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1.
Cell Mol Life Sci ; 81(1): 291, 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38970683

RESUMO

Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.


Assuntos
Actinas , Junções Aderentes , Placofilinas , Proteína rhoA de Ligação ao GTP , Placofilinas/metabolismo , Placofilinas/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Junções Aderentes/metabolismo , Humanos , Actinas/metabolismo , Queratinócitos/metabolismo , Queratinócitos/citologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Transdução de Sinais , Fibras de Estresse/metabolismo , Células Cultivadas , Animais
4.
Cell Rep ; 42(1): 112031, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36689330

RESUMO

Plakophilin 3 (PKP3) is a component of desmosomes and is frequently overexpressed in cancer. Using keratinocytes either lacking or overexpressing PKP3, we identify a signaling axis from ERK to the retinoblastoma (RB) protein and the E2F1 transcription factor that is controlled by PKP3. RB and E2F1 are key components controlling G1/S transition in the cell cycle. We show that PKP3 stimulates the activity of ERK and its target RSK1. This inhibits expression of the transcription factor RUNX3, a positive regulator of the CDK inhibitor CDKN1A/p21, which is also downregulated by PKP3. Elevated CDKN1A prevents RB phosphorylation and E2F1 target gene expression, leading to delayed S phase entry and reduced proliferation in PKP3-depleted cells. Elevated PKP3 expression not only increases ERK activity but also captures phosphorylated RB (phospho-RB) in the cytoplasm to promote E2F1 activity and cell-cycle progression. These data identify a mechanism by which PKP3 promotes proliferation and acts as an oncogene.


Assuntos
Placofilinas , Proteína do Retinoblastoma , Animais , Camundongos , Divisão Celular , Citoplasma/metabolismo , Fator de Transcrição E2F1/metabolismo , Receptores ErbB/metabolismo , Fosforilação , Placofilinas/genética , Placofilinas/metabolismo , Proteína do Retinoblastoma/metabolismo , Fase S , Transdução de Sinais
5.
J Invest Dermatol ; 142(2): 272-274, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34799122

RESUMO

Single gene disorders are ideally suited to establish robust genotype‒phenotype correlations and provide excellent opportunities to understand molecular pathomechanisms with relevance to complex disorders. The observation that patients diagnosed with the same causative mutation can present with phenotypic disease variability illustrates the significant role of disease modifiers and warns against oversimplification. In a new article in the Journal of Investigative Dermatology, Zimmer et al. (2021) analyze two mutations located in the desmoglein (DSG) 1 transmembrane domain (TMD) and find that both mutants fail to assemble into desmosomes owing to reduced membrane trafficking and lipid raft targeting. One mutation maintained normal protein expression levels and turnover relative to those of wild-type (WT) DSG1, and behaved as a dominant negative. The second mutant showed reduced stability and increased turnover compared with WT DSG1 as well as reduced desmosome size and abundance. A full understanding of the TMD of DSG1 requires cell biological approaches, underscoring the value of cell biology in biomedical research in general.


Assuntos
Desmogleína 1 , Desmossomos , Desmogleína 1/genética , Desmossomos/genética , Humanos , Microdomínios da Membrana , Mutação
6.
Front Cell Dev Biol ; 9: 745670, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34631720

RESUMO

Desmosomes are intercellular junctions, which preserve tissue integrity during homeostatic and stress conditions. These functions rely on their unique structural properties, which enable them to respond to context-dependent signals and transmit them to change cell behavior. Desmosome composition and size vary depending on tissue specific expression and differentiation state. Their constituent proteins are highly regulated by posttranslational modifications that control their function in the desmosome itself and in addition regulate a multitude of desmosome-independent functions. This review will summarize our current knowledge how signaling pathways that control epithelial shape, polarity and function regulate desmosomes and how desmosomal proteins transduce these signals to modulate cell behavior.

7.
Mol Cancer ; 20(1): 88, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34116687

RESUMO

BACKGROUND: Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. METHODS: We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. RESULTS: We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. CONCLUSION: Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.


Assuntos
Carcinoma de Células Escamosas/patologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Neoplasias Bucais/patologia , Invasividade Neoplásica/genética , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Dasatinibe/farmacologia , Humanos , Neoplasias Bucais/genética , Invasividade Neoplásica/patologia , Transcriptoma
8.
J Cell Sci ; 133(8)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32122945

RESUMO

Desmosome remodeling is crucial for epidermal regeneration, differentiation and wound healing. It is mediated by adapting the composition, and by post-translational modifications, of constituent proteins. We have previously demonstrated in mouse suprabasal keratinocytes that plakophilin (PKP) 1 mediates strong adhesion, which is negatively regulated by insulin-like growth factor 1 (IGF1) signaling. The importance of PKP3 for epidermal adhesion is incompletely understood. Here, we identify a major role of epidermal growth factor (EGF), but not IGF1, signaling in PKP3 recruitment to the plasma membrane to facilitate desmosome assembly. We find that ribosomal S6 kinases (RSKs) associate with and phosphorylate PKP3, which promotes PKP3 association with desmosomes downstream of the EGF receptor. Knockdown of RSKs as well as mutation of an RSK phosphorylation site in PKP3 interfered with desmosome formation, maturation and adhesion. Our findings implicate a coordinate action of distinct growth factors in the control of adhesive properties of desmosomes through modulation of PKPs in a context-dependent manner.


Assuntos
Desmossomos , Placofilinas , Animais , Adesão Celular , Desmossomos/metabolismo , Camundongos , Fosforilação , Placofilinas/genética , Placofilinas/metabolismo , Proteínas Quinases S6 Ribossômicas
9.
J Invest Dermatol ; 139(9): 1854-1856, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31445572

RESUMO

Hemidesmosomes and focal adhesions attach keratinocytes to the dermis and act as bidirectional signaling centers to control epidermal renewal. Pora and colleagues (Pora et al., 2019) demonstrate that in migrating primary human keratinocytes, hemidesmosomes cluster as ordered arrays consisting of multiple chevrons, flanked by actin-associated focal adhesions. These and related findings have implications for wound healing, cancer invasion, blistering skin diseases, and skin aging.


Assuntos
Hemidesmossomos , Envelhecimento da Pele , Adesões Focais , Humanos , Queratinócitos , Cicatrização
10.
Sci Rep ; 9(1): 5610, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948763

RESUMO

The mature mammalian myocardium contains composite junctions (areae compositae) that comprise proteins of adherens junctions as well as desmosomes. Mutations or deficiency of many of these proteins are linked to heart failure and/or arrhythmogenic cardiomyopathy in patients. We firstly wanted to address the question whether the expression of these proteins shows an age-dependent alteration in the atrium of the human heart. Right atrial biopsies, obtained from patients undergoing routine bypass surgery for coronary heart disease were subjected to immunohistology and/or western blotting for the plaque proteins plakoglobin (γ-catenin) and plakophilin 2. Moreover, the Z-band protein cypher 1 (Cypher/ZASP) and calcium handling proteins of the sarcoplasmic reticulum (SR) like phospholamban, SERCA and calsequestrin were analyzed. We noted expression of plakoglobin, plakophilin 2 and Cypher/ZASP in these atrial preparations on western blotting and/or immunohistochemistry. There was an increase of Cypher/ZASP expression with age. The present data extend our knowledge on the expression of anchoring proteins and SR regulatory proteins in the atrium of the human heart and indicate an age-dependent variation in protein expression. It is tempting to speculate that increased expression of Cypher/ZASP may contribute to mechanical changes in the aging human myocardium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao Cálcio/genética , Átrios do Coração/patologia , Proteínas com Domínio LIM/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Arritmias Cardíacas/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/patologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Feminino , Átrios do Coração/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Proteínas com Domínio LIM/metabolismo , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Sarcômeros/metabolismo , Retículo Sarcoplasmático/metabolismo , Transcriptoma/genética , gama Catenina/genética , gama Catenina/metabolismo
11.
Cell Mol Life Sci ; 76(17): 3465-3476, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30949721

RESUMO

Plakophilins (Pkp) are desmosomal plaque proteins crucial for desmosomal adhesion and participate in the regulation of desmosomal turnover and signaling. However, direct evidence that Pkps regulate clustering and molecular binding properties of desmosomal cadherins is missing. Here, keratinocytes lacking either Pkp1 or 3 in comparison to wild type (wt) keratinocytes were characterized with regard to their desmoglein (Dsg) 1- and 3-binding properties and their capability to induce Dsg3 clustering. As revealed by atomic force microscopy (AFM), both Pkp-deficient keratinocyte cell lines showed reduced membrane availability and binding frequency of Dsg1 and 3 at cell borders. Extracellular crosslinking and AFM cluster mapping demonstrated that Pkp1 but not Pkp3 is required for Dsg3 clustering. Accordingly, Dsg3 overexpression reconstituted cluster formation in Pkp3- but not Pkp1-deficient keratinocytes as shown by AFM and STED experiments. Taken together, these data demonstrate that both Pkp1 and 3 regulate Dsg membrane availability, whereas Pkp1 but not Pkp3 is required for Dsg3 clustering.


Assuntos
Adesão Celular , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Placofilinas/genética , Animais , Anisomicina/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Desmogleína 1/genética , Desmogleína 3/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Microscopia de Força Atômica , Placofilinas/deficiência , Placofilinas/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
J Cell Sci ; 131(10)2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29678907

RESUMO

Desmosomes are essential for strong intercellular adhesion and are abundant in tissues exposed to mechanical strain. At the same time, desmosomes need to be dynamic to allow for remodeling of epithelia during differentiation or wound healing. Phosphorylation of desmosomal plaque proteins appears to be essential for desmosome dynamics. However, the mechanisms of how context-dependent post-translational modifications regulate desmosome formation, dynamics or stability are incompletely understood. Here, we show that growth factor signaling regulates the phosphorylation-dependent association of plakophilins 1 and 3 (PKP1 and PKP3) with 14-3-3 protein isoforms, and uncover unique and partially antagonistic functions of members of the 14-3-3 family in the regulation of desmosomes. 14-3-3γ associated primarily with cytoplasmic PKP1 phosphorylated at S155 and destabilized intercellular cohesion of keratinocytes by reducing its incorporation into desmosomes. In contrast, 14-3-3σ (also known as stratifin, encoded by SFN) interacted preferentially with S285-phosphorylated PKP3 to promote its accumulation at tricellular contact sites, leading to stable desmosomes. Taken together, our study identifies a new layer of regulation of intercellular adhesion by 14-3-3 proteins.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Desmossomos/metabolismo , Exorribonucleases/metabolismo , Placofilinas/metabolismo , Proteínas 14-3-3/genética , Biomarcadores Tumorais/genética , Adesão Celular , Citoplasma/metabolismo , Desmossomos/genética , Exorribonucleases/genética , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Placofilinas/genética
13.
Proc Natl Acad Sci U S A ; 115(13): 3344-3349, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531090

RESUMO

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19INK4d functions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19INK4d controls cell cycle progression. We delineate how the stepwise phosphorylation of p19INK4d Ser66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19INK4d This dissociates the CDK6-p19INK4d inhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19INK4d is ubiquitinated and degraded. Our findings reveal how signaling-dependent p19INK4d unfolding contributes to the irreversibility of G1/S transition.


Assuntos
Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p19/química , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Desdobramento de Proteína , Divisão Celular , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteólise , Transdução de Sinais
14.
J Cell Sci ; 130(19): 3374-3387, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28808088

RESUMO

We here report a novel function of the armadillo protein p0071 (also known as PKP4) during transport mediated by the KIF3 transport complex. Secretion of chromogranin A and matrix metallopeptidase 9 from pancreatic neuroendocrine tumor cells or pancreatic cancer cells, respectively, was substantially reduced following knockdown of p0071. Vesicle tracking indicated that there was impaired directional persistence of vesicle movement upon p0071 depletion. This suggests a disturbed balance between plus- and minus-end directed microtubule transport in cells lacking p0071. p0071 directly interacts with the KIF3 motor subunit KIF3B. Our data indicate that p0071 also interacts with the kinesin cargo adaptor protein KAP3 (also known as KIFAP3) acting as a stabilizing linker between KIF3B and its KAP3 cargo-binding entity. Thus, p0071 is required for directional vesicle movement and secretion of different KIF3-transported carriers, thereby regulating the transport of intracellular membrane vesicles along microtubules.


Assuntos
Cinesinas/metabolismo , Placofilinas/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Placofilinas/genética , Transporte Proteico/fisiologia , Vesículas Secretórias/genética
15.
Artigo em Inglês | MEDLINE | ID: mdl-28096266

RESUMO

Adherens junctions (AJs) and desmosomes connect the actin and keratin filament networks of adjacent cells into a mechanical unit. Whereas AJs function in mechanosensing and in transducing mechanical forces between the plasma membrane and the actomyosin cytoskeleton, desmosomes and intermediate filaments (IFs) provide mechanical stability required to maintain tissue architecture and integrity when the tissues are exposed to mechanical stress. Desmosomes are essential for stable intercellular cohesion, whereas keratins determine cell mechanics but are not involved in generating tension. Here, we summarize the current knowledge of the role of IFs and desmosomes in tissue mechanics and discuss whether the desmosome-keratin scaffold might be actively involved in mechanosensing and in the conversion of chemical signals into mechanical strength.


Assuntos
Citoesqueleto/metabolismo , Desmossomos/metabolismo , Filamentos Intermediários/metabolismo , Engenharia Tecidual/métodos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Epitélio/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinas/metabolismo , Microtúbulos/metabolismo , Domínios Proteicos , Transdução de Sinais , Estresse Mecânico
16.
J Invest Dermatol ; 136(10): 2022-2029, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27375112

RESUMO

Desmosomes are cell-cell adhesive structures essential for tissue integrity of the epidermis and the heart. Their constituents belong to multigene families giving rise to desmosomes of variable composition. So far, the functional significance of context-dependent composition in desmosome formation, dynamics, or stability during epidermal differentiation is incompletely understood. In this comparative study, we have uncovered unique and partially antagonistic functions of plakophilins 1 and 3 that are both expressed in the murine epidermis. These plakophilins differ in their localization patterns and kinetics during de novo desmosome formation and are regulated by distinct mechanisms. Moreover, plakophilin 3-containing desmosomes are more dynamic than desmosomes that contain predominantly plakophilin 1. Further, we show that Ca(2+)-independence of desmosomes strictly depends on plakophilin 1, whereas elevated levels of plakophilin 3 prevent the formation of hyperadhesive desmosomes in a protein kinase C alpha-dependent manner, even in the presence of plakophilin 1. Our study demonstrates that the balance between plakophilins 1 and 3 determines the context-dependent properties of epidermal desmosomes. In this setting, plakophilin 1 provides stable intercellular cohesion that resists mechanical stress, whereas plakophilin 3 confers dynamics as required during tissue homeostasis and repair. Our data have implications for the role of plakophilins in carcinogenesis.


Assuntos
Cálcio/metabolismo , Adesão Celular/fisiologia , Desmossomos/metabolismo , Placofilinas/metabolismo , Animais , Linhagem Celular , Células Epidérmicas , Humanos , Camundongos , Camundongos Knockout , Proteína Quinase C-alfa/metabolismo
17.
J Invest Dermatol ; 136(7): 1471-1478, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27033150

RESUMO

Desmosomes mediate strong intercellular adhesion through desmosomal cadherins that interact with intracellular linker proteins including plakophilins (PKPs) 1-3 to anchor the intermediate filaments. PKPs show overlapping but distinct expression patterns in the epidermis. So far, the contribution of individual PKPs in differentially regulating desmosome function is incompletely understood. To resolve the role of PKP1 we ablated the PKP1 gene. Here, we report that PKP1(-/-) mice were born at the expected mendelian ratio with reduced birth weight, but they otherwise appeared normal immediately after birth. However, their condition rapidly declined, and the mice died within 24 hours, developing fragile skin with lesions in the absence of obvious mechanical trauma. This was accompanied by sparse and small desmosomes. Newborn PKP1(-/-) mice showed disturbed tight junctions with an impaired inside-out barrier, whereas the outside-in barrier was unaffected. Keratinocytes isolated from these mice showed strongly reduced intercellular cohesion, delayed tight junction formation, and reduced transepithelial resistance and reduced proliferation rates. Our study shows a nonredundant and essential role of PKP1 in desmosome and tight junction function and supports a role of PKP1 in growth control, a function that is crucial in wound healing and epidermal carcinogenesis.


Assuntos
Desmossomos/metabolismo , Epiderme/patologia , Placofilinas/fisiologia , Junções Íntimas/metabolismo , Animais , Animais Recém-Nascidos , Carcinogênese , Adesão Celular , Proliferação de Células , Epiderme/metabolismo , Camundongos , Camundongos Knockout , Placofilinas/genética , Pele/metabolismo , Pele/patologia , Cicatrização
18.
Cell Commun Adhes ; 21(1): 25-42, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24460199

RESUMO

The regulation of adhesion and growth is important for epithelial function and dysfunction. ß-catenin (armadillo in Drosophila) is the prototype of a multifunctional molecule that regulates cell adhesion via adherens junctions and cell signaling via LEF/TCF transcription factors. Desmosomal armadillo proteins comprise plakoglobin and the plakophilins 1, 2, and 3. These proteins are essential for building up the desmosome and linking the desmosomal cadherins to keratin filaments. High expression of plakophilins in desmosomes correlates with strong intercellular cohesion and is essential for tissue integrity under mechanical stress. However, like ß-catenin, these proteins have diverse non-desmosomal functions, for example, in regulating actin organization, protein synthesis, and growth control. In line with these functions, their de-regulated expression with up- as well as down-regulation has been connected to cancer and metastasis. Now, recent evidence sheds light on the post-translational regulation and provides an explanation for how de-regulation of plakophilins can contribute to cancer.


Assuntos
Desmossomos/metabolismo , Placofilinas/metabolismo , Animais , Adesão Celular , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Placofilinas/química , Placofilinas/genética , Transdução de Sinais , beta Catenina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
19.
J Cell Sci ; 127(Pt 1): 60-71, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24163434

RESUMO

p0071 is an intercellular junction protein of the p120 catenin family. We have identified Rab11a as a novel interaction partner of p0071. p0071 interacted preferentially with active Rab11a. Knockdown experiments revealed an interdependent regulation of both proteins. On the one hand, p0071 depletion induced a perinuclear accumulation of Rab11, suggesting a role of p0071 in the anterograde transport of Rab11 from the pericentrosomal region to the plasma membrane but not in retrograde transport. p0071 as well as Rab11 depletion increased transferrin receptor recycling indicating that p0071-induced Rab11 mislocalization interfered with Rab11 function and shifted recycling from the slow Rab11-dependent pathway to the fast Rab4-dependent pathway. When p0071 or Rab11 depletion was combined with a Rab4 knockdown the effect was reversed. On the other hand, Rab11a depletion increased p0071 recycling to cell contacts thereby identifying p0071 as a Rab11 cargo protein. This correlated with increased intercellular adhesion. Thus, we propose that p0071 has a key role in regulating recycling through the Rab11-dependent perinuclear recycling compartment, and links the regulation of adherens junctions to recycling to allow dynamic modulation of intercellular adhesion.


Assuntos
Junções Aderentes/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Placofilinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Endossomos/metabolismo , Regulação da Expressão Gênica , Humanos , Placofilinas/antagonistas & inibidores , Placofilinas/genética , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
20.
RNA ; 19(11): 1483-96, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24062571

RESUMO

Loss of fragile X mental retardation protein (FMRP) causes synaptic dysfunction and intellectual disability. FMRP is an RNA-binding protein that controls the translation or turnover of a subset of mRNAs. Identifying these target transcripts is an important step toward understanding the pathology of the disease. Here, we show that FMRP regulates actin organization and neurite outgrowth via the armadillo protein p0071. In mouse embryonic fibroblasts (MEFs) lacking FMRP (Fmr1-), the actin cytoskeleton was markedly reorganized with reduced stress fibers and F-actin/G-actin ratios compared to fibroblasts re-expressing the protein. FMRP interfered with the translation of the p0071 mRNA in a 3'-UTR-dependent manner. Accordingly, FMRP-depleted cells revealed elevated levels of p0071 protein. The knockdown of p0071 in Fmr1- fibroblasts restored stress fibers and an elongated cell shape, thus rescuing the Fmr1- phenotype, whereas overexpression of p0071 in Fmr1+ cells mimicked the Fmr1- phenotype. Moreover, p0071 and FMRP regulated neurite outgrowth and branching in a diametrically opposed way in agreement with the negative regulation of p0071 by FMRP. These results identify p0071 as an important and novel FMRP target and strongly suggest that impaired actin cytoskeletal functions mediated by an excess of p0071 are key aspects underlying the fragile X syndrome.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neuritos/metabolismo , Placofilinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Placofilinas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
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