The cells that make up biological organisms comprise numerous components. Many are proteins, the sequence of which is encoded genetically. Proteins carry small, localized, electrostatic charges that force each molecule to adopt a three-dimensional configuration specific for that protein. This configuration determines its function. Several mechanisms have evolved by which the configuration and function of proteins can be altered. Some are irreversible, resulting in permanent structural and functional change. Others involve temporary reactions catalysed by opposing enzyme classes. These permit reversible changes in cell functioning and potentially operate as 'on/off' or 'more/less' switches. Protein phosphorylation and dephosphoryla-tion are the most common reversible post-translational modifications. They are used ubiquitously in biological systems to alter cell functioning in response to signals arising from the extracellular environment.
Phosphorylative regulation involves a complex series of events (Fig. 1). A protein is phos-phorylated when the terminal phosphate is transferred from adenosine triphosphate (ATP) to a hydroxyl group within the protein. This requires, first, that ATP be complexed to a divalent cation, usually magnesium (Mg2+). A second requirement is the involvement of an enzyme, a protein kinase, which catalyses the phosphate transfer. The protein is dephosphorylated when the phosphate molecule is removed via hydrolysis, a reaction catalysed by enzymes known as protein phosphatases. As phosphate molecules are negatively charged, their addition to, or deletion from, a protein alters the distribution of electrostatic forces between that section of the protein and other sections. This can induce a change in the protein's three-dimensional configuration and modify its functional state. Phosphorylative changes are typically rapid and reversible, persisting only until a further enzyme reverses the initial change. They can be maintained, however, extending and/or amplifying a response to the signal initially responsible for enzyme activation.
The brain is a rich source of kinases, phosphatases and substrate proteins, many of which are unique to neural tissue,204 and phosphorylation is implicated in many brain processes and mediates numerous nerve cell responses (reviewed in refs. 68 and 194). Indeed, the number of brain proteins regulated by phosphorylation appears to increase at the same rate as new proteins are described and it is difficult to locate a well-characterized protein not regulated either directly or indirectly by this process. Most of the events that are fundamental to normal operations in the brain, and to synaptic transmission in particular, are regulated by phosphorylation. For example, it seems likely that all voltage-gated ion channels may be subject to some form of phosphorylative regulation (reviewed in ref. 135). In some cases phosphorylation plays a mediatory role, being an obligatory step in the opening or closing of a channel. More commonly, phosphorylation is modulatory, altering channel sensitivity to factors responsible for opening or closing the channel, altering the time course of channel opening, and so forth. Neurotrans-mitter synthesis typically also proceeds only following phosphorylation of rate-limiting enzymes, and several Ca2+-dependent kinases and phosphatases act to both mediate and modulate the process of neurotransmitter release.160 Postsynaptic effector molecules strongly implicated in important functional changes in the brain are similarly regulated by phosphorylation. These include many ligand-gated ion channels and G-protein-coupled receptors (reviewed in ref. 101). The consequences of phosphorylation vary with each particular system but include desensitisation, internalisation and activation.
While changes in acute pre- and post-synaptic events may underlie permanent information storage, most theorists argue that they are more likely to maintain transient changes in synaptic efficacy, with other, more stable, mechanisms being responsible for permanent storage.2 6 Consistent with this, formation of permanent memories depends on synthesis of new proteins208 and changes in gene expression.45 Phosphorylation and dephosphorylation are intimately involved in initiating transcription and translation, with over 20 critical proteins being regulated by these processes.212 The same processes also regulate the fibrillar cytoskeletal proteins that interact to dynamically determine the shape of a cell at any given time.150 Phosphorylative
Figure 1. Schematic representation of the processes of protein phosphorylation and dephosphorylation.
regulation of one cytoskeletal element, microtubule-associated protein-2 (MAP2), has been proposed to play a particularly important role in memory formation.97 MAP2 phosphorylation may act as a trigger for the incorporation of new cytoskeletal proteins, perhaps 'tagging' recently activated sites to allow specificity of plastic changes. This may, in turn, lead to rearrangement and enlargement of critical synaptic areas.216
Finally, numerous enzymes responsible for regulating all aspects of brain functioning are controlled, either directly or indirectly, by phosphorylation and dephosphorylation. Indeed, many kinases and phosphatases, in addition to being regulated by other means, are themselves regulated by phosphorylation.131 Others are subject to regulation by specific proteins, some of which are inhibitory and some of which target the enzymes to specific cellular locations.207 Many of these proteins are, in turn, regulated by phosphorylation and dephosphorylation. Complex sequences of phosphorylative events often act either competitively, additively or syn-ergistically to bring about extremely subtle, rapid, and precise changes in cell functioning. These may be transient or persist indefinitely according to the requirements of the organ-ism.204
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