Dataset Information

Light-regulated sampling of protein tyrosine kinase activity.

ABSTRACT: Protein tyrosine kinases serve as key mediators of signaling pathways, biochemical highways that control various aspects of cell behavior. Although fluorescent reporters of tyrosine kinases have been described, these species can suffer immediate phosphorylation upon exposure to the cellular milieu, thereby hindering a detailed analysis of kinase activity as a function of the cell cycle or exposure to environmental stimuli. The first example of a light-regulated tyrosine kinase reporter is described herein, which allows the investigator to control when kinase activity is sampled. In addition, the set of sensors created in this study contain different fluorophores, each with its own unique photophysical properties, thereby furnishing an array of choices that can be used in combination with other intracellular probes.

SUBMITTER: Wang Q

PROVIDER: S-EPMC2660521 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Light-regulated sampling of protein tyrosine kinase activity.

Wang Qunzhao Q Dai Zhaohua Z Cahill Sean M SM Blumenstein Michael M Lawrence David S DS

Journal of the American Chemical Society 20061101 43

Protein tyrosine kinases serve as key mediators of signaling pathways, biochemical highways that control various aspects of cell behavior. Although fluorescent reporters of tyrosine kinases have been described, these species can suffer immediate phosphorylation upon exposure to the cellular milieu, thereby hindering a detailed analysis of kinase activity as a function of the cell cycle or exposure to environmental stimuli. The first example of a light-regulated tyrosine kinase reporter is descri ...[more]

PMID: 17061870

Similar Datasets

Project description:BackgroundProtein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other.MethodologyWe used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles.Results and conclusionReproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.

Dataset Information

Light-regulated sampling of protein tyrosine kinase activity.

Publications

Light-regulated sampling of protein tyrosine kinase activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets