Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinasemediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies. [Cancer Res 2009;69(14):5987-95]
Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.Lactococcus lactis is a gram-positive lactic acid bacterium used for the manufacture of fermented dairy products (2). The milk fermentation process is susceptible to infection by bacteriophages found in raw milk (3,19,(32)(33)(34) or by induction of prophages from lysogenic starter strains (19). The phage infection results in lysis of the bacteria, leading to production delays, variations in the taste and texture of the products, or even complete failure of fermentation. To minimize economic losses by phage infections, a variety of precautions are used (35,36). Lactococcal phages fall into three prevalent groups of DNA homology, 936-, c2-and P335-like phages (32-34). Characteristics of these phages include a double-stranded DNA genome and a long noncontractile tail. The 936 and P335 groups have a small isometric head, while members of the c2 group have a prolate head.We describe here the generation of phage-neutralizing monoclonal single-domain antibody fragments (V H H) derived from cameloid heavy-chain antibodies. In the blood of Camelidae, a high proportion of the immunoglobulins consists of homodimers of only heavy chains, devoid of light chains (17). As described in this and other papers, it is possible to elicit good immune response in camelids against complex protein mixtures, phages, or even whole organisms (26). Genes encoding V H H fragments that bind to these complex protein mixtures can be selected easily. In such libraries of binders, there is a high probability of finding V H Hs that block essential biological processes, mainly because of the long CDR3, which can block active centers (27).It was demonstrated that after immunization of a llama with lactococcal bacteriophage p2 (936 group) the fraction of heavy-chain antibodies contained about 10-fold higher neutralizing activity than conventional antibodies. We generated a phage display library (31, 3...
Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET. Receptor tyrosine kinases (RTKs)2 are key molecules for the recognition and transduction of external signals across the plasma membrane. Thus, RTKs play an essential role in regulating fundamental cellular and biological processes in the cell, such as cell proliferation, cell cycle, cell migration, metabolism, and survival (1). Once stimulated by their cognate ligands, activation of the kinase domain leads to trans-phosphorylation of specific tyrosine residues within the activation segment of the kinase domain and the C-terminal tail. These phosphotyrosine residues serve as docking sites for adaptor and signaling proteins that contain Src homology domain 2 (SH2) or phosphotyrosine binding (PTB) domains triggering the activation of intracellular signaling cascades (1, 2). The selective and specific recognition of short peptide motifs in RTKs by proteins containing modular interaction domains has been an area of intense study over recent years in the kinase signaling field (3). However, whether RTKs are able to directly phosphorylate and activate downstream targets and whether mechanisms in trans by cytoplasmic kinases can regulate activation and signaling by RTKs remain to be evaluated.RET (rearranged during transfection) is an RTK expressed and required during early development for the formation of neural crest-derived lineages, kidney organogenesis, and spermatogenesis (4 -6). To date, a family of glial cell-derived neurotrophic factor (GDNF) ligands, which includes GDNF, AR...
A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.
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