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The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function. 总被引:4,自引:0,他引:4
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Q Sheng D Denis M Ratnofsky T M Roberts J A DeCaprio B Schaffhausen 《Journal of virology》1997,71(12):9410-9416
Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T. 相似文献
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The carboxy-terminal cysteine of the tetraspanin L6 antigen is required for its interaction with SITAC, a novel PDZ protein
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Borrell-Pagès M Fernández-Larrea J Borroto A Rojo F Baselga J Arribas J 《Molecular biology of the cell》2000,11(12):4217-4225
PDZ domains are protein modules that mediate protein-protein interactions. Here, we present the identification and characterization of a protein similar to the recently identified PDZ-containing protein TACIP18, which we have named SITAC (similar to TACIP18). SITAC is preferentially expressed in cells of the digestive tract, associated with intracellular membranes. Despite the high degree of sequence identity between the PDZ domains of TACIP18 and those of SITAC, none of the known ligands of the former shows interaction with the latter, as judged by two-hybrid analysis. SITAC interacts with peptides containing bulky hydrophobic amino acids two positions upstream of the C-terminal residue. Surprisingly, SITAC also shows interaction with peptides ending in C, a previously unacknowledged ability of PDZ domains. The sequence -Y-X-C-COOH, bound in vitro by SITAC, is present in the member of the tetraspanin superfamily, the L6 antigen. Coimmunoprecipitation experiments show that SITAC interacts with L6A, but not with an L6A C-terminal mutant, confirming the capacity of SITAC to interact with proteins ending in C. Confocal analysis shows that the interaction between L6A and SITAC is necessary for the precise colocalization of both molecules in the same subcellular compartment. In summary, the characterization of the protein SITAC has unveiled novel sequences recognized by PDZ domains, and it suggests that L6A is a natural ligand of this PDZ protein. 相似文献
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The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development. 相似文献
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Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein. 总被引:16,自引:12,他引:4
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Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35S-labeled herpes simplex virus type 1-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure. Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the alpha-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an alpha-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an alpha-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5. 相似文献
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The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Ras-guanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucose-induced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain. 相似文献
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Modha R Campbell LJ Nietlispach D Buhecha HR Owen D Mott HR 《The Journal of biological chemistry》2008,283(3):1492-1500
Protein kinase C-related kinase 1 (PRK1 or PKN) is involved in regulation of the intermediate filaments of the actin cytoskeleton, as well as having effects on processes as diverse as mitotic timing and apoptosis. It is activated by interacting with the Rho family small G proteins and arachidonic acid or by caspase cleavage. We have previously shown that the HR1b of PRK1 binds exclusively to Rac1, whereas the HR1a domain binds to both Rac1 and RhoA. Here, we have determined the solution structure of the HR1b-Rac complex. We show that HR1b binds to the C-terminal end of the effector loop and switch 2 of Rac1. Comparison with the HR1a-RhoA structure shows that this part of the Rac1-HR1b interaction is homologous to one of the contact sites that HR1a makes with RhoA. The Rac1 used in this study included the C-terminal polybasic region, which is frequently omitted from structural studies, as well as the core G domain. The Rac1 C-terminal region reverses in direction to interact with residues in switch 2, and the polybasic region itself interacts with residues in HR1b. The interactions with HR1b do not prevent the polybasic region being available to contact the negatively charged membrane phospholipids, which is considered to be its primary role. This is the first structural demonstration that the C terminus of a G protein forms a novel recognition element for effector binding. 相似文献
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Sakurai-Yageta M Recchi C Le Dez G Sibarita JB Daviet L Camonis J D'Souza-Schorey C Chavrier P 《The Journal of cell biology》2008,181(6):985-998
Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases. 相似文献
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Yang X Yu RH Calmettes C Moraes TF Schryvers AB 《The Journal of biological chemistry》2011,286(52):45165-45173
Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA. 相似文献
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Joohyun Ryu Sung Goo Park Phil Young Lee Sayeon Cho Do Hee Lee Gwang Hoon Kim Jeong-Hoon Kim Byoung Chul Park 《Biochemical and biophysical research communications》2013,430(2):610-615
Anterior Gradient 2 (AGR2), an ER stress-inducible protein, has been reported to be localized in endoplasmic reticulum (ER) and its level is elevated in numerous metastatic cancers. Recently, it has been demonstrated that AGR2 is involved in the control of ER homeostasis. However, the molecular mechanism how AGR2 regulates ER stress response remains unclear. Herein we show that AGR2 homo-dimerizes through an intermolecular disulfide bond. Moreover, dimerization of AGR2 attenuates ER stress-induced cell death through the association with BiP/GRP78. Thus, these results suggest that dimerization of AGR2 is crucial in mediating the ER stress signaling pathway. 相似文献
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Huang Y Biswas C Klos Dehring DA Sriram U Williamson EK Li S Clarke F Gallucci S Argon Y Burkhardt JK 《Journal of immunology (Baltimore, Md. : 1950)》2011,187(11):5952-5963
The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs. 相似文献
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Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site-specific recombination at the dif locus of Escherichia coli. We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition. 相似文献