RESUMO
Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4(-/-)) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.
Assuntos
Transformação Celular Neoplásica , Leucemia Mieloide Aguda/patologia , NADPH Oxidases/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , NADPH Oxidase 4 , NADPH Oxidases/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/análise , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/análiseRESUMO
Fms-like tyrosine kinase-3 is a commonly mutated gene in acute myeloid leukemia, with about one-third of patients carrying an internal-tandem duplication of the juxtamembrane domain in the receptor (FLT3-ITD). FLT3-ITD exhibits altered signaling quality, including aberrant activation of STAT5. To identify genes affecting FLT3-ITD-mediated STAT5 signaling, we performed an esiRNA-based RNAi screen utilizing a STAT5-driven reporter assay. Knockdowns that caused reduced FLT3-ITD-mediated STAT5 signaling were enriched for genes encoding proteins involved in protein secretion and intracellular protein transport, indicating that modulation of protein transport processes could potentially be used to reduce constitutive STAT5 signaling in FLT3-ITD-positive cells. The relevance of KDELR1, a component involved in the Golgi-ER retrograde transport, was further analyzed. In FLT3-ITD-expressing leukemic MV4-11 cells, downregulation of KDELR1 resulted in reduced STAT5 activation, proliferation and colony-forming capacity. Stable shRNA-mediated depletion of KDELR1 in FLT3-ITD-expressing 32D cells likewise resulted in reduced STAT5 signaling and cell proliferation. Importantly, these cells also showed a reduced capacity to generate a leukemia-like disease in syngeneic C3H/HeJ mice. Together our data suggest intracellular protein transport as a potential target for FLT3-ITD driven leukemias, with KDELR1 emerging as a positive modulator of oncogenic FLT3-ITD activity.
Assuntos
Genoma , Proteínas/fisiologia , Interferência de RNA , Transdução de Sinais/fisiologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Sequência de Bases , Primers do DNA , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT5/metabolismoRESUMO
The class III receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) regulates normal hematopoiesis and immunological functions. Nonetheless, constitutively active mutant FLT3 (FLT3-ITD) causally contributes to transformation and is associated with poor prognosis of acute myeloid leukemia (AML) patients. Histone deacetylase inhibitors (HDACi) can counteract deregulated gene expression profiles and decrease oncoprotein stability, which renders them candidate drugs for AML treatment. However, these drugs have pleiotropic effects and it is often unclear how they correct oncogenic transcriptomes and proteomes. We report here that treatment of AML cells with the HDACi LBH589 induces the ubiquitin-conjugating enzyme UBCH8 and degradation of FLT3-ITD. Gain- and loss-of-function approaches show that UBCH8 and the ubiquitin-ligase SIAH1 physically interact with and target FLT3-ITD for proteasomal degradation. These ubiquitinylating enzymes though have a significantly lesser effect on wild-type FLT3. Furthermore, physiological and pharmacological stimulation of FLT3 phosphorylation, inhibition of FLT3-ITD autophosphorylation and analysis of kinase-inactive FLT3-ITD revealed that tyrosine phosphorylation determines degradation of FLT3 and FLT3-ITD by the proteasome. These results provide novel insights into antileukemic activities of HDACi and position UBCH8, which have been implicated primarily in processes in the nucleus, as a previously unrecognized important modulator of FLT3-ITD stability and leukemic cell survival.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Western Blotting , Linhagem Celular , Separação Celular , Citometria de Fluxo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hidrólise , Imunoprecipitação , Mutação , Fosforilação , Tirosina/metabolismo , Tirosina Quinase 3 Semelhante a fms/genéticaAssuntos
Transformação Celular Neoplásica , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Humanos , Camundongos , Camundongos Endogâmicos C3H , RNA Interferente Pequeno/genética , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Anticarcinógenos/farmacologia , Colo/citologia , Neoplasias do Colo/patologia , Células Epiteliais/efeitos dos fármacos , Proteínas de Neoplasias/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Adenocarcinoma/enzimologia , Adenoma/enzimologia , Butiratos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Colo/enzimologia , Neoplasias do Colo/enzimologia , Regulação para Baixo , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Flavonoides/farmacologia , Humanos , Lisofosfolipídeos/farmacologia , Malus/química , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Polifenóis , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/farmacologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Chá/química , Transcrição Gênica/efeitos dos fármacos , Transfecção , Regulação para Cima/efeitos dos fármacosRESUMO
Among activating Flt3 mutations that have been shown in 25-30% of acute myeloid leukaemia (AML) Flt3-internal tandem duplication (ITD) mutations are predominant. We investigated the influence of all-trans-retinoic acid (ATRA) and granulocyte colony stimulating factor (G-CSF) for their effects on differentiation and apoptosis in human cell lines with different Flt3 variants (THP-1 versus MV4-11 and MOLM13) dependent on the inhibition of Flt3 tyrosine kinase by the bis(lH-2-indolyl)methanone D-65476. While myeloid differentiation was not observed in both Flt3-ITD cell lines (MV4-11 and MOLM13), we demonstrate an enhanced proapoptotic effect of D-65476 in the presence of ATRA that was restricted to the Flt3-ITD expressing cells. The combined treatment with ATRA and D-65476 also led to a pronounced down-regulation of surviv in on mRNA and protein level in Flt3-ITD but not in Flt3 wildtype expressing cells (THP-1). Surprisingly, there was no differential expression of important proteins like Bcl-X(L), Bcl-2 or Bax that might explain enhanced apoptosis. Furthermore, Akt phosphorylation after stimulation with Flt3 ligand dependent on D-65476 was not affected by pretreatment with ATRA. We suggest that regulation of inhibitors of apoptosis might play a crucial role how ATRA can increase the proapoptotic effect of Flt3 inhibitors in myeloid leukemia cells expressing Flt3-ITD. This effect can potentially be exploited for the treatment of Flt3-ITD positive acute myeloid leukemia.
Assuntos
Apoptose/efeitos dos fármacos , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Tretinoína/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Mutação , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/biossíntese , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
FLT3 is a receptor tyrosine kinase that may play a role in a significant proportion of leukemias. In addition to being aberrantly expressed in acute leukemias, activating mutations of the FLT3 gene have been found in patients with AML, myelodysplastic syndrome (MDS) and more rarely, ALL. Internal tandem duplications (ITDs) of the FLT3 gene have been detected in 17-34% of patients with AML and portend a poor prognosis for these patients. FLT3 receptors containing ITD mutations (FLT3/ITDs) are constitutively activated in the absence of FLT3 ligand (FL) stimulation leading to the activation of downstream signaling proteins, including ERK and STAT 5. FLT3 activity, therefore, is a logical target for therapeutic intervention. AG1296 is a tyrosine kinase inhibitor of the tyrphostin class that shows inhibitory activity for wild-type FLT3, in addition to the PDGF and c-KIT receptors. We examined the inhibitory effects of AG1296 on FLT3/ITDs isolated from AML patients in the IL-3-dependent cell line, Ba/F3, as well as in primary leukemia samples from AML patients. Immunoprecipitation and immunoblotting analyses demonstrated that FLT3/ITDs were constitutively phosphorylated in the absence of FL. The auto-phosphorylation of FLT3/ITDs was inhibited by AG1296 with an IC(50) of approximately 1 microM. FLT3/ITDs were associated with constitutive phosphorylation of ERK, STAT 5A, STAT 5B, CBL, VAV and SHP2 in Ba/F3 cells. The phosphorylation of these downstream signaling molecules was suppressed in a dose-responsive fashion by AG1296. AG1296 inhibited IL-3 independent growth and induced apoptosis in Ba/F3 cells transformed by FLT3/ITDs. AG1296 also inhibited FLT3 auto-phosphorylation, and induced a cytotoxic effect, in primary AML cells. These findings suggest that inhibiting the activity of FLT3 may have a therapeutic value in some leukemias expressing FLT3/ITDs.
Assuntos
Inibidores Enzimáticos/farmacologia , Duplicação Gênica , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Sequência de Aminoácidos , Apoptose , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , Regulação para Baixo , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Tirfostinas/farmacologia , Tirosina Quinase 3 Semelhante a fmsAssuntos
Carcinoma de Células Escamosas/patologia , Técnicas de Cocultura/métodos , Dissecação/métodos , Terapia a Laser/métodos , Neoplasias Bucais/patologia , Células Estromais/patologia , Separação Celular/instrumentação , Separação Celular/métodos , Dissecação/instrumentação , Fibroblastos/patologia , Humanos , Terapia a Laser/instrumentação , Lasers , RNA Neoplásico/isolamento & purificação , Células Tumorais CultivadasRESUMO
Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias.
Assuntos
Inibidores Enzimáticos/farmacologia , Células-Tronco Hematopoéticas/enzimologia , Indóis/farmacologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Becaplermina , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-3/farmacologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-sis , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transfecção , Tirosina Quinase 3 Semelhante a fmsRESUMO
A new class of simple synthetic antimitotic compounds based on 2-aroylindoles was discovered. (5-Methoxy-1H-2-indolyl)-phenylmethanone (1) as well as analogous 3-fluorophenyl- (36) and 3-methoxyphenyl (3) derivatives displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. In in vitro assays with tubulin isolated from bovine brain, in general antiproliferative activity correlated with inhibition of tubulin polymerization. Thus, the antimitotic activity of 2-aroylindoles is explained by interference with the mitotic spindle apparatus and destabilization of microtubules. In contrast to colchicine, vincristine, nocodazole, or taxol, 1 did not significantly affect the GTPase activity of beta-tubulin. Interestingly, selected compounds inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. In xenograft experiments, 1 was highly active after oral administration at 200 mg/kg against the human amelanocytic melanoma MEXF 989 in athymic nude mice. We conclude, that 2-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.
Assuntos
Antineoplásicos/síntese química , Indóis/síntese química , Tubulina (Proteína)/química , Alantoide/irrigação sanguínea , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Biopolímeros , Bovinos , Córion/irrigação sanguínea , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2/efeitos dos fármacos , GTP Fosfo-Hidrolases/química , Humanos , Técnicas In Vitro , Indóis/química , Indóis/farmacologia , Melanoma/tratamento farmacológico , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Relação Estrutura-Atividade , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17-27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas do Leite , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Tirfostinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Fosforilação , Fator de Transcrição STAT5 , Transativadores/metabolismo , Tirosina/metabolismo , Tirosina Quinase 3 Semelhante a fmsRESUMO
p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.
Assuntos
Moléculas de Adesão Celular/química , Fosfoproteínas/química , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Animais , Sítios de Ligação , Western Blotting , Células COS , Cateninas , Linhagem Celular , Citoplasma/química , Citoplasma/metabolismo , Análise Mutacional de DNA , Eletroforese em Gel Bidimensional , Fator de Crescimento Epidérmico/metabolismo , Deleção de Genes , Genes Dominantes , Glutationa Transferase/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação , Fenilalanina/química , Fosforilação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transfecção , Tirosina/metabolismo , Vanadatos/farmacologia , delta CateninaRESUMO
Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.
Assuntos
Proteínas Tirosina Fosfatases/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Comunicação Celular/fisiologia , Dimerização , Humanos , Modelos Animais , Fosforilação , Receptores Proteína Tirosina Quinases/química , Especificidade por SubstratoRESUMO
Src-family kinase expression was measured in 52 human mammary tumor (T) specimens compared with non-tumor (NT) tissue from the same patient by enzymatic assays employing a Src-kinase family-specific peptide substrate and by immunoblotting with an antibody recognizing the Src-family kinases Src, Fyn, and Yes. In the T specimens, the mean enzymatic activity was moderately elevated (T: 160 fmol ATP min-1 mg-1; NT: 115 fmol ATP min-1 mg-1) with 25 tumor samples having higher activity than the corresponding NT tissue, 17 having lower activity, and no activity detectable in ten T/NT pairs. Immunoblotting revealed clearly elevated expression in 25 tumor tissues and no differences or expression below the detection limit in the remaining T/NT pairs. The data are in agreement with a possible role of Src-family kinases for the biology of mammary carcinoma.
Assuntos
Neoplasias da Mama/enzimologia , Mama/enzimologia , Quinases da Família src/metabolismo , Idoso , Sequência de Aminoácidos , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Dados de Sequência MolecularRESUMO
Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.
Assuntos
Células Epiteliais/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases , Receptor trkA/fisiologia , Transdução de Sinais/fisiologia , Células 3T3 , Animais , Linhagem Celular , Epididimo/citologia , Células Epiteliais/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptor trkA/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Domínios de Homologia de srcRESUMO
Several studies have suggested that morphogenesis and patterning in hydra are regulated through pathways involving protein kinase C (PKC). Nevertheless, the complete signal system for regeneration in hydra is still not completely understood. Using inhibitors of different signalling pathways we are dissecting this system. We found that sphingosine (2 microM), staurosporine (0.1 microM), PP1/AGL1872 (1 microM) and H7 (25 microM) were able to inhibit head but not foot regeneration. The inhibition was reversible. When the inhibitor was replaced with hydra medium the animals continue their regeneration in a normal way. The exception was PP1/AGL1872, in this case the animals regenerated only one or two tentacles. These results imply that head and foot regeneration are independent processes and they are not directly related as has been proposed. Sphingosine and PP1/AGL1872 inhibit the transcription of ks1, an early regeneration gene, at 24 and 48 h of treatment. Sphingosine 2 microM arrested the cells on the G1 phase of the cell cycle, but 1 microM of PP1/AGL1872 did not. The regeneration was not affected if the animals were exposed to inhibitors of human growth factor receptors. We propose that head regeneration in hydra may be regulated at least by two pathways, one going through PKC and the other through Src. The first pathway could be related to cellular proliferation and the second one to cellular differentiation.
Assuntos
Proteínas de Transporte/farmacologia , Hydra/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Benzoquinonas , Ciclo Celular/efeitos dos fármacos , Humanos , Hydra/efeitos dos fármacos , Lactamas Macrocíclicas , Proteína Quinase C/antagonistas & inibidores , Quinonas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Rifabutina/análogos & derivados , Esfingosina/farmacologia , Estaurosporina/farmacologiaRESUMO
G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.
Assuntos
Bradicinina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Receptor Cross-Talk , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , HumanosAssuntos
Receptores ErbB/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Receptores ErbB/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos da radiação , Elongação Traducional da Cadeia Peptídica/efeitos da radiação , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/efeitos da radiação , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Members of the structurally diverse family of beta-carbolines have previously been shown to exhibit a wide range of biological activities. A novel synthetic strategy for generation of beta-carbolines was developed, allowing imido-beta-carbolines to be created in three steps from known compounds. The compounds were screened for inhibition of platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation in Swiss 3T3 fibroblasts. A number of the newly synthesized beta-carbolines with moderate to potent inhibitory activity were revealed. The most active derivative, 2,3-dihydro-8,9-dimethoxy-5-(2-methylphenyl)-1H,6H-pyrrolo[3, 4-c]pyrido¿3,4-bindole-1,3-dione 2ee, inhibited purified PDGF receptor kinase and PDGF-receptor autophosphorylation in intact cells with IC(50) values of 0.4 and 2.6 microM, respectively. Dione 2ee also inhibited PDGF-stimulated DNA synthesis in Swiss 3T3 fibroblasts with an IC(50) of 3.2 microM. The compound had no effect on Src or epidermal growth factor (EGF) receptor kinase activity and a six-seven-fold higher IC(50) for inhibition of basic fibroblast growth factor (bFGF)-stimulated tyrosine phosphorylation or Kit/stem cell factor (SCF) receptor autophosphorylation, indicating a reasonable extent of kinase specificity. Thus, beta-carbolines present a new lead of tyrosine kinase inhibitors with the capacity to selectively interfere with PDGF receptor signal transduction and PDGF-dependent cell growth.