RESUMEN
The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/efectos adversos , Pirimidinas/efectos adversos , Enfermedades Vasculares/inducido químicamente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Endotelio Vascular/citología , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana EdadAsunto(s)
Resistencia a Antineoplásicos , Eosinofilia/complicaciones , Trastornos Mieloproliferativos/tratamiento farmacológico , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Animales , Proteínas Portadoras/genética , Línea Celular , Humanos , Lactante , Ratones , Mutación , Trastornos Mieloproliferativos/complicaciones , Trastornos Mieloproliferativos/genética , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Análisis de Secuencia de ADN , Transducción GenéticaRESUMEN
MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.
Asunto(s)
Apoptosis , Aurora Quinasa A/metabolismo , Resistencia a Antineoplásicos , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Transducción de Señal , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Azepinas/farmacología , Línea Celular Tumoral , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirimidinas/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC(50) values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.
Asunto(s)
Mastocitos/efectos de los fármacos , Mastocitosis/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Estaurosporina/análogos & derivados , Adulto , Anciano , Basófilos/efectos de los fármacos , Basófilos/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Liberación de Histamina/efectos de los fármacos , Humanos , Masculino , Mastocitos/fisiología , Mastocitosis/patología , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-kit/metabolismo , Estaurosporina/farmacologíaRESUMEN
Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-ß-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.
Asunto(s)
Imidazoles/farmacología , Necrosis/tratamiento farmacológico , Piridazinas/farmacología , Pirimidinas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Sulfonamidas/farmacología , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteína de Dominio de Muerte Asociada a Fas/genética , Células HEK293 , Células HT29 , Humanos , Indazoles , Células Jurkat , Células L , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Breast cancer is genetically heterogeneous, and recent studies have underlined a prominent contribution of epigenetics to the development of this disease. To uncover new synthetic lethalities with known breast cancer oncogenes, we screened an epigenome-focused short hairpin RNA library on a panel of engineered breast epithelial cell lines. Here we report a selective interaction between the NOTCH1 signaling pathway and the SUMOylation cascade. Knockdown of the E2-conjugating enzyme UBC9 (UBE2I) as well as inhibition of the E1-activating complex SAE1/UBA2 using ginkgolic acid impairs the growth of NOTCH1-activated breast epithelial cells. We show that upon inhibition of SUMOylation NOTCH1-activated cells proceed slower through the cell cycle and ultimately enter apoptosis. Mechanistically, activation of NOTCH1 signaling depletes the pool of unconjugated small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 leading to increased sensitivity to perturbation of the SUMOylation cascade. Depletion of unconjugated SUMO correlates with sensitivity to inhibition of SUMOylation also in patient-derived breast cancer cell lines with constitutive NOTCH pathway activation. Our investigation suggests that SUMOylation cascade inhibitors should be further explored as targeted treatment for NOTCH-driven breast cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Receptor Notch1/genética , Transducción de Señal/genética , Activación Transcripcional , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Técnicas de Cocultivo , Citometría de Flujo , Humanos , Microscopía Fluorescente , Interferencia de ARN , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Salicilatos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/efectos de los fármacos , Sumoilación/genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-specific expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identified as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Indoles/farmacología , Espectrometría de Masas , Melanocitos/efectos de los fármacos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Oncogenes , Pirroles/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Sunitinib , TransfecciónRESUMEN
Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteómica , Pirimidinas/farmacología , Receptor con Dominio Discoidina 1 , Receptores con Dominio Discoidina , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Quinasas Quinasa Quinasa PAM , Proteínas Quinasas/fisiología , Quinona Reductasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Receptores Mitogénicos/antagonistas & inhibidoresRESUMEN
BACKGROUND: The multikinase inhibitor dasatinib exerts growth-inhibitory effects in patients with imatinib-resistant chronic myeloid leukaemia (CML). In first clinical trials, side effects of dasatinib, 140 mg daily, were reported to be mild and tolerable. PATIENTS AND METHODS: We examined the side effect profile in 16 patients with imatinib-resistant CML who received 140 mg dasatinib daily in our center. RESULTS: Dasatinib produced substantial and sometimes severe or even life-threatening side effects with > or = 10% body weight loss (6/16 patients), pleural effusions grade II or higher (12/16) and infectious complications (12/16), including atypical infections not seen in imatinib-treated patients. One patient developed Epstein-Barr-Virus-positive mucosal leucoplakia, one died from pneumonia caused by pneumocystis carinii and three patients developed a skin-cancer. Most events were recorded within the first 2 years of therapy, only skin tumours developed after the second year. In ex vivo experiments performed in dasatinib-treated patients, transient suppression of IgE-dependent activation of blood basophils and TcR-dependent activation of T-lymphocytes was found. Moreover, in drug-binding studies, dasatinib was found to bind to several key kinase-targets of the immune system including Lyn and Btk, in mast cell, basophil, B-cell and T-cell lines. CONCLUSION: Dasatinib acts not only anti-neoplastic in CML but may also act as an immunosuppressive agent when applied at 140 mg daily, and produces frequent pleural effusions and weight loss in advanced CML.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antineoplásicos/efectos adversos , Inmunosupresores/efectos adversos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Linfocitos/inmunología , Pirimidinas/efectos adversos , Tiazoles/efectos adversos , Adulto , Anciano , Antígenos de Neoplasias/efectos de los fármacos , Antineoplásicos/administración & dosificación , Basófilos/efectos de los fármacos , Basófilos/inmunología , Dasatinib , Femenino , Citometría de Flujo , Humanos , Inmunoglobulinas/sangre , Inmunoglobulinas/efectos de los fármacos , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proteoma/análisis , Pirimidinas/administración & dosificación , Tiazoles/administración & dosificaciónRESUMEN
The detailed molecular mechanism of action of second-generation BCR-ABL tyrosine kinase inhibitors, including perturbed targets and pathways, should contribute to rationalized therapy in chronic myeloid leukemia (CML) or in other affected diseases. Here, we characterized the target profile of the dual SRC/ABL inhibitor bosutinib employing a two-tiered approach using chemical proteomics to identify natural binders in whole cell lysates of primary CML and K562 cells in parallel to in vitro kinase assays against a large recombinant kinase panel. The combined strategy resulted in a global survey of bosutinib targets comprised of over 45 novel tyrosine and serine/threonine kinases. We have found clear differences in the target patterns of bosutinib in primary CML cells versus the K562 cell line. A comparison of bosutinib with dasatinib across the whole kinase panel revealed overlapping, but distinct, inhibition profiles. Common among those were the SRC, ABL and TEC family kinases. Bosutinib did not inhibit KIT or platelet-derived growth factor receptor, but prominently targeted the apoptosis-linked STE20 kinases. Although in vivo bosutinib is inactive against ABL T315I, we found this clinically important mutant to be enzymatically inhibited in the mid-nanomolar range. Finally, bosutinib is the first kinase inhibitor shown to target CAMK2G, recently implicated in myeloid leukemia cell proliferation.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Células K562/efectos de los fármacos , Leucemia Mieloide de Fase Acelerada/enzimología , Proteínas de Neoplasias/antagonistas & inhibidores , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinolinas/farmacología , Compuestos de Anilina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Dasatinib , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Perfilación de la Expresión Génica , Humanos , Células K562/enzimología , Leucemia Mieloide de Fase Acelerada/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Nitrilos/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirimidinas/farmacología , Quinolinas/química , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Tiazoles/farmacología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The NUP214-ABL1 fusion kinase has recently been identified in 6% of patients with T-cell acute lymphoblastic leukemia. In contrast to the more common oncogenic ABL1 fusion BCR-ABL1, NUP214-ABL1 localizes to the nuclear pore complexes and has attenuated transforming properties in hematopoietic cells and in mouse bone marrow transplant models. We have performed a thorough biochemical comparative analysis of NUP214-ABL1 and BCR-ABL1 and show that, despite their common tyrosine kinase domain, the two fusion proteins differ in many critical catalytic properties. NUP214-ABL1 has lower in vitro tyrosine kinase activity, which is in agreement with the absence of phosphorylation on its activation loop. NUP214-ABL1 was more sensitive to imatinib (Glivec) than BCR-ABL1 in vitro and in cells, indicating a different activation state and conformation of the two ABL1 fusion kinases. Using a peptide array, we identified differences in the spectrum and efficiency of substrate peptide phosphorylation and a differential involvement of Src kinases in downstream signaling. These results clearly indicate that different fusion partners of the same kinase can determine not only localization, but also critical functional properties of the enzyme such as inhibitor sensitivity and substrate preference, with subsequent differences in downstream signaling effectors and likely consequences in disease pathogenesis.
Asunto(s)
Proteínas de Fusión bcr-abl/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Benzamidas , Dasatinib , Activación Enzimática/fisiología , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica , Humanos , Mesilato de Imatinib , Técnicas In Vitro , Células K562 , Leucemia Eritroblástica Aguda/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Fosforilación , Piperazinas/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Transducción de Señal/fisiología , Especificidad por Sustrato , Tiazoles/farmacologíaRESUMEN
The BCR-ABL oncogenic tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, cells are selected in some patients with BCR-ABL kinase domain mutations that render decreased drug sensitivity. In addition, some patients express deletion mutants of BCR-ABL, apparently due to missplicing. Most commonly these deletion mutants lack a significant portion of the kinase domain that includes the P-loop. We describe a screen for such mutations in patients with CML and demonstrate that they are not oncogenic and are catalytically inactive. We hypothesized that coexpressing BCR-ABL deletion mutants has a dominant-negative effect on the native form through heterocomplex formation. However, upon coexpression of native and deletion mutant BCR-ABL in Ba/F3 cells, growth factor independence is maintained and signaling is activated normally. Despite this, these cells have increased imatinib sensitivity compared to cells expressing only native BCR-ABL. Thus, it will be important to investigate the prognostic impact of coexpression of deletion mutants in CML patients during imatinib treatment.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación/genética , Eliminación de Secuencia , Adulto , Anciano , Benzamidas , Proliferación Celular , Células Cultivadas , Resistencia a Antineoplásicos , Femenino , Estudios de Seguimiento , Humanos , Mesilato de Imatinib , Immunoblotting , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Fosforilación , Piperazinas/uso terapéutico , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Conformación Proteica , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Tirosina/metabolismoRESUMEN
The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.
Asunto(s)
Simulación por Computador , Modelos Moleculares , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas/química , Dominios Homologos src/fisiología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Bioensayo , Proteína Tirosina Quinasa CSK , Pollos , Activación Enzimática/genética , Activación Enzimática/fisiología , Estabilidad de Enzimas/fisiología , Mutagénesis Sitio-Dirigida , Fosforilación , Conformación Proteica , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-hck , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Tirosina/metabolismo , Familia-src QuinasasRESUMEN
c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner.
Asunto(s)
Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/metabolismo , Western Blotting , Catálisis , Línea Celular , Activación Enzimática , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Fosfotirosina/fisiología , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Relación Estructura-ActividadRESUMEN
Tyrosine phosphorylation is usually associated with cytoplasmic events. Yet, over the years, many reports have accumulated on tyrosine phosphorylation of individual molecules in the nucleus, and several tyrosine kinases and phosphatases have been found to be at least partially nuclear. The question arises as to whether nuclear tyrosine phosphorylation represents a collection of loose ends of events originating in the cytoplasm or if there may be intranuclear signaling circuits relying on tyrosine phosphorylation to regulate specific processes. The recent discovery of a mechanism causing nuclear tyrosine phosphorylation has prompted us to review the cumulative evidence for nuclear tyrosine phosphorylation pathways and their possible role. While we found that no complex nuclear function has yet been shown to rely upon intranuclear tyrosine phosphorylation in an unambiguous fashion, we found a very high number of compelling observations on individual molecules that suggest underlying networks linking individual events. A systematic proteomics approach to nuclear tyrosine phosphorylation should help chart possible interaction pathways.
Asunto(s)
Núcleo Celular/metabolismo , Tirosina/metabolismo , Transporte Activo de Núcleo Celular , Animales , Ciclo Celular/fisiología , Núcleo Celular/genética , Citoplasma/metabolismo , Reparación del ADN , Humanos , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , ARN/metabolismo , Recombinación Genética , Transducción de Señal/fisiologíaRESUMEN
Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.
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Miopatías Estructurales Congénitas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Fosfatasas/genética , Animales , Línea Celular , Regulación hacia Abajo , Humanos , Pruebas de Precipitina , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , TransfecciónRESUMEN
The catalytic activity of Src family tyrosine kinases is inhibited by intramolecular interactions between the regulatory SH3 and SH2 domains and the catalytic domain. In the inactive state, the critical alphaC-helix in the catalytic domain is positioned such that the formation of the Glu 310-Lys 295 salt bridge is precluded, Tyr 416 in the activation loop is unphosphorylated, and the SH2 and SH3 domains are unavailable for interactions with other proteins. We found that phosphorylation of the activation loop or mutation of the loop preceding the alphaC-helix activates Src and increases the accessibility of the SH3 domain for ligands. Interaction of the alphaC-helix with the activation loop is a central component of this regulatory system. Our data suggest a bidirectional regulation mechanism in which the regulatory domains inhibit Src activity, and Src activity controls the availability of the regulatory domains. By this mechanism, Src family kinases can be activated by proteins phosphorylating or changing the conformation of the catalytic domain. Once active, Src family kinases become less prone to regulation, implying a positive feedback loop on their activity.
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Dominio Catalítico/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Dominios Homologos src/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión , Línea Celular , Pollos , Activación Enzimática , Retroalimentación , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Modelos Moleculares , Mutación/genética , Péptidos/metabolismo , Fosforilación , Fosfotirosina/genética , Fosfotirosina/metabolismo , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/genética , Transfección , Dominios Homologos src/genéticaRESUMEN
The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, we constructed a constitutively active and nuclear form of the protein. We found that active nuclear Abl efficiently phosphorylate c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, both proteins interacted via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activated nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signalling models.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Programas Informáticos , Especificidad por Sustrato , Transfección , Tirosina , Dominios Homologos srcRESUMEN
Studies of the actin-based motility of the intracellular pathogens Listeria monocytogenes and Shigella flexneri have provided important insight into the events occurring at the leading edges of motile cells. Like the bacteria Listeria and Shigella, vaccinia virus, a relative of the causative agent of smallpox, uses actin-based motility to spread between cells. In contrast to Listeria or Shigella, the actin-based motility of vaccinia is dependent on an unknown phosphotyrosine protein, but the underlying mechanism remains obscure. Here we show that phosphorylation of tyrosine 112 in the viral protein A36R by Src-family kinases is essential for the actin-based motility of vaccinia. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor protein Nck and the recruitment of the Ena/VASP family member N-WASP to the site of actin assembly. We also show that Nck and N-WASP are essential for the actin-based motility of vaccinia virus. We suggest that vaccinia virus spreads by mimicking the signalling pathways that are normally involved in actin polymerization at the plasma membrane.
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Actinas/fisiología , Lectinas Tipo C , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal , Virus Vaccinia/fisiología , Animales , Células HeLa , Humanos , Lectinas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Imitación Molecular , Fosforilación , Mutación Puntual , Receptores de Superficie Celular/fisiología , Tirosina/metabolismo , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
The activity of the c-Src tyrosine kinase is regulated through intramolecular interactions between the catalytic and SH2/SH3 domains. However, the exact mechanism by which this occurs remains obscure. In the crystal structure of c-Src, the peptide that links the SH2 and catalytic domain (SH2-CD linker) is sandwiched between the latter and the SH3 domain. A residue in the linker, Leu 255, inserts its side chain into a deep hydrophobic pocket present on the surface of the catalytic domain. To investigate the possible regulatory role of this prominent interaction, we mutated Leu 255 to different hydrophobic residues. We found that the length and 'bulkiness' of the side chain had a profound influence on c-Src regulation. Src-L255V was highly active but showed reduced SH3 accessibility in vitro as well as an altered localization in vivo when compared to other deregulated forms of Src. Our analyses lead us to suggest that the Leu 255-pocket interaction is a critical component of the intramolecular inhibition mechanism of Src family kinases.