ABSTRACT
Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.
Subject(s)
Hepacivirus , Oncogene Proteins v-abl , Gene Knockdown Techniques , HEK293 Cells , Hepacivirus/physiology , Hepatitis C , Humans , Oncogene Proteins v-abl/genetics , Oncogene Proteins v-abl/metabolism , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Assembly/genetics , Virus Replication/geneticsABSTRACT
OBJECTIVES: Leukemia is one of the severe cancer types all around the globe. Even though some chemotherapeutic drugs are available for treating leukemia, they have various side effects. As an alternative approach, herbal drugs are focused on current research to overcome leukemia. The present work was conducted to investigate the antileukemic mechanism of active phytochemical vitexin, which was isolated from ethno-medicine (Prosopis cineraria leaf) used by traditional healers of West Bengal, India. METHODS: Antiproliferative mechanisms of selected phyto-compound against K-562 cells were evaluated using cellular uptake, morphological changes, DNA fragmentation, mitochondrial membrane potential and signaling pathways analysis. KEY FINDINGS: Vitexin exhibited cytotoxicity by reducing mitochondrial membrane potential (32.40%) and causing DNA fragmentation (84.15%). The western blotting study indicated inhibition of cell survival proteins (BCR, ABL, H-RAS, N-RAS, K-RAS and RAF) and expression of apoptotic proteins (p38, BAX and caspase-9) in leukemia cells upon treatment with vitexin. CONCLUSIONS: Based on the results, presently investigated phyto-compound vitexin could be considered for developing safe and natural drugs to treat leukemia after conducting suitable preclinical and clinical trials.
Subject(s)
Apigenin/pharmacology , Oncogene Proteins v-abl/metabolism , Prosopis , Proto-Oncogene Proteins c-bcr/metabolism , raf Kinases/metabolism , ras Proteins/metabolism , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Survival/drug effects , DNA Fragmentation/drug effects , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Membrane Potential, Mitochondrial/drug effects , Phytochemicals/pharmacology , Signal Transduction/drug effectsABSTRACT
Targeting mitochondrial metabolism has emerged as a treatment option for cancer patients. The ABL tyrosine kinases promote metastasis, and enhanced ABL signaling is associated with a poor prognosis in lung adenocarcinoma patients. Here we show that ABL kinase allosteric inhibitors impair mitochondrial integrity and decrease oxidative phosphorylation. To identify metabolic vulnerabilities that enhance this phenotype, we utilized a CRISPR/Cas9 loss-of-function screen and identified HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway and target of statin therapies, as a top-scoring sensitizer to ABL inhibition. Combination treatment with ABL allosteric inhibitors and statins decreases metastatic lung cancer cell survival in vitro in a synergistic manner. Notably, combination therapy in mouse models of lung cancer brain metastasis and therapy resistance impairs metastatic colonization with a concomitant increase in animal survival. Thus, metabolic combination therapy might be effective to decrease metastatic outgrowth, leading to increased survival for lung cancer patients with advanced disease.
Subject(s)
Apoptosis/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Lung Neoplasms/drug therapy , Oncogene Proteins v-abl/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Allosteric Regulation/drug effects , Allosteric Regulation/genetics , Animals , Apoptosis/genetics , Cell Line, Tumor , Drug Synergism , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Nude , Neoplasm Metastasis , Oncogene Proteins v-abl/genetics , Oncogene Proteins v-abl/metabolism , Signal Transduction/genetics , Xenograft Model Antitumor AssaysABSTRACT
Imatinib, a drug used for the treatment of chronic myeloid leukemia and other cancers, works by blocking the catalytic site of pathological constitutively active Abl kinase. While the binding pose is known from X-ray crystallography, the different steps leading to the formation of the complex are not well understood. The results from extensive molecular dynamics simulations show that imatinib can primarily exit the known crystallographic binding pose through the cleft of the binding site or by sliding under the αC helix. Once displaced from the crystallographic binding pose, imatinib becomes trapped in intermediate states. These intermediates are characterized by a high diversity of ligand orientations and conformations, and relaxation timescales within this region may exceed 3-4 ms. Analysis indicates that the metastable intermediate states should be spectroscopically indistinguishable from the crystallographic binding pose, in agreement with tryptophan stopped-flow fluorescence experiments.
Subject(s)
Imatinib Mesylate/chemistry , Molecular Dynamics Simulation , Oncogene Proteins v-abl/chemistry , Protein Kinase Inhibitors/chemistry , Binding Sites/drug effects , Crystallography, X-Ray , Humans , Imatinib Mesylate/pharmacology , Oncogene Proteins v-abl/antagonists & inhibitors , Oncogene Proteins v-abl/metabolism , Protein Kinase Inhibitors/pharmacologyABSTRACT
Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib's high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib's half-maximal inhibitory concentration (IC50). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients.
Subject(s)
Antineoplastic Agents/pharmacology , Imatinib Mesylate/pharmacology , Neoplasms/enzymology , Oncogene Proteins v-abl/genetics , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Drug Resistance, Neoplasm , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Oncogene Proteins v-abl/chemistry , Oncogene Proteins v-abl/metabolismABSTRACT
Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.
Subject(s)
Oncogene Proteins v-abl/metabolism , Transcription, Genetic , YY1 Transcription Factor/chemistry , YY1 Transcription Factor/metabolism , Benzamides/pharmacology , Conserved Sequence , Gene Knockout Techniques , Gene Silencing , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Imatinib Mesylate/pharmacology , MCF-7 Cells , Oncogene Proteins v-abl/genetics , Phosphorylation , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Tyrosine/chemistryABSTRACT
The incidence of melanoma is increasing, particularly in young women, and the disease remains incurable for many because of its aggressive, metastatic nature and its high rate of resistance to conventional, targeted, and immunological agents. Cathepsins are proteases that are critical for melanoma progression and therapeutic resistance. Intracellular cathepsins cleave or degrade proteins that restrict cancer progression, whereas extracellular cathepsins directly cleave the extracellular matrix and activate proinvasive proteases in the tumor microenvironment. Cathepsin secretion is markedly increased in cancer cells. We investigated the signaling pathways leading to increased cathepsin secretion in melanoma cells. We found that the nonreceptor tyrosine kinases Abl and Arg (Abl/Arg) promoted the secretion of cathepsin B and cathepsin L by activating transcription factors (namely, Ets1, Sp1, and NF-κB/p65) that have key roles in the epithelial-mesenchymal transition (EMT), invasion, and therapeutic resistance. In some melanoma cell lines, Abl/Arg promoted the Ets1/p65-induced secretion of cathepsin B and cathepsin L in a kinase-independent manner, whereas in other melanoma lines, Abl/Arg promoted the kinase-dependent, Sp1/Ets1/p65-mediated induction of cathepsin L secretion and the Sp1/p65-mediated induction of cathepsin B secretion. As an indication of clinical relevance, the abundance of mRNAs encoding Abl/Arg, Sp1, Ets1, and cathepsins was positively correlated in primary melanomas, and Abl/Arg-driven invasion in culture and metastasis in vivo required cathepsin secretion. These data suggest that drugs targeting Abl kinases, many of which are FDA-approved, might inhibit cathepsin secretion in some melanomas and potentially other aggressive cancers harboring activated Abl kinases.
Subject(s)
Cathepsins/metabolism , Cysteine Proteases/metabolism , Melanoma/enzymology , Oncogene Proteins v-abl/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Cathepsins/genetics , Cell Line, Tumor , Cysteine Proteases/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Mice, Nude , Neoplasm Invasiveness , Neoplasm Metastasis , Oncogene Proteins v-abl/genetics , Protein-Tyrosine Kinases/genetics , Pyrimidines/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Xenograft Model Antitumor Assays/methodsABSTRACT
Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Membrane Proteins/chemistry , Nuclear Proteins/chemistry , Oncogene Proteins v-abl/chemistry , Proto-Oncogene Proteins c-fyn/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Conserved Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oncogene Proteins v-abl/metabolism , Phosphorylation , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-fyn/metabolism , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , ZebrafishABSTRACT
Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα13, is critical for actin cytoskeletal reorganization, cell migration, cell proliferation, and apoptosis. Previously, we have shown that Gα13 is essential for both G-protein-coupled receptor and receptor tyrosine kinase-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα13 directly interacts with Abl tyrosine kinase, which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα13-induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα13 controls actin cytoskeletal reorganization.
Subject(s)
Actin Cytoskeleton/metabolism , Cell Movement/physiology , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , GTP-Binding Protein alpha Subunits, G12-G13/metabolism , Oncogene Proteins v-abl/metabolism , Animals , Cells, Cultured , Embryo, Mammalian/cytology , Fibroblasts/cytology , GTP-Binding Protein alpha Subunits, G12-G13/genetics , Mice , Mice, Knockout , Oncogene Proteins v-abl/genetics , Signal Transduction , Spheroids, Cellular , Wound HealingABSTRACT
Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.
Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA Breaks, Double-Stranded , Gene Editing/methods , Precursor Cells, B-Lymphoid/metabolism , Recombinational DNA Repair , Animals , Apoptosis/drug effects , CRISPR-Associated Proteins/metabolism , Cell Line, Transformed , DNA End-Joining Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , Genotype , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Imatinib Mesylate/pharmacology , Mice, Inbred C57BL , Oncogene Proteins v-abl/antagonists & inhibitors , Oncogene Proteins v-abl/genetics , Oncogene Proteins v-abl/metabolism , Phenotype , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinational DNA Repair/drug effectsABSTRACT
BACKGROUND: Saraphi (Mammea siamensis) is a Thai traditional herb. In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. MATERIALS AND METHODS: The flowers of M. siamensis were extracted using ethanol. The ethanol flower extract was further fractionated with hexane, ethyl acetate, and methanol. Cytotoxic effects were measured by the MTT assay. Bcr/Abl and WT1 protein levels after treatments were determined by Western blotting. The total cell number was determined via the typan blue exclusion method. RESULTS: The hexane fraction showed the strongest cytotoxic activity on Molt4 and K562 cells, with IC50 values of 2.6 and 77.6 µg/ml, respectively. The hexane extract decreased Bcr/Abl protein expression in K562 cells by 74.6% and WT1 protein expressions in Molt4 and K562 cells by 68.4 and 72.1%, respectively. Total cell numbers were decreased by 66.2 and 48.7% in Molt4 and K562 cells, respectively. Mammea E/BB (main active compound) significantly decreased both Bcr/Abl and WTlprotein expressions by 75 and 49.5%, respectively when compared to vehicle control. CONCLUSION: The hexane fraction from M. siamensis flowers inhibited cell proliferation via the suppression of WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The active compound may be mammea E/BB. Extracts from M. siamensis flowers show promise as naturally occurring anti-cancer drugs.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Fusion Proteins, bcr-abl/metabolism , Leukemia/drug therapy , Mammea , Phytotherapy , Plant Extracts/therapeutic use , WT1 Proteins/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Cell Proliferation , Flowers , Humans , K562 Cells , Leukemia/metabolism , Medicine, Traditional , Oncogene Proteins v-abl/metabolism , Plant Extracts/pharmacology , Proto-Oncogene Proteins c-bcr/metabolism , ThailandABSTRACT
BACKGROUND: MicroRNAs (miRNAs) have emerged as crucial factors that regulate proliferation and apoptosis of cardiac c-kit+ cells. Although much is known about their role in maintaining cardiac c-kit+ cell pluripotency, the mechanisms by which they affect cell fate decisions that are an essential part of the repair of heart failure remain poorly understood. METHODS: Cardiac c-kit+ cells were obtained from Balb/c mice and cultured in vitro. Lentiviral vectors of miR199a-3p, its corresponding anti-miRNA, or short hairpin RNA against Cables1 were transfected into cells. The proliferation of cardiac c-kit+ cells was evaluated using EdU and flow cytometry. Furthermore, we examined cell apoptosis by flow cytometry under treatment with 200nM angiotensin II for 48 h. The levels of miR199a-3p and Cables1 mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to examine the expression of Cables1 and P53 proteins. RESULTS: We demonstrated a significantly decreased expression of miR199a-3p in heart failure samples compared with healthy donors. Meanwhile, we identified miR199a-3p as a proliferation- and apoptosis-associated regulator impacted through Cdk5 and Abl enzyme substrate 1 (CABLES1) targeting, and also attributed their repression to P53 protein expression. We further demonstrated that P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. CONCLUSION: Collectively, our findings uncover one new mechanism by which P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. Therefore, miR199a-3p and P53 are coupled through CABLES1 and comprise a novel negative feedback loop that likely contributes to cardiac c-kit+ cell proliferation and apoptosis.
Subject(s)
Apoptosis/physiology , Carrier Proteins/metabolism , Cell Proliferation/physiology , Cyclins/metabolism , Heart Failure/metabolism , MicroRNAs/metabolism , Myocytes, Cardiac/physiology , Phosphoproteins/metabolism , Tumor Suppressor Protein p53/metabolism , Aged , Angiotensins/pharmacology , Animals , Apoptosis/drug effects , Carrier Proteins/genetics , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 5/metabolism , Cyclins/genetics , Feedback, Physiological , Female , HEK293 Cells , Heart Failure/genetics , Heart Failure/pathology , Humans , Male , Mice , Mice, Inbred BALB C , MicroRNAs/genetics , Middle Aged , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Oncogene Proteins v-abl/metabolism , Phosphoproteins/genetics , Proto-Oncogene Proteins c-kit/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.
Subject(s)
B-Lymphocytes/metabolism , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/chemistry , Sequence Homology, Amino Acid , V(D)J Recombination/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , CRISPR-Cas Systems/genetics , DNA Damage , DNA Repair , DNA-Binding Proteins/metabolism , Gene Deletion , Gene Editing , Gene Rearrangement, B-Lymphocyte , Immunoglobulins/genetics , Ku Autoantigen/metabolism , Models, Biological , Oncogene Proteins v-abl/metabolismABSTRACT
Cyclophilin A (CypA) is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. CrkII influences cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binding site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly upregulated.
Subject(s)
Cyclophilin A/pharmacology , Oncogene Proteins v-abl/metabolism , Proto-Oncogene Proteins c-crk/metabolism , Signal Transduction/drug effects , Amino Acid Sequence , Blotting, Western , Calorimetry , Cell Line, Tumor , Cell Movement/drug effects , Humans , Molecular Sequence DataABSTRACT
The DNA damage response network stimulates microRNA (miRNA) biogenesis to coordinate repair, cell cycle checkpoints, and apoptosis. The multistep process of miRNA biogenesis involves the cleavage of primary miRNAs by the microprocessor complex composed of the ribonuclease Drosha and the RNA binding protein DGCR8. We found that the tyrosine kinase ABL phosphorylated DGCR8, a modification that was required for the induction of a subset of miRNAs after DNA damage. Focusing on the miR-34 family, ABL stimulated the production of miR-34c, but not miR-34a, through Drosha/DGCR8-dependent processing of primary miR-34c (pri-miR-34c). This miRNA-selective effect of ABL required the sequences flanking the precursor miR-34c (pre-miR-34c) stem-loop. In pri-miRNA processing, DGCR8 binds the pre-miR stem-loop and recruits Drosha to the miRNA. RNA cross-linking assays showed that DGCR8 and Drosha interacted with pri-miR-34c, but we found an inverse correlation between ABL-stimulated processing and DGCR8 association with pri-miR-34c. When coexpressed in HEK293T cells, ABL phosphorylated DGCR8 at Tyr(267). Ectopic expression of a Y267F-DGCR8 mutant reduced the recruitment of Drosha to pri-miR-34c and prevented ABL or Drosha from stimulating the processing of pri-miR-34c. In mice engineered to express a nuclear import-defective mutant of ABL, miR-34c, but not miR-34a, expression was reduced in the kidney, and apoptosis of the renal epithelial cells was impaired in response to cisplatin. These results reveal a new pathway in the DNA damage response wherein ABL-dependent tyrosine phosphorylation of DGCR8 stimulates the processing of selective primary miRNAs.
Subject(s)
DNA Damage , MicroRNAs/metabolism , Oncogene Proteins v-abl/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA-Binding Proteins/metabolism , Animals , Humans , Mice , MicroRNAs/genetics , Oncogene Proteins v-abl/genetics , Phosphorylation/physiology , RNA-Binding Proteins/geneticsABSTRACT
RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.
Subject(s)
Oncogene Proteins v-abl/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction/physiology , Amino Acid Substitution , Animals , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mutation, Missense , Oncogene Proteins v-abl/chemistry , Oncogene Proteins v-abl/genetics , Phosphorylation/genetics , Proto-Oncogene Mas , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Rats , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolism , rab5 GTP-Binding Proteins/chemistry , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolism , raf Kinases/chemistry , raf Kinases/genetics , raf Kinases/metabolismABSTRACT
BACKGROUND: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.
Subject(s)
B-Lymphocytes/physiology , Mutation/genetics , Nuclear Proteins/genetics , Severe Combined Immunodeficiency/genetics , Adolescent , Adult , Alleles , B-Lymphocytes/radiation effects , Cell Line, Transformed , Child , Child, Preschool , DNA Mutational Analysis , DNA Repair/genetics , DNA-Binding Proteins , Endonucleases , Heterozygote , Histones/metabolism , Humans , Infant , Infant, Newborn , Male , Oncogene Proteins v-abl/genetics , Oncogene Proteins v-abl/metabolism , Phenotype , Radiation Tolerance/genetics , Radiation, Ionizing , V(D)J Recombination/genetics , Young AdultABSTRACT
Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.
Subject(s)
Proto-Oncogene Proteins c-crk/metabolism , Signal Transduction , Tyrosine/metabolism , src Homology Domains , Amino Acid Sequence , Animals , Blotting, Western , Cell Line, Tumor , Chromatography, Liquid , HEK293 Cells , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Oncogene Proteins v-abl/metabolism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-crk/genetics , Sequence Homology, Amino Acid , Tandem Mass Spectrometry , Tyrosine/geneticsABSTRACT
The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.
Subject(s)
Oncogene Proteins v-abl/physiology , src Homology Domains/physiology , Enzyme Activation/physiology , Fusion Proteins, bcr-abl/physiology , Oncogene Proteins v-abl/metabolism , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcr/physiologyABSTRACT
Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli, and induce actin-filled membranous pedestals but do not invade the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL, and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and platelet-derived growth factor (PDGF) receptor (PDGFR) kinases, as well as p38 MAP kinase, have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula but are not required for late intimate bacterial attachment or high inoculum infections.