ABSTRACT
Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the trans-Golgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGN-derived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitin-dependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Carrier Proteins/metabolism , Cullin Proteins/metabolism , Microfilament Proteins/metabolism , Protein Transport , Ubiquitin-Protein Ligases/metabolism , Actins/genetics , Actins/metabolism , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/genetics , Animals , COS Cells , Carrier Proteins/genetics , Cell Line , Chlorocebus aethiops , Cullin Proteins/genetics , Golgi Apparatus/metabolism , HEK293 Cells , HeLa Cells , Humans , Lysine/metabolism , Microfilament Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , trans-Golgi Network/metabolismABSTRACT
BACKGROUND: The axonal tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Paired helical filaments of tau and extracellular plaques containing beta-amyloid are found in the brain of Alzheimer's disease (AD) patients. RESULTS: Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human tau under the control of a neuron-specific HuC promoter. Approximately ten neuronal cells expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording, in order to evaluate the neurotoxicity induced by tau-GFP proteins. Expression of tau-GFP was observed to cause high levels of neuronal death. However, multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing tau-GFP from death. Treatment with chemical compounds that exert anti-oxidative or neurotrophic effects also resulted in a similar protective effect and maintained human tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8. CONCLUSIONS: The novel finding of this study is that we established an expression system expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording to evaluate the neurotoxicity induced by tau-GFP proteins. This system may serve as an efficient in vivo imaging platform for the discovery of novel drugs against tauopathy.
Subject(s)
Frontotemporal Dementia/metabolism , Neurons/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , tau Proteins/metabolism , Animals , Animals, Genetically Modified , Cell Death , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 17/metabolism , Disease Models, Animal , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/pathology , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , Humans , Neurons/pathology , Zebrafish/genetics , Zebrafish Proteins/genetics , tau Proteins/geneticsABSTRACT
Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. Here, we show that TRAF6 E3 ubiquitin ligase and A20 deubiquitinase coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The ROS-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of VPS34-UVRAG complex, thereby stimulating autophagy. Notably, expression of the ATG9A ubiquitination mutants impairs ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreases IRF-3 phosphorylation in LPS-stimulated macrophages. Our findings provide important insights into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.
Subject(s)
TNF Receptor-Associated Factor 6 , Ubiquitin-Protein Ligases , Autophagy/physiology , Oxidative Stress , TNF Receptor-Associated Factor 6/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. Here, we discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1+ vesicles during starvation-induced autophagy. We further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. Our findings highlight the important role of PTPN9 in the regulation of ATG16L1+ autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status.Abbreviations: csw: corkscrew; EBSS: Earle's balanced salt solution; ERGIC: ER-Golgi intermediate compartment; ESCRT: endosomal sorting complexes required for transport; mop: myopic; NSF: N-ethylmaleimide-sensitive factor; PAS: phagophore assembly site; PolyQ: polyglutamine; PtdIns3P: phosphatidylinositol-3-phosphate; PTK: protein tyrosine kinase; PTM: posttranslational modification; PTP: protein tyrosine phosphatase; PTPN23/HD-PTP: protein tyrosine phosphatase non-receptor type 23; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; STX7: syntaxin 7; STX8: syntaxin 8; STX17: syntaxin 17; VAMP3: vesicle associated membrane protein 3; VAMP7: vesicle associated membrane protein 7; VTI1B: vesicle transport through interaction with t-SNAREs 1B; YKT6: YKT6 v-SNARE homolog; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.
Subject(s)
Autophagosomes , Autophagy-Related Proteins , Macroautophagy , Protein Tyrosine Phosphatases, Non-Receptor , Qb-SNARE Proteins , Autophagosomes/metabolism , Autophagy/physiology , Autophagy-Related Proteins/metabolism , HeLa Cells , Humans , Membrane Fusion , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Qb-SNARE Proteins/metabolismABSTRACT
This study describes the synthesis and structure-activity relationships of a series of furazan-3,4-diamide analogs. 1,2,5-Oxadiazole ring and electron-withdrawing substituent on the phenyl ring are proposed to be the important elements which contribute to a significant extent maximal potency of anti-proliferation effect.
Subject(s)
Antineoplastic Agents/chemical synthesis , Diamide/analogs & derivatives , Diamide/chemical synthesis , Furans/chemical synthesis , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Diamide/therapeutic use , Furans/therapeutic use , Humans , Structure-Activity RelationshipABSTRACT
In this study, highly invasive tumor cell lines (designated A431-I, -II and -III) derived from parental A431 tumor cells (A431-P) were isolated by three successive passages through a Boyden chamber with matrigel-coated membrane support. The invasive potential and the activity of secreted MMP-9 of each sub-line increased significantly compared to the A431-P (A431-III > A431-II > A431-I) as evidenced by the in vitro invasion assay, gelatin zymography and immunoblotting analyses. RT-PCR results also revealed the elevated expression of MMP-9 in A431-III. We further characterized the A431-III sub-line and found these cells exhibited a greater potential for attachment and spreading on fibronectin-coated substratum and for migration. The A431-III cells displayed multiple cytoplasmic extensions with focal contacts (vinculin-positive staining) during cell spreading within 30 min. We also noticed an increase in FAK phosphorylation, but no significant change in FAK protein level in the A431-III sub-line compared to those of A431-P cells. Together, these results demonstrate that the greater invasion potential exhibited by the highly invasive A431-III subline is likely attributed to an increased ability for attachment, spreading and migration, as well as increased MMP activity. Thus, A431-P and the highly invasive A431-III sub-line could be an excellent model for studying the mechanism of cancer metastasis.
Subject(s)
Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Neoplasms/enzymology , Neoplasms/pathology , Cell Adhesion/physiology , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/physiology , Diffusion Chambers, Culture , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Neoplasm Invasiveness , Neoplasms/metabolism , Phosphorylation , Vinculin/metabolismABSTRACT
In the present study, the zebrafish epo cDNA was cloned. The encoded protein displays 90%, 55% and 32% identity to the Epo from carp, fugu and human, respectively. Through RT-PCR, the expression of zepo mRNA was mainly in the heart and liver. In the COS-1 cell transfection experiments, the recombinant zEpo-HA protein was efficiently secreted into the culture medium as a glycoprotein and the carbohydrate moiety can be cleaved by the treatment of peptide-N-glycosidase F (PNGase F). Using the morpholino approach, we showed that zepo morphants displayed severe anemia leading to high mortality during development. Such an effect can be significantly rescued by zepo RNA. Furthermore, in the absence of functional zEpo, the expression of specific markers for adult globin genes, such as alphaA1- and betaA1-globin, but not the embryonic betae1-globin, was affected.
Subject(s)
Erythropoietin/genetics , Erythropoietin/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Erythroid Cells/metabolism , Erythropoietin/chemistry , Gene Expression Profiling , Gene Expression Regulation, Developmental , Hemoglobins/biosynthesis , Molecular Sequence Data , Organ Specificity , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Zebrafish/embryology , Zebrafish Proteins/chemistryABSTRACT
Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.
Subject(s)
Autophagy-Related Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/embryology , Drosophila/physiology , Gastrointestinal Tract/embryology , Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Animals , Autophagy-Related Proteins/genetics , Cell Cycle Proteins/metabolism , Drosophila/genetics , Drosophila Proteins/genetics , Eye Proteins/metabolism , Gene Knockout Techniques , Homeostasis , Membrane Proteins/geneticsABSTRACT
Regarding the involvement of cyclooxygenase-2 (COX-2)-independent pathways in celecoxib-mediated antineoplastic effects, the following two issues remain outstanding: identity of the non-COX-2 targets and relative contributions of COX-2-dependent versus -independent mechanisms. We use a close celecoxib analog deficient in COX-2-inhibitory activity, DMC (4-[5-(2,5-dimethylphenyl)-3(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide), to examine the premise that Akt signaling represents a major non-COX-2 target. Celecoxib and DMC block Akt activation in PC-3 cells through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1) with IC(50) of 48 and 38 micro M, respectively. The consequent effect on Akt activation is more pronounced (IC(50) values of 28 and 20 micro M, respectively), which might be attributed to the concomitant dephosphorylation by protein phosphatase 2A. In serum-supplemented medium, celecoxib and DMC cause G(1) arrest, and at higher concentrations, they induce apoptosis with relative potency comparable with that in blocking Akt activation. Moreover, the effect of daily oral celecoxib and DMC at 100 and 200 mg/kg on established PC-3 xenograft tumors is assessed. Celecoxib at both doses and DMC at 100 mg/kg had marginal impacts. However, a correlation exists between the in vitro potency of DMC and its ability at 200 mg/kg to inhibit xenograft tumor growth through the inhibition of Akt activation. Analysis of the tumor samples indicates that a differential reduction in the phospho-Akt/Akt ratio was noted in celecoxib- and DMC-treated groups vis-à-vis the control group. Together, these data underscore the role of 3-phosphoinositide-dependent protein kinase-1/Akt signaling in celecoxib-mediated in vitro antiproliferative effects in prostate cancer cells.
Subject(s)
Cyclooxygenase Inhibitors/pharmacology , Isoenzymes/physiology , Prostaglandin-Endoperoxide Synthases/physiology , Prostatic Neoplasms/drug therapy , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/physiology , Signal Transduction/physiology , Sulfonamides/pharmacology , 3-Phosphoinositide-Dependent Protein Kinases , Apoptosis/drug effects , Celecoxib , Cell Division/drug effects , Cell Line, Tumor , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , G1 Phase/drug effects , Humans , Male , Membrane Proteins , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-akt , PyrazolesABSTRACT
A highly invasive Du145-III subline was isolated by three successive passages of the parental Du145 prostate tumor cell line (Du145-P) through a Boyden chamber with matrigel-coated membrane support. Du145-III cells showed great invasion potential based on their increased ability to spread/migrate and enhanced expression/secretion of the matrix metalloproteinase 9 (MMP9). Du145-III cells exerted vasculogenic mimicry (VM) properties, reminiscent of endothelial cell characteristics and expressed elevated levels of cancer stem cell (CSC) markers, including Nanog, Sox2, CD44 and ABCG2 and ability to self-renew. Of prominence, MMP9 was required for the induction of VM and for increased stemness in Du145-III cells. Using Du145-III as a model, the effects of dietary flavonoids, luteolin and quercetin, were evaluated on stemness and invasion capacity of Du145-III cells in relation to JNK signaling pathway activation. These flavonoids depressed the malignancy of highly invasive Du145-III cells, VM, anchorage-independent spheroid formation and expression of certain CSC markers. Since luteolin and quercetin were able to target CSC cells and prevent cancer cell invasiveness, may serve as potential anti-angiogenesis and anti-metastasis agents.
Subject(s)
Diet , Luteolin/pharmacology , Neoplastic Stem Cells/drug effects , Prostatic Neoplasms/pathology , Quercetin/pharmacology , Humans , Luteolin/chemistry , Male , Quercetin/chemistryABSTRACT
Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Cyclins/physiology , Motor Neurons/physiology , Neural Stem Cells/physiology , Animals , COS Cells , Cell Proliferation , Chlorocebus aethiops , Gene Expression , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Mice , Neurogenesis , Zebrafish/embryologyABSTRACT
Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbalpha and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Mucrocetin is a GPIbalpha agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbalpha antagonist) on GPIbalpha, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 A (1 A=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four alphabeta heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer 'head-to-tail' disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the alpha-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the beta-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb-IX-V complex.
Subject(s)
Crotalid Venoms/chemistry , Models, Molecular , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIb-IX Complex/agonists , Reptilian Proteins , Trimeresurus , Viper Venoms/chemistry , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Carrier Proteins/pharmacology , Cloning, Molecular , Crotalid Venoms/genetics , Crotalid Venoms/pharmacology , Crystallography, X-Ray , Humans , Molecular Sequence Data , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIb-IX Complex/antagonists & inhibitors , Sequence Homology, Amino Acid , Viper Venoms/genetics , Viper Venoms/pharmacologyABSTRACT
The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.
Subject(s)
Intercellular Signaling Peptides and Proteins/genetics , Lateral Line System/physiology , Morphogenesis/genetics , Myelin Proteins/genetics , Receptors, Cell Surface/genetics , Wnt Signaling Pathway/genetics , Zebrafish Proteins/genetics , Animals , Embryo, Nonmammalian/physiology , Embryonic Development/genetics , Gene Expression Regulation, Developmental/genetics , Nogo Proteins , Signal Transduction/genetics , ZebrafishABSTRACT
The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.
Subject(s)
Collagen Type II/genetics , Notochord/metabolism , Transcription Factors/physiology , Zebrafish Proteins/physiology , Zebrafish/genetics , Amino Acid Sequence , Animals , Body Patterning/genetics , Cloning, Molecular , Collagen Type II/metabolism , Gene Expression Regulation, Developmental , Molecular Sequence Data , Sequence Alignment , Sequence DeletionABSTRACT
The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Amino Acid Substitution , Antibodies, Monoclonal , Forkhead Transcription Factors/chemistry , Forkhead Transcription Factors/metabolism , HEK293 Cells , Humans , Models, Biological , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Phosphothreonine/chemistry , Protein Interaction Domains and Motifs , Protein Multimerization , RNA Interference , RNA, Small Interfering/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
BACKGROUND: Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth. CONCLUSIONS/SIGNIFICANCE: Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.
Subject(s)
Embryo, Nonmammalian/cytology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Neurites/metabolism , Pseudopodia/metabolism , Serine/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolism , Zebrafish/embryology , Amino Acid Sequence , Animals , COS Cells , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Chlorocebus aethiops , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/metabolism , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Membrane Glycoproteins/genetics , Molecular Sequence Data , Nerve Growth Factor/pharmacology , Neurites/drug effects , PC12 Cells , Phosphorylation/drug effects , Pseudopodia/drug effects , Rats , Signal Transduction/drug effects , Zebrafish Proteins/geneticsABSTRACT
In human tumors, fibronectin (FN) expression is positively associated with tumor metastatic potential and matrix metalloproteinase (MMP) secretion. Additionally, tissue transglutaminase (TG2) is implicated as playing an important role in tumor progression, and acts as a co-receptor for integrin-mediated cell binding to FN. This study explored the involvement of FN and TG2 in cancer cell metastasis using the recently established highly invasive A431-III subline. A431-III cells expressed significantly higher levels of FN and TG2 as compared to the parental line (A431-P). Knockdown of endogenous FN by small interfering RNA (siRNA) resulted in dramatic suppression of the migratory and invasive activity, and the secreted MMP-9 activity (but not MMP-2) in A431-III subline. Exogenous administration of FN to A431-III cells also increased the secreted activity of MMP-9 but not MMP-2. Interestingly, knockdown of TG2 by siRNA dramatically reduced the cell attachment, migration and invasion, and the secretion of MMP-9 and MMP-1 (but not MMP-2 and MMP-3) in A431-III cells as compared to A431-P cells. Furthermore, A431-III cells exhibited increased association of integrin ß1 and ß3 with FN and TG2, and knockdown of TG2 markedly suppressed integrin ß1 interaction with FN. Together, this study suggests that FN and TG2 facilitate the metastatic activity of A431 tumor cells, and this may be partly attributed to TG2 enhancement of the association of FN and ß integrin. In addition, the combined targeting of TG2 and FN may be an effective therapeutic strategy for cancer displaying increased expression of both proteins.
Subject(s)
Fibronectins/biosynthesis , Integrin beta1/biosynthesis , Matrix Metalloproteinase 1/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Transglutaminases/biosynthesis , Cell Line, Tumor , Fibronectins/genetics , Fibronectins/metabolism , GTP-Binding Proteins , Gene Knockdown Techniques , Humans , Integrin beta1/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis , Protein Glutamine gamma Glutamyltransferase 2 , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Transglutaminases/genetics , Transglutaminases/metabolism , Up-RegulationABSTRACT
Human synuclein family consists of alpha-, beta-, and gamma-synucleins. Here, we cloned three genes, sncb, sncga and sncgb from zebrafish. They encode beta-, gamma1-, and gamma2-synucleins, respectively. The zSyn-beta, zSyn-gamma1, and zSyn-gamma2 proteins display 69%, 47%, and 50% identity to human beta-synuclein and gamma-synuclein, respectively. By reverse transcriptase-polymerase chain reaction, we demonstrated that sncb and sncga mRNA were abundant in brain and eye, while sncgb expression was moderate in brain, kidney, ovary and testis. The 1.8-kb 5'-upstream/promoter region of the sncga gene was sufficient to direct green fluorescent protein (GFP) expression in the central nervous system and cranial ganglions. A transgenic line, Tg(sncga:GFP), was generated and its GFP expression is similar to that of endogenous sncga mRNA. Moreover, this line also labels the habenular complex and the domain of GFP expression is larger in the left than in the right habenula. Thus, this line can be used to study sncga gene regulation and for left-right asymmetry study in zebrafish brain.
Subject(s)
Gene Expression Regulation, Developmental/genetics , Habenula/metabolism , Synucleins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Aging/physiology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cloning, Molecular , Conserved Sequence , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Habenula/embryology , Habenula/growth & development , Humans , Molecular Sequence Data , Phylogeny , Promoter Regions, Genetic/genetics , Protein Binding , Sequence Alignment , Synucleins/chemistry , Synucleins/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.