For many proteins, a change in charge and conformation due to the addition or removal of phosphate groups results in alterations in their intrinsic functional activity. Although proteins are covalently modified in many other ways—for example, by ADP ribosylation, acylation (acetylation, myristoylation), carboxymethylation, and glycosylation—none of these mechanisms appear to be as widespread and readily subject to regulation by physiological stimuli as phosphorylation. Indeed, protein phosphorylation/dephosphorylation represents a pathway of fundamental importance in the chemistry of biological regulation (see Nestler et al. 2001). Virtually all types of extracellular signals are known to produce many of their diverse physiological effects by regulating the state of phosphorylation of specific proteins in the cells that they target.
The phosphate group provides an unwieldy negative charge that often interacts with the catalytic and other regions of enzymes. The addition of a phosphate often results in conformational changes in proteins. In the case of enzymes, this change may increase (more commonly) or decrease the affinity of an enzyme for its substrate. Thus, phosphorylation may result in a change in kinase activity, a change in phosphatase activity, or the marking of a protein for cleavage by proteases. The catalytic activity of an enzyme can be switched on or off, or an ion channel can be opened or closed. For many other proteins, phosphorylation-induced changes in charge and conformation result in alterations in the affinity of the proteins for other molecules. For example, phosphorylation alters the affinity of numerous enzymes for their cofactors and end-product inhibitors, phosphorylation of receptors can alter their affinity for G proteins, and phosphorylation of some nuclear transcription factors alters their DNA-binding properties. Therefore, phosphorylation can produce varied effects on cellular physiology and ultimately can have major behavioral manifestations.
Protein kinases are classified by the residues that they phosphorylate, with the two major classes being serine/threonine kinases and tyrosine kinases. Most phosphorylation (>95%) of proteins occurs on serine residues, a small amount (about 3%-4%) on threonine residues, and very little (0.1%) on tyrosine residues (but, as discussed earlier, the tyrosine kinases can be very important for neurotrophic signaling). In all cases, the kinases catalyze the transfer of the terminal (7) phosphate group of ATP to the hydroxyl moiety in the respective amino acid residue, a process that requires Mg2+. Within cells, protein kinases often form sequential pathways, whereby one kinase phosphorylates/activates another, which phosphorylates/activates another kinase, and so forth. In this manner, signals can be propagated within cells, allowing ample opportunity (see below) for the signal to be altered by other intracellular signals, often in a cell type-specific manner, allowing for considerable "fine-tuning."
Although clearly playing critical roles in modulating the function of proteins by catalyzing the cleavage of the phosphoester bond, protein phosphatases have not been as extensively studied as kinases. In the CNS, phosphatases often function as a molecular "off switch," thereby decreasing the activity of enzymes and the intracellular signaling pathways they control. However, it is clear that protein phosphatases are much more than simple off switches. Thus, in an elegant series of studies, Greengard and associates demonstrated that a major CNS phosphoprotein, known by the acronym DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa), brings about many of its long-term neuroplastic effects by regulating the activity of a protein phosphatase (protein phosphatase-1; PP-1) (for a review, see Greengard 2001a). Thus, they demonstrated that the DARPP-32/PP-1 pathway integrates information from a variety of neurotransmitters and produces a coordinated response involving numerous downstream physiological effectors. DARPP-32 phosphorylation by PKA is regulated by the actions of various neurotransmitters, principally dopamine acting at D1 receptors but also a variety of other neurotransmitters (Greengard 2001b; Nestler et al. 2001). Phospho-DARPP-32, by inhibiting the activity of PP-1, acts in a synergistic manner with different protein kinases (primarily PKA and PKC) to increase the level of phosphorylation of various downstream effector proteins and thereby long-term neuronal adaptations that have also been implicated in the actions of drugs of abuse and antidepressants (Greengard 2001b; Nestler et al. 2001).
While propagation of signals may be very immediate, even short-term phosphorylation of many types of proteins can have long-term effects, resulting in "molecular and cellular memory." Indeed, various forms of learning and memory are known to be regulated, in large part, by phosphorylation events. Short-term memory may involve the phosphorylation of presynaptic or postsynaptic proteins in response to synaptic activity, a process that results in transient facilitation or inhibition of synaptic transmission. Long-term memory may involve phosphorylation of proteins that play a role in the regulation of gene expression, which would result in more permanent modifications of synaptic transmission, potentially via structural brain changes (Malenka and Nicoll 1999). As discussed, long-term potentiation, one of the most extensively studied models of learning and memory, is believed to be initiated through short-term changes in Ca2+-dependent protein phosphorylation and maintained by long-term changes in gene expression. There is also growing appreciation that protein phosphatases play a critical role in the extinction of memory. Thus, abundant data now suggest that rather than representing a passive process, "forgetting" is more an active process of memory erasure (discussed in Genoux et al. 2002). In an elegant series of behavioral studies using transgenic mice, Genoux et al. (2002) provided strong evidence that PP-1 is involved in forgetting rather than in preventing the encoding of memory. Although the precise mechanisms by which PP-1 brings about these effects remain to be fully elucidated, these investigators postulate that CaMKII and the GluR1 subunit of the AMPA receptor play important roles. These findings may have major implications for the ultimate development of agents that could be used to facilitate the erasure of traumatic memories—for example, in PTSD.
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