Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent development of recombinant antibody technology has enabled fusion of recombinant antibody fragment with fluorescent proteins for various applications such as flow cytometry, fluorescence immunoassay, and fluorescent microscopy. In this study, we generated various forms of green fluorescence protein (EGFP)-fused anti-c-Met antibody fragment. Among these fusion proteins, EGFP fusion to the light chain showed high expression in a soluble form of protein in E. coli, and high binding activity to c-Met. A feasibility of the constructs was further examined by replacing the Fab gene by a Fab library of catalytic subunit of protein kinase A (PKA) to construct the Fab library in EGFP fused form. We also constructed the conventional Fab library. After a series of biopanning, we found that the binding capability of EGFP-anti-PKA Fab was comparable with anti-PKA Fab. Sequence analysis of the selected clones showed > or =99% identity in amino acid sequence and shared the same CDR sequence. These results demonstrate that EGFP fusion to the light chain using our vector system does not influence the selection of reactive Fab and that this vector system is useful for EGFP fusion to Fab to develop a one-step detection system.
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PMID:Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment. 1567 5

Following organ injury, morphogenic epithelial responses can vary depending on local cell density. In the present study, the role of cell confluence in determining the responsiveness of renal epithelial cells to the dedifferentiating morphogenic signals of hepatocyte growth factor (HGF) was examined. Increasing confluence resulted in a greater tendency of cells to organize into epithelial tubes and a significant decrease in migratory responsiveness to HGF. Analysis of downstream signaling revealed that the HGF receptor c-Met was equally activated in confluent and nonconfluent cells following HGF stimulation but that phosphoinositide 3-kinase-dependent activation of Akt and Rac were selectively diminished in confluent cells. In nonconfluent cells treated with HGF, the high level of Akt activation resulted in inhibitory phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) and increased beta-catenin nuclear signaling. In contrast, confluent cells, in which HGF-stimulated Akt activation was diminished, displayed less inhibitory phosphorylation of GSK-3beta and less nuclear signaling by beta-catenin. Overexpression of beta-catenin (SA), which cannot be phosphorylated by GSK-3beta and targeted for ubiquitination, significantly increased migration in fully confluent cells. Thus, cells maintained at high confluence selectively downregulate signaling events such as Rac activation and beta-catenin-dependent transcription that would otherwise promote cell dedifferentiation and migration.
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PMID:Cell confluence regulates hepatocyte growth factor-stimulated cell morphogenesis in a beta-catenin-dependent manner. 1703 Jun 2

The Wnt/beta-catenin pathway plays an important role in embryonic liver development, morphogenesis, and organogenesis. Here, we report on the activation of beta-catenin during early postnatal liver growth. Modulation of beta-catenin expression was studied in CD-1 mice livers over a time course of 0 to 30 postnatal days (PD) and 3 mo. Increases in total and active beta-catenin were observed in developing livers from PD 5 to 20. A concomitant increase in the beta-catenin-transcription factor (TCF) complex along with nuclear and cytoplasmic beta-catenin was also evident, which coincided with ongoing hepatocyte proliferation by PCNA immunohistochemistry. This activation of beta-catenin was multifactorial, including cyclical inhibition of glycogen synthase kinase-3beta, suppression of casein kinase-IIalpha, and a transient increase in beta-catenin gene expression. Coprecipitation experiments revealed the formation of the beta-catenin-cadherin complex at PD 5, whereas adequate beta-catenin-c-Met complex at the hepatocyte membrane did not form until PD 20, which might be contributing to the free beta-catenin pool during early postnatal growth. Furthermore, beta-catenin liver-specific knockout mice exhibited smaller livers at PD 30, secondary to diminished hepatocyte proliferation. These data indicate that the activation of beta-catenin is critical for early postnatal liver growth and development.
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PMID:beta-Catenin is critical for early postnatal liver growth. 1733 75

It has been proposed that the metastasis suppressor CD82/KAI-1, which is a member of the tetraspanin superfamily, regulates biological activity by associating with cell surface receptors or proteins. We show a novel association between CD82 and the hepatocyte growth factor (HGF) receptor c-Met. Although ectopic expression of CD82 in nonsmall cell lung carcinoma cells did not affect the tyrosine phosphorylation of c-Met, these cells showed significant suppression of HGF-induced lamellipodial protrusion and cell migration. CD82 selectively attenuated c-Met signaling via the Ras-Cdc42/Rac and the phosphatidylinositol 3-kinase/Cdc42/Rac pathways. In contrast, another c-Met signaling pathway that involves phosphatidylinositol 3-kinase/Akt and phosphatidylinositol 3-kinase/mitogen activated protein kinase was not affected by CD82. Signaling adapter proteins for c-Met, such as Grb2 and p85, exhibited reduced association with c-Met in cells that ectopically expressed CD82. These results indicate that the CD82-c-Met complex inhibits HGF-induced cancer cell migration by the inactivation of small GTP-binding proteins of the Rho family via c-Met adapter proteins.
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PMID:Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration. 1762 32

In recent years, the T1799A B-type Raf kinase (BRAF) mutation in thyroid cancer has received enthusiastic investigation, and significant progress has been made toward understanding its tumorigenic role and clinical significance. Among various thyroid tumors, this mutation occurs uniquely in papillary thyroid cancer (PTC), the most common endocrine malignancy, and some apparently PTC-derived anaplastic thyroid cancers. Many studies have found this mutation to be associated with those clinicopathological characteristics of PTC that are conventionally known to predict tumor progression and recurrence, including, for example, old patient age, extrathyroidal invasion, lymph node metastasis, and advanced tumor stages. Direct association of BRAF mutation with the clinical progression, recurrence, and treatment failure of PTC has also been demonstrated. The BRAF mutation has even been correlated with PTC recurrence in patients with conventionally low-risk clinicopathological factors. Some molecular mechanisms determining BRAF mutation-promoted progression and the aggressiveness of PTC have recently been uncovered. These include the down-regulation of major tumor suppressor genes and thyroid iodide-metabolizing genes and the up-regulation of cancer-promoting molecules, such as vascular endothelial growth factor, matrix metalloproteinases, nuclear transcription factor kappaB, and c-Met. Thus, BRAF mutation represents a novel indicator of the progression and aggressiveness of PTC. Significant advances have also occurred in the preclinical testing of new therapeutic strategies targeting the MAPK pathway aberrantly activated by BRAF mutation and other related mutations. New mitogen extracellular kinase (MEK) inhibitors developed recently are particularly promising therapeutic agents for thyroid cancer. With these advances, it has become clearer that BRAF mutation will likely have significant impact on the clinical management of PTC.
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PMID:BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. 1794 Jan 85

Activity of receptor tyrosine kinases (RTK) in colorectal cancer (CRC) is associated with enhanced tumor growth and a poorer prognosis. In addition, cyclooxygenase-2 (COX-2) expression contributes to tumor growth and invasion. COX-2 inhibitors exhibit important anticarcinogenic potential against CRC, but the molecular mechanism underlying this effect and the relation with RTK signaling remain the subject of intense research effort. Therefore, the rapid effects of COX-2 inhibition in CRC on the complement of all cellular kinases were investigated using a kinase substrate peptide array, Western blotting, transfection, small interfering RNA assays, and CRC cell lines. The resulting alterations in the kinome profile revealed that celecoxib, a selective COX-2 inhibitor, impairs phosphorylation of substrates for the RTKs c-Met and insulin-like growth factor receptor, resulting in decreased downstream signaling. The decrease in c-Met activation is accompanied with an increase in glycogen synthase kinase 3beta kinase activity together with a rapid increase in phosphorylation of beta-catenin. In agreement, a significant reduction of beta-catenin-T-cell factor-dependent transcription is observed both with celecoxib and selective inhibition of c-Met phosphorylation by small molecules. Hence, corepression of c-Met-related and beta-catenin-related oncogenic signal transduction seems a major effector of celecoxib in CRC, which provides a rationale to use c-Met inhibitors and celecoxib analogous to target c-Met and Wnt signaling in a therapeutic setting for patients with CRC.
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PMID:Cyclooxygenase-2 inhibition inhibits c-Met kinase activity and Wnt activity in colon cancer. 1828 98

Growth factors accelerate G0 to S progression in the cell cycle, however, the roles of growth factors in other cell cycle phases are largely unknown. Here, we show that treatment of HeLa cells with hepatocyte growth factor (HGF) at G2 phase induced the G2/M transition delay as evidenced by FACS analysis as well as by mitotic index and time-lapse analyses. Growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) also induced G2/M transition delay like HGF. HGF treatment at G2 phase causes a delayed activation of cyclin B1-associated kinase and a diminished nuclear translocation of cyclin B1. Either U0126, a MAPK kinase (MEK) inhibitor, or kinase-dead mutant of ribosomal S6 kinase (RSK) abolished the delay. Additionally, knockdown of RSK1, but not RSK2, with siRNA abrogated the delay, indicating that the extracellular-regulated protein kinase (ERK)-RSK1 mediates the HGF-induced delay. We further found that the delay in G2/M transition of cells expressing oncogenic HGF receptor, M1268T, was abolished by RSK1 knockdown. Intriguingly, we observed that HGF induced chromosomal segregation defects, and depletion of RSK1, but not RSK2, aggravated these chromosomal aberrations. Taken together, the ERK-RSK1 activation by growth factors delays G2/M transition and this might be required to maintain genomic integrity during growth factor stimulation.
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PMID:The ERK-RSK1 activation by growth factors at G2 phase delays cell cycle progression and reduces mitotic aberrations. 1845 Apr 23

Marine alkaloid meridianin G derivatives, substituted on the pyrimidine ring by aryl groups, were evaluated for their kinase inhibitory potencies and their in-vitro antiproliferative activities. The derivatives were tested toward a panel of nine protein kinases (KDR, IGF-1R, c-Met, RET, c-Src, c-Abl, PKA, CDK2/cyclin A, and HER-1) and their in-vitro antiproliferative activities were evaluated toward a human fibroblast primary culture and two human solid cancer cell lines (MCF-7 and PA 1). Despite weak kinase inhibitory potencies, high in-vitro antiproliferative activities were found for compounds 5, 7, 12, and 14, which do not interfere with the PA 1 cell cycle and may be considered as direct cytolysis or apoptosis inducers.
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PMID:In-vitro antiproliferative activities and kinase inhibitory potencies of meridianin derivatives. 1869 90

The irradiated fibroblast-induced response of non-irradiated neighboring cells is called 'radiation-induced bystander effect', but it is unclear in non-irradiated human squamous cell carcinoma (SCC) cells. The present study shows that irradiated fibroblasts promoted the invasive growth of T3M-1 SCC cells, but not their apoptosis, more greatly than non-irradiated fibroblasts, using collagen gel invasion assay, immunohistochemistry and Western blot. The number of irradiated fibroblasts decreased to about 30% of that of non-irradiated fibroblasts, but irradiated fibroblasts increased the growth marker ki-67 display of SCC cells more greatly than non-irradiated fibroblasts. Irradiated fibroblasts did not affect the apoptosis marker ss-DNA expression of SCC cells. Irradiated fibroblasts enhanced the display of the following growth-, invasion- and motility-related molecules in SCC cells more greatly than non-irradiated fibroblasts: c-Met, Ras, mitogen-activated protein kinase (MAPK) cascade (Raf-1, MEK-1 and ERK-1/2), matrix metalloproteinase-1 and -9, laminin 5 and filamin A. Irradiated fibroblasts, but not non-irradiated ones, formed irradiation-induced foci (IRIF) of the genomic instability marker p53-binding protein 1 (53BP1) and expressed transforming growth factor-beta1 (TGF- beta1). Irradiated fibroblasts in turn enabled SCC cells to enhance 53BP1 IRIF formation more extensively than non-irradiated fibroblasts. Finally, effects of irradiated fibroblasts on growth and apoptosis of another HEp-2 SCC cell type were similar to those of T3M-1. These results suggest that irradiated fibroblasts promotes invasion and growth of SCC cells by enhancement of invasive growth-related molecules above through TGF- beta1-mediated bystander mechanism, in which irradiated fibroblast-induced genomic instability of SCC cells may be involved.
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PMID:Irradiated fibroblast-induced bystander effects on invasive growth of squamous cell carcinoma under cancer-stromal cell interaction. 1901 71

The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show approximately 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.
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PMID:Chaperone over-expression in Escherichia coli: apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates. 1903 47


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