ABSTRACT
The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.
Subject(s)
ADP-Ribosylation Factors , Neoplasms , Humans , ADP-Ribosylation Factors/metabolism , Chlorobenzenes , Pyrazoles , GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factor 1/metabolismABSTRACT
Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.
Subject(s)
Epidermal Growth Factor , Sarcoma , Cell Adhesion , Cell Line , Child , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Phosphorylation , Sarcoma/geneticsABSTRACT
Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.
Subject(s)
Actin Cytoskeleton , Actins , Adaptor Proteins, Signal Transducing , GTPase-Activating Proteins , ADP-Ribosylation Factors/metabolism , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , GTPase-Activating Proteins/metabolism , Lysine/metabolism , Nerve Tissue Proteins/metabolism , Protein Binding , Protein DomainsABSTRACT
Suppressors of cytokine signaling (SOCS) exhibit diverse antiinflammatory effects. Since ROS acts as a critical mediator of inflammation, we have investigated the anti-inflammatory mechanisms of SOCS via ROS regulation in monocytic/macrophagic cells. Using PMA-differentiated monocytic cell lines and primary BMDMs transduced with SOCS1 or shSOCS1, the LPS/TLR4-induced inflammatory signaling was investigated by analyzing the levels of intracellular ROS, antioxidant factors, inflammasome activation, and pro-inflammatory cytokines. The levels of LPS-induced ROS and the production of pro-inflammatory cytokines were notably down-regulated by SOCS1 and up-regulated by shSOCS1 in an NAC-sensitive manner. SOCS1 up-regulated an ROS-scavenging protein, thioredoxin, via enhanced expression and binding of NRF-2 to the thioredoxin promoter. SOCS3 exhibited similar effects on NRF-2/thioredoxin induction, and ROS downregulation, resulting in the suppression of inflammatory cytokines. Notably thioredoxin ablation promoted NLRP3 inflammasome activation and restored the SOCS1-mediated inhibition of ROS and cytokine synthesis induced by LPS. The results demonstrate that the anti-inflammatory mechanisms of SOCS1 and SOCS3 in macrophages are mediated via NRF-2-mediated thioredoxin upregulation resulting in the downregulation of ROS signal. Thus, our study supports the anti-oxidant role of SOCS1 and SOCS3 in the exquisite regulation of macrophage activation under oxidative stress. [BMB Reports 2020; 53(12): 640-645].
Subject(s)
Inflammasomes/metabolism , Suppressor of Cytokine Signaling 1 Protein/metabolism , Animals , Anti-Inflammatory Agents/immunology , Cytokines/analysis , Cytokines/metabolism , Humans , Inflammation/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Promoter Regions, Genetic/genetics , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Suppressor of Cytokine Signaling 1 Protein/physiology , Suppressor of Cytokine Signaling Proteins/metabolism , Suppressor of Cytokine Signaling Proteins/physiology , THP-1 Cells , Thioredoxins/metabolism , Thioredoxins/pharmacology , Toll-Like Receptor 4ABSTRACT
Water from the waterlines of dental units is often contaminated with bacteria but there have been few studies accurately assessing the diversity of these bacterial populations. The aim of our study was to assess the bacterial diversity present in water collected from dental unit waterlines using the Illumina MiSeq. Water was collected from two separate dental units located in a dental hospital and two units found in two separate private clinics in Gangneung-si, Korea. From the four water samples that were analyzed, a total of 233 bacterial genera were identified. The most abundant genera were Sphingomonas (25%), Halomonas (20%), Reyranella (8%), and Novosphingobium (6%). Halomonas was more prevalent in the two dental units located at the dental hospital, while Reyranella and Sphingomonas were more commonly found in the private dental clinics. Only 19 of the 233 identified genera were common between water samples from all dental units. Opportunistic pathogens were shown to account for 7.7% of the total bacterial genera identified. Our results have demonstrated that there is a wide assortment of bacterial genera present in dental unit waterlines.
Subject(s)
Bacteria/classification , Dental Equipment/microbiology , Water Microbiology , Bacteria/genetics , Bacteria/pathogenicity , Bacterial Load , Equipment Contamination , Genetic Variation , High-Throughput Nucleotide Sequencing , Humans , Republic of Korea , Sequence Analysis, DNAABSTRACT
Susceptibility testing of bacteria to disinfecting chemical agents isolated from dental unit waterlines (DUWL) is necessary for the development of effective disinfectant products. However, until now, susceptibility tests for chemical agents, which are components of DUWL disinfectant products, have not been conducted on bacteria isolated from DUWL water. The aim of this study was to evaluate and compare the susceptibilities of DUWL isolates in planktonic and biofilm states to cetylpyridinium chloride, as well as to the four chemical agents currently used for DUWL management. A total of 56 isolates, including 12 genera, were identified by 16S rDNA sequencing, and one strain of each genus was selected for susceptibility testing. A total of 12 isolates were used for the susceptibility tests. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the planktonic state and the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) for the biofilm state using microtiter plates. MIC, MBC, MBIC, and MBEC of the 12 isolates for ethanol were the highest, followed by sodium hypochlorite, hydrogen peroxide, and chlorhexidine. Similar to chlorhexidine, the lowest MIC, MBC, MBIC, and MBEC were found in cetylpyridinium chloride. The susceptibilities of the isolates for sodium hypochlorite and ethanol were similar in the planktonic and biofilm states. For hydrogen peroxide and chlorhexidine, the MBIC and MIC were similar, but MBEC was 256 times higher than MBC. The MBIC and MBEC of isolates for cetylpyridinium chloride were 128 and 256 times higher than the MIC and MBC, respectively. As far as we know, this was the first study reporting the susceptibility of DUWL isolates to cetylpyridinium chloride and chemical agents used for disinfecting DUWLs. Cetylpyridinium chloride, for which the DUWL isolates showed the highest susceptibility, could be used for disinfecting DUWLs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Dental Equipment/microbiology , Disinfectants/pharmacology , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Biofilms/drug effects , Biofilms/growth & development , DNA, Bacterial/genetics , Genome, Viral/genetics , Microbial Sensitivity Tests , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner's 2A (R2A) for 10 days, and were subsequently stored at -70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 105 CFU cm-2 and 10-14 µm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.
Subject(s)
Biofilms/growth & development , Dental Equipment/microbiology , Equipment Contamination , Bacteria/drug effects , Bacteria/growth & development , Bacterial Physiological Phenomena , Bioreactors , Centers for Disease Control and Prevention, U.S. , Colony Count, Microbial , Disinfection/methods , Polyurethanes , Reproducibility of Results , United States , Water MicrobiologyABSTRACT
RATIONALE: Acute mesenteric vein thrombosis (MVT) is defined as new-onset thrombosis of the mesenteric vein without evidence of collateralization, finally resulting in extensive intestinal infarction. MVT may be idiopathic or be caused by conditions responsible for thrombophilia and acquired risk factors. To date, there have been few reports of MVT after trauma. Herein we describe our experiences treating three patients with MVT. PATIENT CONCERNS: Case 1 was a 44-year-old man with transverse colon mesenteric hematoma after blunt abdominal trauma. Case 2 was a 55-year-old man with jejunal transection after a traffic accident. Case 3 was a 26-year-old man presented with multiple abdominal stab bowel injury. DIAGNOSES: A 1-week follow-up abdominal computed tomography scan showed superior mesenteric vein thrombosis in all of three patients. INTERVENTIONS: All patients were treated with anticoagulant for 3 or 6 months. OUTCOMES: MVTs were completely resolved without any complications. LESSONS: If early diagnosis and treatment could be available, anticoagulation alone might be adequate for the treatment of SMVT associated with trauma. Early anticoagulation in patients with acute SMVT may avoid the grave prognosis observed in patients with arterial thrombosis.
Subject(s)
Abdominal Injuries/complications , Mesenteric Vascular Occlusion/etiology , Venous Thrombosis/etiology , Wounds, Nonpenetrating/complications , Acute Disease , Adult , Humans , Male , Mesenteric Vascular Occlusion/drug therapy , Mesenteric Veins , Middle Aged , Venous Thrombosis/drug therapyABSTRACT
PURPOSE: We analyze the outcomes of open repair (OR) in patients with ruptured abdominal aortic aneurysm (RAAA) according to the anatomic suitability for endovascular aneurysm repair (EVAR). MATERIALS AND METHODS: We reviewed retrospectively all consecutive RAAA patients who underwent OR from January 2005 to March 2014. All suspected patients underwent preoperative computed tomography (CT). Outcomes were major morbidities and mortality. Multivariate analysis was performed by using logistic regression adjusted by controlled variables; gender, Hardman index, maximal aneurysmal diameter, rupture type, perioperative transfusion requirement, and perioperative urinary output. RESULTS: Among 54 consecutive patients with RAAA who underwent OR, 45 patients were included after exclusion of 9 patients (7, suprarenal; 1, infected; 1, inflammatory). Preoperative CT showed 27% (12/45) EVAR-suitable patients. Hostile neck anatomy was found in 88% (29/33) among unsuitable anatomy (UA) (n=33). The maximal aneurysmal diameter was statistically larger (83.1±21.0 mm vs. 68.8±12.3 mm, P=0.032) in the UA group. The 30-day mortality was 28.9% (13/45; 33% vs. 17% in UA group vs. suitable anatomy [SA] group, P=0.460; adjusted P=0.445). UA group had more patients with cardiac morbidity (55% vs. 25%, P=0.079; adjusted P=0.032; odds ratio, 12.914; 95% confidence interval, 1.238-134.675). There was no statistical difference in survival rate between SA and UA groups (74.1%, 74.1%, and 74.1% vs. 60.6%, 55.6%, and 32.4% at 1-, 3- and 5-year, respectively; P=0.145). CONCLUSION: In this study, relatively unfavorable outcomes were found in the EVAR-unsuitable group after OR in RAAA patients. However, unsuitable anatomy did not influence patient survival after OR by multivariate analysis.
ABSTRACT
Focal adhesions (FAs) are dynamic structures that connect the actin cytoskeleton with the extracellular matrix. At least six ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), including ARAP2 (an Arf6 GAP), are implicated in regulation of FAs but the mechanisms for most are not well defined. Although Rac1 has been reported to function downstream of Arf6 to control membrane ruffling and cell migration, this pathway has not been directly examined as a regulator of FAs. Here we test the hypothesis that ARAP2 promotes the growth of FAs by converting Arf6·GTP to Arf6·GDP thereby preventing the activation of the Rho family GTP-binding protein Rac1. Reduced expression of ARAP2 decreased the number and size of FAs in cells and increased cellular Arf6·GTP and Rac1·GTP levels. Overexpression of ARAP2 had the opposite effects. The effects of ARAP2 on FAs and Rac1 were dependent on a functional ArfGAP domain. Constitutively active Arf6 affected FAs in the same way as did reduced ARAP2 expression and dominant negative mutants of Arf6 and Rac1 reversed the effect of reduced ARAP2 expression. However, neither dominant negative Arf6 nor Rac1 had the same effect as ARAP2 overexpression. We conclude that changes in Arf6 and Rac1 activities are necessary but not sufficient for ARAP2 to promote the growth of FAs and we speculate that ARAP2 has additional functions that are effector in nature to promote or stabilize FAs.
Subject(s)
ADP-Ribosylation Factors/metabolism , Carrier Proteins/physiology , GTPase-Activating Proteins/physiology , Signal Transduction , rac1 GTP-Binding Protein/metabolism , ADP-Ribosylation Factor 6 , Animals , Carrier Proteins/metabolism , Cell Adhesion , Cell Line , Focal Adhesions , GTPase-Activating Proteins/metabolism , Gene Expression Regulation , Glutathione Transferase/metabolism , HeLa Cells , Humans , Microscopy, Confocal/methods , Models, Genetic , Mutation , RNA, Small Interfering/metabolismABSTRACT
BACKGROUND INFORMATION: ARAP1 is an Arf (ADP-ribosylation factor)-directed GAP (GTPase-activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1. RESULTS: Here we report that ARAP1 associates with the CIN85 (Cbl-interacting protein of 85 kDa). Arg86 and Arg90 of ARAP1 and the SH3 (Src homology 3) domains of CIN85 are necessary for the interaction. We found that a mutant of ARAP1 with reduced affinity for CIN85 does not efficiently rescue the effect of reduced ARAP1 expression on EGFR trafficking to the early endosome. Reduced expression of CIN85 has a similar effect as reduced expression of ARAP1 on traffic of the EGFR. Cbl proteins regulate the endocytic trafficking of the EGFR by mediating ubiquitination of the EGFR. Overexpression of ARAP1 reduced ubiquitination of the EGFR by Cbl and slowed Cbl-dependent degradation of the EGFR. Reduced expression of ARAP1 accelerated degradation of EGFR but did not affect the level of ubiquitination of the receptor that was detected. CONCLUSION: ARAP1 interaction with CIN85 regulates endocytic trafficking of the EGFR and affects ubiquitination of EGFR. We propose a model in which the ARAP1-CIN85 complex drives exit of EGF-EGFR-Cbl complex from a pre-early endosome into a pathway distinct from the early endosome/lysosome pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Endocytosis , ErbB Receptors/metabolism , GTPase-Activating Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Carrier Proteins/genetics , Epidermal Growth Factor/metabolism , ErbB Receptors/genetics , GTPase-Activating Proteins/genetics , HeLa Cells , Humans , Molecular Sequence Data , Protein Binding , Protein TransportABSTRACT
PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin delta-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg(105) residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr(231) in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.
Subject(s)
Carrier Proteins/metabolism , Down-Regulation , ErbB Receptors/genetics , GTPase-Activating Proteins/metabolism , Neoplasm Proteins/physiology , Protein-Tyrosine Kinases/physiology , Binding Sites , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Humans , Neoplasm Proteins/metabolism , Phosphorylation/physiology , Protein Binding , Protein-Tyrosine Kinases/metabolism , Signal TransductionABSTRACT
ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3))-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P(3) binding. Here, we tested the hypothesis that PtdIns(3,4,5)P(3)-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P(3), but with relatively low affinity (approximately 1.6 microm), and the PH domains did not mediate PtdIns(3,4,5)P(3)-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P(3) binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P(3), the subsequent production of which triggers enzymatic activity.
Subject(s)
ADP-Ribosylation Factors/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Membrane/metabolism , GTPase-Activating Proteins/metabolism , Phosphatidylinositol Phosphates/metabolism , ADP-Ribosylation Factors/genetics , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins/genetics , Chlorocebus aethiops , Endocytosis , Epidermal Growth Factor/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/genetics , HeLa Cells , Humans , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
Signaling through the EGF receptor is regulated by endocytosis. ARAP1 is a protein with Arf guanosine triphosphatase-activating protein (GAP) and Rho GAP domains. We investigated the role of ARAP1 in EGF receptor endocytic trafficking. Following EGF treatment of cells, ARAP1 rapidly and transiently associated with the edge of the cell and punctate structures containing Rab5, rabaptin 5 and EGFR but not early embryonic antigen 1 (EEA1). EGF associated with the ARAP1-positive punctate structures prior to EEA1-positive early endosomes. Recruitment of ARAP1 to the punctate structures required active Rab5 and an additional signal from EGFR. Decreasing ARAP1 levels with small interfering RNA accelerated association of EGF with EEA1 endosomes and degradation of EGFR. Phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun-amino-terminal kinase (JNK) was diminished and more transient in cells with reduced levels of ARAP1 than in controls. Based on these findings, we propose that ARAP1 regulates the endocytic traffic of EGFR and, consequently, the rate of EGFR signal attenuation.
Subject(s)
Carrier Proteins/metabolism , Endocytosis , ErbB Receptors/metabolism , GTPase-Activating Proteins/metabolism , Carrier Proteins/genetics , Epidermal Growth Factor/metabolism , GTPase-Activating Proteins/genetics , Gene Expression Regulation , Genes, Reporter/genetics , HeLa Cells , Humans , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
Arf GTP-binding proteins and Rho-family GTPases play key roles in regulating membrane remodeling and cytoskeletal reorganization involved in cell movement. Several studies have implicated neurotrophins and their receptors as upstream activators of these small GTP-binding proteins, however, the mechanisms and the cell type specificity of this neurotrophin activity are still under investigation. Here we describe the rationale and protocols used for the dissection of an NT3 activated pathway that leads to the specific activation of Arf6 and Rac1.
Subject(s)
ADP-Ribosylation Factors/metabolism , Neurons/metabolism , Neurotrophin 3/physiology , rac1 GTP-Binding Protein/metabolism , ADP-Ribosylation Factor 6 , Animals , Cell Line , Enzyme Activation , Humans , Mice , Mice, Mutant StrainsABSTRACT
Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane Structures/enzymology , Proto-Oncogene Proteins pp60(c-src)/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Motifs , Animals , Cell Line, Tumor , Cortactin/metabolism , GTPase-Activating Proteins/metabolism , Humans , Mice , Mutant Proteins/metabolism , NIH 3T3 Cells , Phosphopeptides/metabolism , Phosphorylation , Protein Binding , Protein Isoforms/metabolism , Protein Structure, Tertiary , Protein Transport , Tyrosine/metabolismABSTRACT
ARAP2 is a protein that contains both ArfGAP and RhoGAP domains. We found that it is a phosphatidylinositol (3,4,5)-trisphosphate-dependent Arf6 GAP that binds RhoA-GTP but lacks RhoGAP activity. In agreement with the hypothesis that ARAP2 mediates effects of RhoA, endogenous ARAP2 associated with focal adhesions (FAs) and reduction of ARAP2 expression, by RNAi, resulted in fewer FAs and actin stress fibers (SFs). In cells with reduced levels of endogenous ARAP2, FAs and SFs could be restored with wild-type recombinant ARAP2 but not mutants lacking ArfGAP or Rho-binding activity. Constitutively active Arf6 also caused a loss of SFs. The Rho effector ROKalpha was ineffective in restoring FAs. Conversely, overexpression of ARAP2 did not restore SFs in cells treated with a ROK inhibitor but induced punctate accumulations of paxillin. We conclude that ARAP2 is an Arf6GAP that functions downstream of RhoA to regulate focal adhesion dynamics.
Subject(s)
ADP-Ribosylation Factors/metabolism , Actins/metabolism , Carrier Proteins/physiology , Cytoskeleton/metabolism , GTPase-Activating Proteins/physiology , rhoA GTP-Binding Protein/metabolism , ADP-Ribosylation Factor 1/metabolism , ADP-Ribosylation Factor 6 , Carrier Proteins/genetics , Cell Line , Focal Adhesions , GTPase-Activating Proteins/genetics , Humans , Models, Molecular , Mutation , Phosphatidylinositol Phosphates/physiology , Protein Binding , Protein Structure, Tertiary , Protein Transport , Stress Fibers/physiologyABSTRACT
Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1-tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.
Subject(s)
ADP-Ribosylation Factors/metabolism , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Receptor, trkC/physiology , Signal Transduction , rac1 GTP-Binding Protein/metabolism , ADP-Ribosylation Factor 6 , Cell Line , Cells, Cultured , Humans , Models, Biological , Protein Isoforms/physiologyABSTRACT
ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Phospholipids/metabolism , ADP-Ribosylation Factor 1/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Binding Sites/genetics , Binding, Competitive/drug effects , Biotin/analogs & derivatives , Biotin/chemistry , Blood Proteins/metabolism , Catalytic Domain , Fluorescence Resonance Energy Transfer , GTPase-Activating Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/pharmacology , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Liposomes/metabolism , Liposomes/pharmacology , Lysine/chemistry , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphatidic Acids/metabolism , Phosphatidylinositol 4,5-Diphosphate/analogs & derivatives , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phospholipase C delta , Phospholipids/pharmacology , Phosphoproteins/metabolism , Plasmids/genetics , Protein Binding/drug effects , Protein Conformation , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Succinimides/chemistry , Type C Phospholipases/genetics , Type C Phospholipases/metabolismABSTRACT
1 The mitochondrial respiratory chain produces reactive oxygen species (ROS) during normal electron transport. Despite producing ROS, mitochondria are vulnerable to oxidative stress. Mitochondrial dysfunction has been associated with many degenerative diseases, making it important to identify compounds that protect mitochondria from ROS-mediated toxicity. Here we report that ciclopirox (CPX) blocks H2O2-induced mitochondrial injury by maintaining mitochondrial transmembrane potential (Deltapsim). 2 CPX completely blocked H2O2-stimulated release of lactate dehydrogenase (a marker of cell death) and decrease in MTT reduction (a marker of mitochondrial function) in adenocarcinoma SK-HEP-1 cells. 3 H2O2 rapidly depolarized the Deltapsim, and CPX blocked this H2O2-stimulated Deltapsim decrease. Similar data were obtained in experiments using mitochondria isolated from rat liver. 4 Furthermore, CPX effectively inhibited H2O2-induced mitochondrial permeability transition pore (MPTP) opening. In de-energized mitochondria, however, CPX did not inhibit Ca2+-evoked MPTP opening, indicating that CPX is not a direct inhibitor of the MPTP. 5 Oxygen consumption studies showed that in the presence of pyruvate and malate CPX restored the rate of state 3 to state 4 respiration decreased by H2O2. Consistent with this, CPX replenished ATP levels lowered by H2O2. 6 The present results indicate that CPX protects SK-HEP-1 cells from H2O2 cytotoxicity by inhibiting Deltapsim decrease and indirectly preventing MPTP opening.