Pyk2 Kinase Inhibitor Screening Assay Kit

Intended Use

The Pyk2 Kinase Inhibitor Screening Assay Kit is designed to measure the activities of recombinant catalytic domain of Pyk2 (Proline-rich Tyrosine Kinase 2) for the rapid and sensitive evaluation of inhibitors or activators. The phosphotyrosine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in recombinant "Tyrosine kinase-substrate-1", which is efficiently phosphorylated by Pyk2 in vitro.

Applications of this kit include:
1) Screening inhibitors or activators of recombinant catalytic domain of Pyk2.
2) Detecting the effects of pharmacological agents on recombinant catalytic domain of Pyk2.

This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage

Upon receipt store all components at 4°C.
Don't expose reagents to excessive light.

Performance Characteristics

The Pyk2 Kinase Inhibitor Screening Assay Kit has been shown to detect the kinase activity of recombinant catalytic domain of Pyk2. The assay shows good linearity of sample response.

General Description

Intracellular signal transduction following extracellular ligation by a wide variety of surface molecules involves the activation and tyrosine phosphorylation of protein tyrosine kinases (PTKs). Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases and tyrosine kinases, is a critical regulatory mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function.
The focal adhesion PTK family consists of the focal adhesion kinase (FAK) and the Pyk2 kinase (also known as RAFTK, CAK-beta and CADTK). Pyk2 kinase can be activated in response to various stimuli, such as TNF-alpha, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic acid, in addition of bradykinin, a neuropeptide hormone that binds to a G protein-coupled receptor and in turn stimulates phosphatidylinositol hydrolysis. Pyk2 kinase is expressed mainly in the central nervous system and in cells derived from hematopoietic lineages, while FAK is widely expressed in various tissues and links transmembrane integrin receptors to intracellular pathways.
Lev et al showed that the Pyk2 protein undergoes rapid tyrosine phosphorylation in response to various stimuli, Pyk2 is also tyrosine phosphorylated following activation of the nicotinic acetylcholine receptor, by membrane depolarization, and by treatment of cells with a calcium ionophore. It has been proposed that Pyk2 may represent an important signaling intermediate between neuropeptide activated receptors or neurotransmitters that increase calcium flux and the downstream signals that regulate neuronal activity. Pyk2 may also provide a mechanism for a variety of short- and long-term calcium-dependent signaling events in the nervous system.
Activation of Pyk2 leads to the modulation of ion channel function and activation of the MAP kinase signaling pathway including p38 cacade. Furthermore, overexpression of Pyk2 led to activation of JNK, and a dominant-negative mutant of Pyk2 interfered with ultraviolet light- or osmotic shock-induced activation of JNK. Pyk2 represents a cell type-specific, stress-sensitive mediator of the JNK signaling pathway.

Measurement of Pyk2 Kinase activity

The protocol generally regarded as most sensitive for the quantitative measurement of Pyk2 kinase activity involves incubation of the Pyk2 kinase sample with substrate, either a natural or synthetic polypeptide (such as poly[Glu,Tyr]4:1), in the presence of Mg2+, Mn2+ and 32P-labeled ATP. The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to precipitate the radiolabeled product. The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is labor-intensive, generates hazardous radioactive waste, and depends on a radioisotope of short half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The Pyk2 Kinase Inhibitor Screening Assay Kit uses a horseradish peroxidase coupled anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-wells ELISA format. This assay provides a non-isotopic, sensitive and specific method to detect kinase activity of recombinant Pyk2 catalytic domain.