, Merlin and Kibra activate the Hippo pathway to repress Drosophila Yki. Stratified squamous epithelia lack an apical domain and thus depend primarily on basal Integrin adhesions to activate YAP/TAZ in basal layer stem cells via multiple postulated signalling mechanisms. Finally, YAP and TAZ retain the ability to sense the apical domain in the columnar epithelial cells lining internal organs such as the lung bronchus, where YAP/TAZ localize to the nucleus in proliferating basal layer stem cells but translocate to the cytoplasm in differentiated columnar cells.


Assuntos
Proteínas de Drosophila/metabolismo , Epitélio/metabolismo , Mecanotransdução Celular/genética , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Drosophila , Humanos , Transdução de Sinais , Proteínas de Sinalização YAP
14.
Cell Rep ; 22(7): 1639-1646, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29444419

RESUMO

Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. Here, we show that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. Our results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia.


Assuntos
Membrana Celular/metabolismo , Polaridade Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Quinases Ativadas por p21/metabolismo , Sequência de Aminoácidos , Animais , Células CACO-2 , Proteínas de Drosophila/metabolismo , Humanos , Camundongos , Fosforilação , Proteína Quinase C/metabolismo , Interferência de RNA , Quinases Ativadas por p21/química
15.
Elife ; 72018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30231971

RESUMO

Squamous cell carcinoma (SCC) can progress to malignant metastatic cancer, including an aggressive subtype known as spindle cell carcinoma (spSCC). spSCC formation involves epithelial-to-mesenchymal transition (EMT), yet the molecular basis of this event remains unknown. The transcriptional co-activator YAP undergoes recurrent amplification in human SCC and overexpression of YAP drives SCC formation in mice. Here, we show that human spSCC tumours also feature strong nuclear localisation of YAP and overexpression of activated YAP (NLS-YAP-5SA) with Keratin-5 (K5-CreERt) is sufficient to induce rapid formation of both SCC and spSCC in mice. spSCC tumours arise at sites of epithelial scratch wounding, where tumour-initiating epithelial cells undergo EMT to generate spSCC. Expression of the EMT transcription factor ZEB1 arises upon wounding and is a defining characteristic of spSCC in mice and humans. Thus, the wound healing response synergises with YAP to drive metaplastic transformation of SCC to spSCC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Fosfoproteínas/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Animais , Núcleo Celular/metabolismo , Epiderme/patologia , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Fatores de Transcrição , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
16.
Dev Cell ; 38(4): 384-98, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27554858

RESUMO

Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.


Assuntos
Junções Aderentes/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Drosophila , Proteínas de Drosophila/genética , Epitélio/crescimento & desenvolvimento , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fosforilação , Ligação Proteica/genética , Estrutura Terciária de Proteína
17.
J Cell Biol ; 204(1): 111-27, 2014 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-24379416

RESUMO

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical-lateral border. Cdc42 and its effector complex Par6-atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6-aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical-lateral border positioning, and apical differentiation.


Assuntos
Células Epiteliais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Junções Íntimas/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células CACO-2 , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Cães , Células Epiteliais/metabolismo , Humanos , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Junções Íntimas/metabolismo
18.
J Cell Biol ; 204(5): 821-38, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24567356

RESUMO

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1-c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/fisiologia , Junções Íntimas/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Proteínas de Membrana/metabolismo , Pressão Osmótica
19.
J Cell Biol ; 198(4): 677-93, 2012 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-22891260

RESUMO

Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.


Assuntos
Adesão Celular/fisiologia , Células Epiteliais/citologia , Proteínas Ativadoras de GTPase/fisiologia , Junções Intercelulares/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas de Capeamento de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células CACO-2 , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Feminino , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Junções Intercelulares/metabolismo , Complexos Multiproteicos/fisiologia , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia
20.
PLoS One ; 7(11): e50188, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185572

RESUMO

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.


Assuntos
Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Pseudópodes/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Colágeno/química , Combinação de Medicamentos , Células Epiteliais/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Fibronectinas/química , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Laminina/química , Cadeias Leves de Miosina/genética , Miosina não Muscular Tipo IIA/genética , Fosforilação , Proteoglicanas/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/patologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genética
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