RESUMO
SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
RESUMO
Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Adesões Focais/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transdução de SinaisRESUMO
The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.
Assuntos
Caseína Quinase Ialfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Sinalização Wnt , Animais , Proteína Axina/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular , Expressão Ectópica do Gene , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismoRESUMO
The function of the FAM83F protein, like the functions of many members of the FAM83 family, is poorly understood. Here, we show that injection of Fam83f mRNA into Xenopus embryos causes axis duplication, a phenotype indicative of enhanced Wnt signalling. Consistent with this, overexpression of FAM83F activates Wnt signalling, whereas ablation of FAM83F from human colorectal cancer (CRC) cells attenuates it. We demonstrate that FAM83F is farnesylated and interacts and co-localises with CK1α at the plasma membrane. This interaction with CK1α is essential for FAM83F to activate Wnt signalling, and FAM83F mutants that do not interact with CK1α fail to induce axis duplication in Xenopus embryos and to activate Wnt signalling in cells. FAM83F acts upstream of GSK-3ß because the attenuation of Wnt signalling caused by loss of FAM83F can be rescued by GSK-3 inhibition. Introduction of a farnesyl-deficient mutant of FAM83F in cells through CRISPR/Cas9 genome editing redirects the FAM83F-CK1α complex away from the plasma membrane and significantly attenuates Wnt signalling, indicating that FAM83F exerts its effects on Wnt signalling at the plasma membrane.
Assuntos
Caseína Quinase Ialfa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Membrana Celular/metabolismo , Desenvolvimento Embrionário/genética , Imunofluorescência , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Prenilação , Ligação Proteica , Transporte Proteico , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.
Assuntos
DNA de Cadeia Simples/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Mesoderma/embriologia , Xenopus laevis/embriologia , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Primers do DNA , Hibridização In Situ , Reação em Cadeia da PolimeraseRESUMO
The animal cap explant is a simple but adaptable tool available to developmental biologists. The use of animal cap explants in demonstrating the presence of mesoderm-inducting activity in the Xenopus embryo vegetal pole is one of many elegant examples of their worth. Animal caps respond to a range of growth factors (e.g., Wnts, FGF, TGF-ß), making them especially useful for studying signal transduction pathways and gene regulatory networks. Explants are also suitable for examining cell behavior and have provided key insights into the molecular mechanisms controlling vertebrate morphogenesis. In this protocol, we outline two methods to isolate animal cap explants from Xenopus laevis, both of which can be applied easily to Xenopus tropicalis The first method is a standard manual method that can be used in any laboratory equipped with a standard dissecting microscope. For labs planning on dissecting large numbers of explants on a regular basis, a second, high throughput method is described that uses a specialized microcautery surgical instrument.
Assuntos
Dissecação/métodos , Embrião não Mamífero/metabolismo , Xenopus laevis/embriologia , Animais , Eletrodos , HumanosRESUMO
DNA replication in the embryo of Xenopus laevis changes dramatically at the mid-blastula transition (MBT), with Y RNA-independent random initiation switching to Y RNA-dependent initiation at specific origins. Here, we identify xNuRD, an MTA2-containing assemblage of the nucleosome remodeling and histone deacetylation complex NuRD, as an essential factor in pre-MBT Xenopus embryos that overcomes a functional requirement for Y RNAs during DNA replication. Human NuRD complexes have a different subunit composition than xNuRD and do not support Y RNA-independent initiation of DNA replication. Blocking or immunodepletion of xNuRD inhibits DNA replication initiation in isolated nuclei in vitro and causes inhibition of DNA synthesis, developmental delay, and embryonic lethality in early embryos. xNuRD activity declines after the MBT, coinciding with dissociation of the complex and emergence of Y RNA-dependent initiation. Our data thus reveal an essential role for a NuRD complex as a DNA replication factor during early Xenopus development.
Assuntos
Replicação do DNA , Desenvolvimento Embrionário , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Óvulo/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Blástula/metabolismo , Extratos Celulares , Células HeLa , Humanos , RNA/metabolismoRESUMO
Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.
Assuntos
Caseína Quinase I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Neoplasias/química , Domínios Proteicos , Caseína Quinase I/química , Caseína Quinase I/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas , Transdução de SinaisRESUMO
Epithelia function as barriers against environmental insults and express the transcription factor aryl hydrocarbon receptor (AhR). However, AhR function in these tissues is unknown. Here we show that AhR regulates multiciliogenesis in both murine airway epithelia and in Xenopus laevis epidermis. In air-exposed airway epithelia, induction of factors required for multiciliogenesis, including cyclin O (Ccno) and Multicilin (Mcidas), is AhR dependent, and air exposure induces AhR binding to the Ccno promoter. Submersion and hypoxic conditions impede AhR-dependent Ccno induction. This is mediated by the persistence of Notch signalling, as Notch blockade renders multiciliogenesis and Ccno induction by AhR independent from air exposure. In contrast to Ccno induction, air exposure does not induce the canonical AhR target cytochrome P450 1a1 (Cyp1a1). Inversely, exposure to AhR ligands induces Cyp1a1 but not Ccno and impeded ciliogenesis. These data indicate that AhR involvement in detoxification of environmental pollutants may impede its physiological role, resulting in respiratory pathology.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclinas/biossíntese , Ciclinas/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Mucosa Respiratória/metabolismo , Poluentes Atmosféricos/farmacocinética , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citocromo P-450 CYP1A1/biossíntese , Epiderme/metabolismo , Regulação da Expressão Gênica , Inativação Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Mucosa Respiratória/citologia , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Proteína Smad1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4/metabolismo , Especificidade por Substrato , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Smad1/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HeLa , Humanos , Camundongos , Fosforilação , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , UbiquitinaçãoRESUMO
The regulated turnover of endoplasmic reticulum (ER)-resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture-based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover.
Assuntos
Ácido Aspártico Endopeptidases/fisiologia , Proteínas de Membrana/metabolismo , Células HeLa , Heme Oxigenase-1/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteólise , Proteômica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , UbiquitinaçãoRESUMO
Tes is a member of an emerging family of proteins sharing a set of protein motifs referred to as PET-LIM domains. PET-LIM proteins such as Prickle regulate cell behavior during gastrulation in Xenopus and zebrafish, and to ask whether Tes is also involved in controlling cell behavior, we isolated its Xenopus orthologue. Xtes is expressed as a maternal transcript that is maintained at low levels until neurula stages when expression is elevated in the head and axial structures. Depletion of Xtes leads to a foreshortened head and severe defects in axis elongation. The anterior defect is due in part to the inhibition of cranial neural crest migration while the defects in elongation may be due to perturbation of expression of XFGF8, Xdelta-1 and Xcad-3 and thereby to disruption of posterior somitogenesis. Finally, we note that simultaneous depletion of Xtes and Xenopus Prickle results in axial defects that are more severe than those resulting from depletion of Xtes alone, suggesting that the two proteins act together to control axial elongation.