BIOLOGICAL OVERVIEW

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II) is one of the major protein kinases coordinating cellular responses to neurotransmitters and hormones. CaM kinase II is so named because of its requirement for calcium-bound calmodulin (CaM) for activation and its ability to phosphorylate and alter the function of a variety of substrates. This discussion will first focus on the effects of CaM kinase II on behavior and learning, and then describe the biochemical events involved. One begins by identifying the target(s) of CaM kinase II.

One target has been identified in Drosophila. Similar defects in both synaptic transmission and associative learning are produced in Drosophila by inhibition of calcium/calmodulin-dependent protein kinase II and mutations in the potassium channel subunit gene ether à go-go (eag), suggesting that EAG is targeted by CaM Kinase in learning. To test whether CaM Kinase influences learning, two behavior assays for learning have been employed: acoustic priming, a measure of nonassociative learning and sensitization, and courtship conditioning, a paradigm known to have characteristics of associative learning.

(1) acoustic priming

Courtship consists of a set of complex stereotyped behaviors, involving olfaction, hearing, vision and locomotion. This behavior was once thought to be completely "hard-wired", but is now known to exhibit significant experience-dependent features. Acoustic priming is an example of such plasticity. Exposure of virgin females to a correct species-specific male courtship song in the presence of wingless courting males (that is, males unable to sing) significantly enhances the speed of subsequently mating. A similar enhancement can be obtained by prestimulating the females with an artificial courtship song and subsequently adding normal winged males. The females appear to retain a memory of the prestimulation and consequently mate faster. This behavior is an example of sensitization: the enhancement of mating speed is the result of stimulation of the female with the male courtship song. The amount and duration of the priming effect appear to be dependent on the same cellular and biochemical mechanisms as other information storage and retrieval processes, since the classic learning mutants dunce, rutabaga and amnesiac are abnormal in this test of acoustic priming. These mutant flies seem to not retain memory of acoustic priming. To test whether learning is altered by an alteration in CaM Kinase activity, a peptide inhibitor of CaM Kinase was expressed from a heat shock promoter, allowing inhibition of CaM Kinase II in the adult female animals subjected to behavioral tests. Although the pathways mediating acoustic priming are intact in flies in which a CaM Kinase inhibitor is expressed, these lines are not normal with respect to their ability to retain the effects of sensitization if delays are introduced between the playing of the courtship song and the presentation of male flies. Such an abnormal loss of retention in flies carrying the CaM Kinase inhibitior takes place over the course of one to three minutes between prestimulation and test (Griffith, 1993b).

(2) courtship conditioning

The second behavioral assay employed was courtship conditioning, an associative learning assay. A normal male will court a virgin female vigorously, but his courtship activity becomes depressed if he is paired with a previously mated (and hence, unresponsive) female. This depression lasts for several hours; during this period, if the male is paired with a virgin female, he takes longer to initiate courtship and spends significantly less time courting. Production of this depression is associative, dependent on aversive chemical cues emitted by the mated female; courting behavior remains depressed in the simultaneous presence of virgin and previously mated females. It is not dependent on visual input, as both blind flies and flies kept in the dark can be conditioned.

In all previously characterized learning mutants of Drosophila, this inhibition of subsequent courtship of a virgin female is absent or decreased, (that is, the mutants show long term memory defects with regard to aversive conditioning), although being paired with a mated female initially leads to a decrease in courtship of that female. Introduction into mutant males of a heat shock activation of a CaM Kinase inhibitor, results in flies that are normal with respect to courtship of virgin females. Such inhibitor expressing lines can be trained by exposure to a mated female, indicating that these lines are responsive to the aversive signals given by a mated female. Nevertheless, the transgenic lines fail to retain the effects of training. When subsequently presented with a virgin female, instead of being inhibited with respect to time of initiation and percentage of time spent in courtship, they initiate rapidly and court vigorously. This failure to modify courtship behavior in an experience-dependent manner is manifested by an increase in courtship activity, again underscoring the fact that the transgenic lines are not simply sick but, rather, have a specific defect in plasticity (Griffith, 1993b).

Subsequent studies were carried out to unravel the relation between CaM kinase II and visual input in the neuronal circuit controlling courtship conditioning. Learning was measured by exposure of male flies to a mated female for 1 hour and then immediately placing the male with an anesthetized virgin female. Exposure to the mated female reduces the level of mating to the virgin female. Memory was tested by measuring a courtship index for the initial 10 minutes of exposure to an anesthetized virgin female after the previous day's training with a mated female. The role of visual input in producing this behavior and the effects of modifying visual input on CaM kinase-dependent memory formation were examined. Inhibition of CaM kinase blocks apparent learning regardless of visual input. Visual input selectively affects the memory phase of courtship conditioning: normal visual input masks (makes up for) the memory effects of inhibition of CaM kinase resulting in the generation of memory without apparent learning, whereas disruption of visual input reveals the CaM kinase-dependence of memory. Visual input is found to be important only during the training period, which implies that vision and CaM kinase are interacting in the formation rather than the retrieval of memory. These results suggest a model for courtship conditioning in which multiple sensory inputs are integrated at a CaM-kinase-dependent neuronal switch to modulate courtship behavior (Joiner, 1997).

What do these defects in learning have to do with potassium channels? At the molecular level, a portion of the putative cytoplasmic domain of Eag is a substrate of calcium/calmodulin-dependent protein kinase II. Flies expressing the inhibitor of CaM Kinase II, as well as flies mutant for eag show supernumary synaptic discharges. Spontaneous discharges occur at frequencies as high as 25 Hz, lasting up to ten seconds after electrical stimulation. These results are in contrast to controls, in which unevoked discharges are never seen. These results suggest a failure of the nerve terminal to repolarize properly after repetitive stimulation, and imply that both CaM kinase II activity and a normal Eag potassium channel subunit are required for repolarization. Thus, an important component of neural and behavioral plasticity may be mediated by modulation of Eag function by calcium/calmodulin-dependent protein kinase II (Griffith, 1994).

Morphological aberrations correlate with to the impaired associative conditioning observed in transformed strains expressing CaM kinase inhibitors. There are increased numbers of nerve terminal branches associated with large varicosities at the nerve terminals of motor neurons in transformed flies. Inhibitory peptide, under to control of a heat-shock promoter, results in morphological effects observed three days after induction of the inhibitor. Excess branching occurs in larger, type I boutons, but not at the smaller type II varicosities. Another striking feature is that inhibition of CaM kinase appears to cause branch misorientation. Frequently, aberrant extra nerve entry points are observed in heat shock flies (Wang, 1994).

Other targets of CaM Kinase II are know in vertebrates. One target is the enzyme tyrosine hydroxylase, the rate-limiting enzyme that catalyzes the hydroxylation of tyrosine to form dopa, a chemical precursor which is subsequently converted to neurotransmitters. Thus CaM kinase II regulates the formation of neurotransmitters. CaM kinase II regulates neural excitability, phosphorylating Synapsin I, a protein which functions in regulating vesicular movement. Vesicles release neurotransmitters at the synapse, and their dynamics is the focus of regulation of neural excitability. CaM kinase targets the AMPA receptor, known to be central to the process of long term potentiation, an experimental analog of learning (Braun, 1995 and references).

CaM kinase is also involved in the regulation of gene expression. The c-fos promoter is targeted by both CREB (Drosophila homolog: dCREB2) and C/EBP (Drosophila homolog: SLBO). Both CREB and C/EBP are targeted by CaM kinase II (Braun, 1995 and references).

Mapping the anatomical circuit of CaM kinase-dependent courtship conditioning

Globally inhibiting CaM kinase activity in Drosophila, using a variety of genetic techniques, disrupts associative memory yet leaves visual and chemosensory perception intact. These studies implicate CaM kinase in the plastic processes underlying learning and memory but do not identify the neural circuitry that specifies the behavior. In this study, the GAL4/UAS binary expression system was used to define areas of the brain that require CaM kinase for modulation of courtship conditioning. The courtship-conditioning assay is divided into two distinct parts. The first is the training period during which the male is in contact with a mated female. The male normally modifies his behavior toward the trainer female during this period. This is the period in which the learning trait is measured. The second part is a test period in which the male is presented with a virgin female. His response to this female is a measure of his memory of associations made during the training period. The CaM kinase-dependent neurons that determine the response to the mated female during conditioning and those involved in formation and expression of memory are located in distinct areas of the brain. This supports the idea that courtship conditioning results in two independent behavioral modifications: a decrement in courtship during the conditioning period and an associative memory of conditioning. This study has allowed the circuit of information flow for a memory process in Drosophila to be genetically determined. The map generated dissects the behavior into multiple components and will provide tools that allow both molecular and electrophysiological access to this circuit (Joiner, 1999).

The anatomical location of CaM kinase-requiring neurons that are used for courtship conditioning was mapped using a total of 18 different P[GAL4] lines to express an inhibitor of CaM kinase in particular parts of the brain. In some of the lines, the expression pattern of GAL4 appears to be isolated within a single neural structure. In other lines, expression is predominantly localized to one or two structures with less intensive expression in other parts of the neuropil. By using, for example, five lines that predominantly express in the mushroom bodies, other areas of non-overlapping expression between the P[GAL4] lines can be ruled out as having a role in the observed phenotypes. Developmental effects can be controlled for using this approach, since there is a difference between the early temporal and spatial patterns of expression of independent lines. The use of multiple lines has also allowed for the determination that there are thresholds of inhibition below which few effects are seen (Joiner, 1999).

These experiments suggest that behavior during the conditioning period is determined by information processing at early synapses in the circuit in the antennal lobes. The main antennal lobes consist of primary sensory neurons that project from the olfactory and gustatory sense organs, as well as second-order intrinsic neurons. Cooling experiments in the honey bee have shown that initial processing of external stimuli occurs in the antennal glomeruli. Incoming information is processed in the first- and second-order neurons of the antennal nerve and lobes and neurons from the maxillary palps. This processing is required to modulate behavior toward the mated female during conditioning (Joiner, 1999).

Memory formation and modification of behavior toward subsequently presented females is determined by information processing deeper in the brain at higher-order synapses. Direct connections from the antennal nerve to the lateral protocerebrum have been described and P[GAL4] inserts (MJ146 and MJ286c) expressing ala (the CaM kinase inhibitor) in the lateral protocerebrum show that second- or third-order processing of this information occurs here. Memory is formed beyond the initial sensory processing centers in the brain at a number of different sites including the mushroom bodies, central complex, and lateral protocerebrum (Joiner, 1999).

The environmental inputs that drive this behavior in the absence of visual input are largely chemosensory. The neuronal circuit begins with chemosensory inputs, which send their processes to the antennal lobes where local inhibitory neurons and projection neurons interact. Of the two tracts from the antenna, mechanosensory and chemosensory, only a subset of the chemosensory input is used in this behavior. From select antennal glomeruli and directly from the antennal nerve, information is transferred to both the lateral protocerebrum and calyces of the mushroom bodies via unilateral connectives. Although the antennal nerve sends projections to both the lateral protocerebrum and indirectly to the central complex, the results presented here imply that processing of conditioning only occurs in the lateral protocerebrum (Joiner, 1999).

The mushroom bodies are used exclusively for memory in CaM kinase-dependent courtship conditioning; they send projections to the lateral protocerebrum. This is underscored by the results of the line 201Y, the only mushroom body line that shows a defect in response to the mated female. The most striking difference between the pattern of expression for 201Y and the other four mushroom-body expressing P[GAL4] lines are two pairs of cells, one in the lateral ventral protocerebrum and the other in the lateral dorsal region. The ventral pair resemble cellular expression of MJ146 and the dorsal pair resemble those of MJ162a. Both of these lines are defective for modulation of the response to the mated female (Joiner, 1999).

Unlike the lateral protocerebrum, the central complex (a neuropil involved in behavior output) is used solely for memory. Although no prominent neural tracts project directly to the central complex, it receives inputs from most areas of the brain, including the antennal nerve, the lateral protocerebrum, and the optic lobes, but not the mushroom bodies. The central complex is also known to be involved with motor output programs, and it is possible that inhibition of CaM kinase in this structure could impair output pathways. A strong argument against this is that global inhibition of CaM kinase has been shown to affect memory formation but not retrieval. In addition, there are no gross motor defects induced by CaM kinase inhibition; in fact failure to remember is associated with an increase in courtship behavior. These results would argue that the memory problems documented here are caused by impairment of formation, not retrieval (Joiner, 1999).

These experiments also demonstrate a difference between courtship conditioning and classical conditioning of odor avoidance in the circuitry and/or biochemistry underlying learning. Expression of activated Galpha_s in the mushroom bodies, but not in the central complex, using a subset of the lines used in this study, disrupts learning of odor avoidance (Connolly, 1996). Memory was not tested in this study. In particular, OK348, which expresses in the fan-shaped body, disrupts memory formation in the courtship-conditioning assay, but not learning in the odor avoidance assay. P-element insertion c232, which expresses in the ellipsoid body, does not affect either behavior. The relative magnitudes of disruption caused by mushroom body expression varies. 201Y, for example, is the least disruptive for classical conditioning, but the most disruptive for courtship conditioning. This biochemically defined circuit is not the equivalent of a connectivity circuit as defined electrophysiologically. The genetic manipulation of signal transduction pathways in discrete areas of the brain that are known to be connected directly or indirectly gives insight into the flow of information, but cannot rule out parallel pathways (Joiner, 1999).

In the courtship-conditioning assay, response to a virgin female is a measure of the male's memory of associations made during the training period. Previous studies have demonstrated that an association between attractive and aversive cues and courtship is required for the male to show intact memory in the test period. In flies in which CaM kinase activity is blocked in the mushroom bodies or parts of the central complex, a memory defect is observed with no change in the male's modification of his behavior during the training period. The decrement in courtship shown by wild-type males during the conditioning hour has been shown to be distinct from, and not necessary for, associative memory formation as assayed during the test period. One conclusion that can be drawn from these studies is that these two behaviors are not interdependent. This idea is supported strongly by the anatomical separation of these two responses, as demonstrated in this study. The decrement in courtship of the mated female is dependent upon intact CaM kinase in antennal lobes, whereas memory formation is dependent upon other brain structures. It is possible that this initial response to the mated female may even represent a form of nonassociative learning. A neural network model accounts for the differences between the phases of the assay in a more integrative way than by simply assigning nonassociative or associative labels to the behaviors (Joiner, 1999).

CaM Kinase II - biochemical activity

Ca2+ may enter the cell through extracellular voltage-senstive Ca2+ channels or through ligand-gated receptor channels. Ca2+ can also enter the cytoplasm from intracellular vesicular Ca2+ stores. Such intracellular stores are released through the action of inositol-3-phosphate, which is generated from lipids through the action of phospholipase-C (PLC). PLC is a target of G-protein coupled receptors. CaM kinase II transduces changes in intracellular free Ca2+ into changes in the phosphorylation state and activity of target proteins involved in neurotransmitter synthesis and release, neuronal plasticity and gene expression. Calmodulin, a ubiquitous enzyme, is the cell's calcium sensor, binding four calcium ions with high affinity. The Ca2+/calmodulin complex activates downstream targets, including CaM kinase II.

Structure/function analyses of the kinase reveal it is kept inactive in its basal state by a regulatory domain that is displaced by the binding of Ca2+/calmodulin. The crystal structures of calmodulin bound to the calmodulin-binding domain of CaM kinase II suggest that calmodulin wraps around the amphipathic calmodulin-binding domain, which then takes on an alpha-helical configuration. In essence, calmodulin may peel away a tight bound inhibitory segment from the active site. Calmodulin thereby activates the kinase by enabling the binding of both ATP and peptide substrates (Schulman, 1995 and references).

The CaM kinase II polypeptide consists of an N-terminal catalytic domain, a central regulatory domain, and a C-terminal association domain that is subject to alternative splicing in vertebrates, but not in Drosophila.

Whereas vertebrate CaM kinase II consists of four distinct classes of CaM kinase, each encoded by a separate gene, and each consisting of multiple isoforms generated by alternative splicing, Drosophila CaM kinase II is coded for by a single gene, whose transcript is subject to alternative splicing. Alternative splicing in the Drosophila enzyme takes place in the mRNA coding for a section near the C-terminus of the putative link segment, which is postulated to join the N-terminal to the C-terminal globules of the polypeptide. Alternative splicing in the Drosophila mRNA does not take place in mRNA coding for the C-terminal domain that is subject to alternative splicing in vertebrates (Griffith, 1993b).

Autophosphorylation provides a critical regulation of CaM kinase II. The autoinhibitory domain of the kinase is disrupted by binding of calmodulin at its C-terminal end, which leads to a de-inhibition of the kinase. The autoinhibitory domain can be further disrupted by phosphorylation of a key residue common to all isoforms. Phosphorylation of this site is not essential for kinase activity, but it does have two important consequences. First, autophosphorylation increases the affinity of the kinase for calmodulin several hundredfold by reducing the dissociation rate of the kinase-calmodulin complex. Second, the presence of a phosphate on this site is itself sufficient to disrupt the autoinhibitory domain, and the kinase retains partial activity even after calmodulin dissociates (Braun, 1995).

The frequency of Ca2+ oscillations or spikes, that is the extent of neural activity, may be decoded by CaM kinase via this autophosphorylation. Calmodulin is essentially trapped by autophosphorylation which converts CaM kinase into a high affinity calmodulin-binding protein. Repetitive stimulation of the kinase may promote recruitment of calmodulin to the kinase so that it becomes increasingly active with each stimulus in a frequency-dependent manner. The association domain at the C-terminal end of CaM kinase of vertebrates contains a variable region that targets isoforms of the kinase to the nucleus or cytoskeleton and assembles the kinase into a decameric structure. Alternative splicing introduces a short nuclear localization signal that targets transfected kinase to the nucleus where it may regulate nuclear functions. The regulatory properties of CaM kinase provide for molecular potentiation of Ca2+ signals and frequency detection whereas its association domain should enable it to decode such Ca2+ fluctuations in the nucleus (Schulman, 1995).

Thus the effects of CaM kinase are felt at different levels of nerve action, including production of neurotransmitters, vesicular release of neurotransmitters, activation of transcription factors known to be involved in the learning process, neural plasticity, as evidenced by axon sprouting, and various learning paradigms.

GENE STRUCTURE

The Drosophila CaM kinase II gene consists of at least 16 exons spanning approximately 20 kilobase pairs. Alternative splicing generates four forms of the enzyme from a single gene. The four forms differ only by amino acid insertions or deletions near the C-terminus of the putative link segment, which is postulated to join the N-terminal to the C-terminal globules of the polypeptide, forming a dumbbell-like shape (Ohsako, 1993).

Bases in 5' UTR - 250

Exons - at least 16

Bases in 3' UTR - 1230 (Ohsako, 1993)

PROTEIN STRUCTURE

A monoclonal antibody against rat brain type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) precipitates three proteins from Drosophila heads with apparent molecular weights similar to those of the subunits of the rat brain kinase. Fly heads also contain a CaM kinase activity that becomes partially independent of Ca2+ after autophosphorylation, as does the rat brain kinase. A Drosophila cDNA encodes an amino acid sequence that is 77% identical to the sequence of the rat alpha subunit. All known autophosphorylation sites are conserved, including the site that controls Ca(2+)-independent activity (Cho, 1991).

The four cDNA sequences encoding Ca2+/calmodulin-dependent protein kinase II encode polypeptides of 490, 509, 516, and 530 amino acids. They are identical to one another except for amino acid insertions or deletions near the carboxyl-terminal of the putative "link" segment. These polypeptides show considerable similarity to rat brain CaM kinase II with more than 70% of the amino acids being identical. The Drosophila adult head contains three major species of CaM kinase II, with molecular masses of 55, 58, and 60 kDa. These cross-react with anti-rat CaM kinase II antibody. An expression study of the four Drosophila cDNA sequences in mammalian cells reveals that the polypeptides of 490, 509, and 530 amino acids that had been predicted from the cDNA sequences correspond to the 55-, 58-, and 60-kDa polypeptides found in the Drosophila head, respectively, and all exhibit enzymatic properties similar to those of rat brain CaM kinase II, including self-regulation (Ohsako, 1993).

Eight different CaM kinase II cDNA sequences, varying only at the junction of the regulatory and association domains of the kinase have been isolated. The diversity of CaM kinase in Drosophila is greater than previously appreciated and is generated by alternative splicing of a single gene (Griffith, 1993b).

Four forms of the Drosophila Ca2+/calmodulin-dependent protein kinase II are generated from a single gene by alternative splicing. A fifth form of the cDNA is maternally derived, and encodes the enzyme expressed in the ovary, unfertilized egg and early embryos. The fifth form is also generated from the gene by alternative splicing and is identical to the cDNA encoding the 530-amino-acid polypeptide, the longest of the four forms previously identified, except that it lacks exon 11. Three splicing derivatives which have lost one amino acid from the 509- and 530-amino-acid polypeptides are also found in 4 to 10 h embryos (Takamatsu, 1994).

Amino Acids - 490, 509, 516 and 530 (Cho, 1991 and Ohsako, 1993)

Structural Domains

See above: biological overview

The database Online Mendelian Inheritance in Man (OMIM) is an excellent source of information about mammalian CaMK2A.

date revised:  20 Dec 96

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