RESUMO
Basal cell adhesion molecule (BCAM), known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobulin superfamily membrane protein that acts as a laminin α5 receptor. The high affinity of BCAM/LU for laminin α5 is thought to contribute to the pathogenesis of sickle red blood cells and to various developmental processes. However, the function of BCAM in carcinogenesis is poorly understood. Based on microarray expression analysis, we found that BCAM was one of the target genes of the oncogenic 14-3-3ß-FBI1/Akirin2 complex, which acts as a transcriptional repressor and suppresses MAPK phosphatase-1 gene expression. To elucidate the detailed function of BCAM in malignant tumors, we established BCAM-expressing hepatoma K2 cells. These cells lost the malignant characteristics of parental cells, such as anchorage-independent growth, migration, invasion, and tumorigenicity. Moreover, luciferase reporter assays and chromatin immunoprecipitation analysis revealed that the 14-3-3ß-FBI1/Akirin2 complex bound to the BCAM promoter and repressed transcription. Thus, these data indicate that BCAM is a suppressive oncoprotein, and that FBI1/Akirin2 is involved in tumorigenicity and metastasis of hepatoma through the downregulation of suppressive oncogenes.
Assuntos
Proteínas 14-3-3/genética , Sistema do Grupo Sanguíneo Lutheran/genética , Proteínas Oncogênicas/genética , Proteínas Repressoras/genética , Proteínas 14-3-3/metabolismo , Animais , Northern Blotting , Western Blotting , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação Neoplásica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema do Grupo Sanguíneo Lutheran/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Transplante HeterólogoRESUMO
No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts.
Assuntos
Quinase 5 Dependente de Ciclina/análise , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Animais , Linhagem Celular , Quinase 1 do Ponto de Checagem , Chlorocebus aethiops , Quinase 5 Dependente de Ciclina/genética , Humanos , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The kinase signaling cascades related to mitogen- and stress-activated protein kinase-1 and -2 (MSK1 and MSK2, respectively) are attractive targets for pharmaceutical intervention, especially for neural injury. Therefore, we have developed a high throughput and cost-effective detection platform for measuring selective activity of MSK1/MSK2 in cells. Through the serial monitoring of both the p38 mitogen-activated protein kinase (stress-activated protein kinase 2B)-MSK1/MSK2- cyclic AMP response element binding protein (CREB)/activating transcription factor 1 (ATF1) pathway and the p38-mammalian heat shock protein 27 (Hsp27) pathway in HeLa cells treated with anisomycin, two selective MSK1 inhibitors showed inhibition of CREB (Ser-133) and ATF1 (Ser-63) phosphorylation and no interference with Hsp-27 phosphorylation (Ser-82). On the other hand, the p38 inhibitor SB-220025 showed equipotent inhibition of CREB/ATF1 and Hsp27 phosphorylation. This study demonstrated that the specific inhibition of a target kinase could be subsequently monitored by a secondary assay that measures the intervention arising from the modulation of off-target kinases. Our established system is applicable to inhibitor screening and drug discovery related to MSK1/MSK2.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Anisomicina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Fosforilação , Plasmídeos/genética , Inibidores da Síntese de Proteínas/farmacologia , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery.
