Intracellular signal transduction following receptor binding by a wide variety of extracellular
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-β, and CADTK). Pyk2 kinase can be activated in response to various stimuli,
such as TNF-α, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic
acid, and by the addition of bradykinin. Pyk2 kinase is expressed mainly in the central nervous system
and in cells derived from hematopoietic lineages (1-3), while FAK is ubiquitously 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
(4). 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 (2, 4).
Activation of Pyk2 leads to the modulation of ion channel function and activation of the MAP kinase
signaling pathway including the p38 cascade (5-7). 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. Thus, Pyk2 represents a cell type-specific, stress-sensitive mediator of
the JNK signaling pathway (8).
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.
The CycLex® Pyk2 Kinase Assay/Inhibitor Screening Kit uses a horseradish peroxidase coupled
anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-well ELISA format. This assay
provides a non-isotopic, sensitive and specific method to detect the effects of inhibitors and activators on
Pyk2 kinase activity.