RESUMO
Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.
Assuntos
Leucemia Experimental/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas Tirosina Quinases/genética , Alelos , Animais , Apoptose/efeitos dos fármacos , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Janus Quinase 2 , Janus Quinase 3 , Células K562 , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , TYK2 QuinaseRESUMO
Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.
Assuntos
Hidroximetil e Formil Transferases/metabolismo , Linfoma Anaplásico de Células Grandes/enzimologia , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleotídeo Desaminases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Antimetabólitos Antineoplásicos/farmacologia , Carbazóis/farmacologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Humanos , Hidroximetil e Formil Transferases/antagonistas & inibidores , Indazóis/farmacologia , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/patologia , Metotrexato/farmacologia , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Nucleotídeo Desaminases/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/análise , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/antagonistas & inibidores , Transcrição GênicaRESUMO
Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Mutação/genética , Fosfotransferases/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Sequência de Aminoácidos , Animais , Benzamidas , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Feminino , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Células Progenitoras Mieloides/citologia , Fosfotirosina/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Especificidade por SubstratoRESUMO
Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteômica/métodos , Tirosina/química , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Células Jurkat , Espectrometria de Massas , Camundongos , Células NIH 3T3 , Peptídeos/química , Fosforilação , Fosfotirosina/química , Transdução de SinaisRESUMO
Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNA/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sirolimo/metabolismo , Animais , Animais Recém-Nascidos , Ciclo Celular/fisiologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Ativação Enzimática , Feminino , Viabilidade Fetal , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/citologia , Miocárdio/patologia , Fenótipo , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
STAT5 is constitutively phosphorylated in leukemic cells in approximately 70% of acute myeloid leukemia (AML) patients. To identify kinase candidates potentially responsible for STAT5 phosphorylation, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) mass spectrometry to detect phosphoproteins in AML cell lines. We established TEL-ARG and BCR-ABL fusion proteins as the mechanism underlying STAT5 phosphorylation in HT-93 and KBM-3 cells, respectively. In addition, we identified a JAK2 pseudokinase domain mutation in HEL cells and using siRNA downregulation, established JAK2 as the kinase responsible for phosphorylating STAT5. This study illustrates the benefit of LC-MS/MS mass spectrometry and siRNA for the identification of novel targets and mutations.
Assuntos
Carcinógenos , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Carcinógenos/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Leucemia Mieloide Aguda/metabolismo , Espectrometria de Massas , Mutação , Proteínas de Neoplasias/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/genética , ProteômicaRESUMO
Our understanding of the mechanisms by which BCR-ABL drives CML is based, in part, on the use of model cell lines such as the K562 cell line. However, the BCR-ABL translocation may occur via a number of different junction points. In addition, CML is a disease of hematopoietic stem cells and, as a result, can give rise to multiple lineages of tumor cells. In this study, we examined the cellular signaling profiles following imatinib mesylate treatment of eight model CML and ALL cell lines that encompass three BCR-ABL junction points and multiple lineages. We used phosphorylation-specific antibodies and flow cytometry to determine the kinase and pathway activation states with each of the cell lines before and after imatinib mesylate exposure. The comparisons of signaling response profiles, junction points and lineages indicate that cell line lineage rather than BCR-ABL junction point may determine cellular response to imatinib mesylate. The large amount of variation observed among the cell lines suggests that further analysis is required to understand the complex signaling profiles present in CML patients.
Assuntos
Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Neoplasias/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Fisiológico/enzimologia , Fosfatases cdc25/metabolismo , Sequência de Aminoácidos/efeitos dos fármacos , Sequência de Aminoácidos/fisiologia , Anticorpos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/fisiologia , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Mitose/efeitos dos fármacos , Mitose/fisiologia , Fosforilação/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fosfatases cdc25/antagonistas & inibidoresRESUMO
Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.
Assuntos
Leucemia Megacarioblástica Aguda/genética , Proteínas de Fusão Oncogênica/fisiologia , Proteínas de Ligação a RNA/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 5 , Humanos , Leucemia Megacarioblástica Aguda/etiologia , Camundongos , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/isolamento & purificação , Proteínas Tirosina Quinases/isolamento & purificação , Receptor de Fator Estimulador de Colônias de Macrófagos/isolamento & purificação , Análise de Sequência de DNA , Translocação Genética , Transplante HeterólogoRESUMO
The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 Bcr-Abl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosine-phosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to the 6 cell lines regardless of cellular background and fusion type, several of which are decreased by imatinib treatment. Comparison of this Bcr-Abl signature with the profile of cells expressing an alternative imatinib-sensitive fusion kinase, FIP1L1-PDGFRalpha, revealed that these kinases signal through different pathways. This phosphoproteomic study of the Bcr-Abl fusion kinase highlights novel disease markers and potential drug-responsive biomarkers and adds novel insight into the oncogenic signals driven by the Bcr-Abl kinase.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Fosfotirosina/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Benzamidas , Biomarcadores Tumorais/análise , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Fusão Oncogênica/metabolismo , Fosfotirosina/análise , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Pirimidinas/uso terapêutico , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
The 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.