RESUMO
Arf GTPase-activating proteins (Arf GAP) play important roles in the formation of the membrane vesicles that traffic between subcellular membranous organelles. The small Arf GTPase-activating protein (SMAP) subfamily of Arf GAPs has two members, SMAP1 and SMAP2, in mammals. The present study investigated whether these two proteins may have an overlapping function in addition to their previously reported distinct functions. Results showed that the presence of either SMAP1 or SMAP2 was sufficient for endocytosis of the transferrin receptor, and that transferrin incorporation was impaired only by the absence of both SMAP1 and SMAP2. This suggests the involvement of both SMAP1 and SMAP2 in transferrin endocytosis. Results also demonstrated a physical association between SMAP1 and SMAP2, which might serve as a basis for a functional interaction, and identified the intramolecular domains responsible for this association.
Assuntos
Endocitose/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Receptores da Transferrina/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Ligação ProteicaRESUMO
Runx1 transcription factor is a key player in the development and function of T cells. Runx1 transcripts consist of two closely related isoforms (proximal and distal Runx1) whose expressions are regulated by different promoters. Which Runx1 isoform is expressed appears to be tightly regulated. The regulatory mechanism for differential transcription is, however, not fully understood. In this study, we investigated the regulation of the proximal Runx1 promoter in T cells. We showed that proximal Runx1 was expressed at a low level in naïve T cells from C57BL/6 mice, but its expression was remarkably induced upon T-cell activation. In the promoter of proximal Runx1, a highly conserved region was identified which spans from -412 to the transcription start site and harbors a NFAT binding site. In a luciferase reporter assay, this region was found to be responsive to T-cell activation through Lck and calcineurin pathways. Mutagenesis studies and chromatin immunoprecipitation assay indicated that the NFAT site was essential for NFAT binding and transactivation of the proximal Runx1 promoter. Furthermore, TCR signaling-induced expression of proximal Runx1 was blocked by treatment of cells with cyclosporin A. Together, these results demonstrate that the calcineurin-NFAT pathway regulates proximal Runx1 transcription upon TCR stimulation.
Assuntos
Calcineurina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Ativação Transcricional , Animais , Sítios de Ligação , Sequência Conservada , Ciclosporina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mutação , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.
Assuntos
Acrossomo/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Espermatogênese , Rede trans-Golgi/metabolismo , Animais , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Modelos Animais de Doenças , Feminino , Marcação de Genes , Humanos , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Proteínas Monoméricas de Montagem de Clatrina/genética , Espermátides/metabolismo , Sintaxina 1/metabolismoRESUMO
The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/deficiência , Síndromes Mielodisplásicas/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Cinética , Lisossomos/metabolismo , Mastócitos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Transporte Proteico , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Baço/patologia , Transferrina/metabolismoRESUMO
The Runx1 transcription factor is abundantly expressed in naive T cells but rapidly downregulated in activated T cells, suggesting that it plays an important role in a naive stage. In the current study, Runx1(-/-)Bcl2(tg) mice harboring Runx1-deleted CD4(+) T cells developed a fatal autoimmune lung disease. CD4(+) T cells from these mice were spontaneously activated, preferentially homed to the lung, and expressed various cytokines, including IL-17 and IL-21. Among these, the deregulation of IL-21 transcription was likely to be associated with Runx binding sites located in an IL-21 intron. IL-17 produced in Runx1-deleted cells mobilized innate immune responses, such as those promoted by neutrophils and monocytes, whereas IL-21 triggered humoral responses, such as plasma cells. Thus, at an initial stage, peribronchovascular regions in the lung were infiltrated by CD4(+) lymphocytes, whereas at a terminal stage, interstitial regions were massively occupied by immune cells, and alveolar spaces were filled with granular exudates that resembled pulmonary alveolar proteinosis in humans. Mice suffered from respiratory failure, as well as systemic inflammatory responses. Our data indicate that Runx1 plays an essential role in repressing the transcription of cytokine genes in naive CD4(+) T cells and, thereby, maintains cell quiescence.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Pneumopatias/imunologia , Ativação Linfocitária/imunologia , Animais , Doenças Autoimunes/mortalidade , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Citocinas/antagonistas & inibidores , Citocinas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/mortalidade , Células Jurkat , Pneumopatias/mortalidade , Pneumopatias/patologia , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologiaRESUMO
The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.
RESUMO
SMAP2 is an Arf GTPase-activating protein that is located and functions on early endosome membranes. In the present study, the trans-Golgi network (TGN) was verified as an additional site of SMAP2 localization based on its co-localization with various TGN-marker proteins. Mutation of specific stretches of basic amino acid residues abolished the TGN-localization of SMAP2. Over-expression of wild-type SMAP2, but not of the mutated SMAP2, inhibited the transport of vesicular stomatitis virus-G protein from the TGN to the plasma membrane. In contrast, this transport was enhanced in SMAP2 (-/-) cells characterized by increased levels of the activated form of Arf. SMAP2 therefore belongs to an ArfGAP subtype that resides on the TGN and functions as a negative regulator of vesicle budding from the organelle.
Assuntos
Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Endossomos/metabolismo , Expressão Gênica , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação , Transporte Proteico , Proteínas do Envelope Viral/metabolismoRESUMO
SMAP1 and SMAP2 proteins constitute a subfamily of the Arf-specific GTPase-activating proteins. Both SMAP proteins bind to clathrin heavy chains and are involved in the trafficking of clathrin-coated vesicles. In cells, SMAP1 regulates Arf6-dependent endocytosis of transferrin receptors from the coated pits of the plasma membrane, whereas SMAP2 regulates Arf1-dependent retrograde transport of TGN38 from the early endosome to the trans-Golgi network. The common and distinct features of SMAP1 and SMAP2 activity provide a valuable opportunity to examine the differential regulation of membrane trafficking by these two proteins. In this chapter, we describe several basic experimental procedures that have been used to study the regulation of membrane trafficking using SMAP proteins, including a GAP assay as well as procedures to study the transport of transferrin receptors and TGN38. In addition, a yeast two-hybrid system is described because of its utility in identifying novel molecules that interact with SMAP.