Bailie et al. [In Vitro Cell Dev. Biol. (1992) 28A, 621-624] reported that primary cultures of rat hepatocytes possess low affinity binding sites for nerve growth factor (NGF). NGF treatment of primary cultures of rat hepatocytes with a maximally effective concentration of NGF (20 ng/ml, 0.8 nM) caused acute phasic activation of Raf-1 and p42MAPkinase, and a smaller sustained activation of B-Raf. The transient increase in Raf-1 and p42MAPkinase activity returned to baseline within ~ 30 min. NGF treatment of hepatocytes did not induce expression of cyclin dependent kinase (cdk) inhibitor proteins, but instead stimulated cdk2 activity and increased [3H]thymidine incorporation into DNA. In contrast to hepatocytes, NGF treatment of PC12 pheochromocytoma cells caused large sustained activations of B-Raf and p42MAPkinase, and a lower phasic activation of Raf-1. The sustained activations of B-Raf and p42MAPkinase were for more than 5 h. Treatment of PC12 cells with NGF increased p21Cip1/WAF-1 expression, reduced cdk2 activity and inhibited DNA synthesis, the opposite to the effects of NGF treatment of hepatocytes. However when p42MAPkinase was chronically activated in hepatocytes, via infection with an inducible oestrogen receptor-Raf-1 fusion protein, expression of p21Cip-1/WAF1 and p16INK4a cdk inhibitor proteins increased, cdk2 activity decreased, and DNA synthesis decreased. Equally, treatment of hepatocytes with 50 mM ethanol elevated the basal activity of p42MAPkinase and temporally extended the ability of NGF treatment to activate p42MAPkinase. Ethanol and NGF co-treatment increased expression of p21Cip-1/WAF1 and p16INK4a cdk inhibitor proteins and decreased hepatocyte DNA synthesis. These data demonstrate that NGF can cause either acute/phasic or sustained activation of the MAP kinase cascade in different cell types. Acute activation of the MAP kinase cascade correlated with increased DNA synthesis. In contrast, sustained activation of the MAP kinase cascade correlated with increased expression of cdk inhibitor proteins, a reduction in cdk activity, and an inhibition of DNA synthesis. These data suggest a general mechanism exists where acute activation of the MAP kinase cascade promotes G1 progression/S phase entry and that chronic activation of the MAP kinase cascade inhibits this process.

Regulation of protein phosphorylation is a primary cellular signaling mechanism. Many cellular responses to internal and external events are mitigated by protein kinase signaling cascades. Dysfunction of protein kinase activity has been linked to a variety of human pathologies, in the areas of cancer, inflammation, metabolism, cell cycle, apoptosis, as well as cardiovascular, neurodegenerative and autoimmune diseases [1-3]. As such, there is an important need for protein kinase activity detection methodologies for researchers engaged in Drug Discovery. A number of different technologies have been employed for the measurement of protein kinase activity, including radioactive methods, luminescent methods, and fluorescent methods. More recently, Homogeneous Time Resolved Fluorescence technology (HTRF®), based on the principle of time-resolved fluorescent resonance energy transfer (TR-FRET), has been developed and applied for the measurement of protein kinase activity in vitro. This technology note describes the development of an HTRF® assay for detection of Syk enzyme activity in a format consistent with the requirements of High-Throughput Screening (HTS) campaigns currently used in drug discovery.

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