Phosphorylation ofproteins by kinases is a central biochemical mechanism to regulate enzyme activity. To become active, kinases have to be first phos-phorylated. This process starts by binding of ATP to an ATP (substrate) binding site of the enzyme. Subsequently, the active site of the enzyme becomes phosphorylated by transfer of energy-rich phosphate. Finally, the activated phosphate group is transferred by the kinase reaction to a target substrate, such as another enzyme or transcription factor, respectively, eventually resulting in a biological response (Figure 2.16).

reaction is competitive, that is, reversible, stochiometric at a 1 :1 relationship and requires higher concentrations of salicylates (>1 mM). The other is an interference with kinase activity by steric interaction with the transfer of the energy-rich phosphate from the ATP-binding site to the active site ofthe kinase, for example, after binding to another binding site, such as an arginine (by analogy with the argininen20 in COX-1). This reaction is noncompetitive and nonstochiometric and probably requires lower concentrations of salicylates (<1 mM).

Inhibition of kinases by salicylates is both a simple and comprehensive explanation for the diversity of salicylate actions on cell function at higher concentrations. The consequences ofkinase inhibition for cell function are then determined by the function ofthe particular phosphorylated target protein or transcription factor, respectively. Kinase activity might be differentially modified by salicy-lates in intact cells as opposed to cell homoge-nates [151], perhaps because of the high millimolar concentrations, which can be used in cell homo-genates but not in intact cells or even tissues in vivo. Myriads of kinases can be inhibited by salicylates and the biological significance of this action is difficult to predict [152].

Nonselective inhibition ofkinases as a mode of action of salicylates was first hypothesized by Frantz and O'Neill [153]. These authors showed that sodium salicylate caused a concentration-dependent, nonselective inhibition of a variety of different transcription factors at millimolar concentrations. This effect was probably due to nonselective inhibition of cellular kinases since an apparently identical response at the same concentrations was also seen in a cellular kinase preparation (Figure 2.17). The hypothesis was that activation of these transcription factors required phosphorylation by ki-nases that were prevented by salicylates.

Inhibition of ATP Bindingto Kinases Yin etal. [154] suggested another more specific mechanism, namely a competitive and specific inhibition by salicylates of ATP-binding to the inhibitory ki-nase-b (IKK-b). This effect was reversible and could be antagonized by increasing the ATP concentra-

Principally, salicylates can interact with these events at several levels: one is the inhibition of substrate (ATP) binding because of chemical analogies between the ring structures of adenine and salicylic acid. This

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