RESUMEN
The phenylalanyl-glycyl-glycyl-alanyl-prolyl (FG-GAP) domain plays an important role in protein-protein interactions, including interaction of integrins with their ligands. Integrin-α FG-GAP repeat-containing protein 2 (Itfg2) is a highly conserved protein in vertebrates that carries two FG-GAP domains, but its role in mammalian physiology is unknown. In this article, we show that Itfg2 is an intracellular protein and it plays a critical role in B cell differentiation and development of autoimmunity. Itfg2-deficient mice displayed a phenotype consistent with retention of B cells in the spleen and had a lower concentration of IgG in the blood when compared with wild-type littermates. Itfg2-deficient splenocytes also showed a defect in cell migration in vitro. After immunization with a thymus-dependent Ag, the absence of Itfg2 caused a shift in B cell maturation from the germinal centers to the extrafollicular regions of the spleen and blocked deposition of Ag-specific plasma cells in the bone marrow. In support of hematopoietic cell intrinsic activity of Itfg2, bone marrow transplantation of Itfg2-deficient cells was sufficient to impair germinal center development in wild-type mice. Furthermore, Itfg2 deficiency exacerbated development of autoimmune disease in MRL/lpr lupus-prone mice. These results identify Itfg2 as a novel contributor to B cell differentiation and a negative regulator of the autoimmune response during lupus.
Asunto(s)
Enfermedades Autoinmunes/genética , Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular/genética , Cadenas alfa de Integrinas/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Secuencia de Bases , Diferenciación Celular/inmunología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Orden Génico , Marcación de Gen , Genotipo , Centro Germinal/inmunología , Centro Germinal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Inmunoglobulinas/sangre , Cadenas alfa de Integrinas/química , Cadenas alfa de Integrinas/metabolismo , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fenotipo , Alineación de Secuencia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.
Asunto(s)
Movimiento Celular/genética , Párpados/citología , Queratinocitos/citología , Quinasas Lim/fisiología , Actinas/metabolismo , Animales , Párpados/embriología , Párpados/patología , Genotipo , Quinasas Lim/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , FosforilaciónRESUMEN
Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98-99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, "substrate trapping" mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.
Asunto(s)
Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Animales , Femenino , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Mutación , Fenotipo , Fosforilación , Esfuerzo Físico , Especificidad por SustratoRESUMEN
An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.
Asunto(s)
Glomérulos Renales/anatomía & histología , Glomérulos Renales/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Antígenos de Superficie/metabolismo , Trasplante de Médula Ósea , Glomerulonefritis/etiología , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Glomerulonefritis por IGA/etiología , Glomerulonefritis por IGA/patología , Glomerulonefritis por IGA/fisiopatología , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Glomérulos Renales/enzimología , Glomérulos Renales/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinasas/deficiencia , Fosfatidilinositol 3-Quinasas/genética , Podocitos/enzimología , Podocitos/patología , Podocitos/fisiología , Insuficiencia Renal/etiología , Insuficiencia Renal/patología , Insuficiencia Renal/fisiopatología , Quimera por TrasplanteRESUMEN
Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/-) mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/-) mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/-) mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Citometría de Flujo , Ratones , Ratones Mutantes , Resonancia Magnética Nuclear Biomolecular , Oogénesis/fisiología , ARN Mensajero/genética , Espermatogénesis/fisiologíaRESUMEN
Tmub1 (C7orf21/HOPS) encodes a protein containing a ubiquitin-like domain. Tmub1 is highly expressed in the nervous system. To study its physiological function, we generated mice with Tmub1 deleted by homologous recombination. The knockout mice were grossly normal and viable. In a comprehensive behavioral testing battery, the only knockout phenotype displayed was a strong increase in home cage locomotor activity during the dark phase (subjective day) of the light:dark (L:D) cycle. There were no changes in activity during the light period. There were no changes in locomotor activity observed in other assays, e.g. novel open-field. The increase in dark phase locomotor activity persisted during a seven day D:D (complete darkness) challenge, and remained largely confined to the normally dark period. Telemetric recording in freely moving subjects for one 24 hr L:D cycle, revealed the same increase in locomotor activity in the dark phase. In addition, EEG analysis showed that the knockout mice exhibited increased waking and decreased NREM & REM times during the dark phase, but the EEG was otherwise normal. Using lacZ as a reporter we found Tmub1 expression prominent in a few brain structures including the thalamus, a region known to drive wakefulness and arousal via its projections to the cortex. We identified calcium modulating cyclophilin ligand CAMLG/CAML as a binding partner by a yeast two-hybrid screen of a brain library. The interaction of Tmub1 and CAMLG was confirmed by co-immunoprecipitation assays in HEK cells. The two proteins were also found to be co-localized to the cytoplasm when expressed in HEK cells. Both Tmub1 and CAMLG have been recently described in the regulation of membrane trafficking of specific receptors. Taken together our results implicate Tmub1 in the regulation of locomotor activity and wakefulness and suggest that Tmub1 binds to and functions together with CAMLG.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/fisiología , Locomoción/fisiología , Proteínas Nucleares/fisiología , Vigilia/fisiología , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cartilla de ADN , Electroencefalografía , Electromiografía , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Unión ProteicaRESUMEN
Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune response and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c expression (Sh2d3c(-/-)) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c(-)(/)(-) splenic B cells were also significantly reduced in vitro compared with wild-type (Sh2d3c(+/+)) cells. In contrast, T cell development and function were largely normal in Sh2d3c(-/-) mice. The thymi of Sh2d3c(-/-) mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c(-/-) mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Transducción de Señal/inmunología , Bazo/inmunología , Bazo/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos T-Independientes/inmunología , Subgrupos de Linfocitos B/patología , Señalización del Calcio/genética , Señalización del Calcio/inmunología , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Femenino , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Transducción de Señal/genética , Bazo/patología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismoRESUMEN
Many shuttling proteins not only function in the nucleus but also control mRNA fates in the cytoplasm. We test whether a link exists between their nuclear association with mRNPs and their cytoplasmic functions using the p37 isoform of hnRNP D, which inhibits the rapid cytoplasmic mRNA decay in NIH3T3 cells. We showed that p37 shuttles between nucleus and cytoplasm, and narrowed down the nuclear import signal to a 50-amino-acid C-terminal domain. A p37 mutant missing this domain, still capable of associating with target mRNAs in vitro, was confined to the cytoplasm, where it was unable to block cytoplasmic mRNA turnover. Introducing heterologous shuttling domains to this mutant, thereby restoring its ability to enter the nucleus, concomitantly restored its cytoplasmic function. Association of p37 with its target mRNAs can only be detected when it can enter the nucleus. Our results suggest that nuclear import of hnRNP D is a prerequisite for it to exert its cytoplasmic function. This study provides a useful model system to elucidate the mechanisms by which "nuclear history" affects cytoplasmic mRNA fates.
Asunto(s)
Núcleo Celular/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/fisiología , Transporte Activo de Núcleo Celular/fisiología , Animales , Ratones , Células 3T3 NIH , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , ARN Mensajero/metabolismoRESUMEN
The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.
Asunto(s)
Presión Sanguínea/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Animales , Secuencia de Bases , Presión Sanguínea/genética , ADN Complementario/genética , Biblioteca de Genes , Técnicas Genéticas , Heterocigoto , Humanos , Hipertensión/terapia , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mutagénesis Insercional/métodos , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Lugares Marcados de Secuencia , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.