The ability of CaMKII to act as a molecular switch, becoming Ca²⁺ independent after activation and autophosphorylation at T287, is critical for experience-dependent synaptic plasticity. This study shows that Caki, the Drosophila homolog of CASK, also known as Camguk, can act as a gain controller on the transition to calcium-independence in vivo. Genetic loss of dCASK significantly increases synapse-specific, activity-dependent autophosphorylation of CaMKII T287. In wild-type adult animals, simple and complex sensory stimuli cause region-specific increases in pT287. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's Calmodulin (CaM) binding domain. Phosphorylation of T306 blocks Ca²⁺/CaM binding, lowering the probability of intersubunit T287 phosphorylation, which requires CaM binding to both the substrate and catalytic subunits. dCASK is the first CaMKII-interacting protein other than CaM found to regulate this plasticity-controlling molecular switch (Hodge, 2006).

Autophosphorylation of CaMKII at a site in the N terminus of its autoregulatory domain (T287 in Drosophila and T286 in mammalian αCaMKII) confers Ca²⁺-independent activity on the enzyme. This switch-like property of the kinase is crucial to its role in long-term potentiation and memory formation in mammals and flies, and generation of the autonomous form of the kinase can be stimulated by a number of different behaviors and activity paradigms. Interestingly, in mice, both mutations that prevent T286 phosphorylation, and transgenes that increase constitutive activity have been shown to block plasticity. This implies that the level of constitutive activity needs to be tightly controlled to remain in a range optimal for learning. This balance has been believed to be exerted by the positive effects of Ca²⁺/CaM and the negative effects of phosphatases. To date, no proteins other than calmodulin have been shown to influence the development of autonomous activity via T287 autophosphorylation (Hodge, 2006).

The function of autophosphorylation in the C terminus of the regulatory domain within the CaM binding region (T306 in Drosophila and T305 in mammalian αCaMKII) has been more mysterious, but also has consequences for plasticity. In the test tube, with purified kinase, this phosphorylation occurs only after T287 phosphorylation and removal of CaM with EGTA because the site is protected from phosphorylation by bound CaM. With purified kinase, this means that pT306 is only found in doubly phosphorylated pT287, pT306 enzyme. Recently, a novel mechanism for phosphorylation of T306 in the absence of pT287 has been described (Lu, 2003). dCASK interacts with the regulatory domain of the kinase and, when the CaM binding site of the kinase is unoccupied (e.g., when synaptic calcium is low), stimulates the kinase to autophosphorylate at T306. This reaction releases CaMKII from the dCASK complex in a form that is incapable of binding Ca²⁺/CaM and has been suggested to provide a mechanism for downregulation of the activatable kinase pool at quiescent synapses. The effects of dCASK on T287 phosphorylation have not been addressed, and modulation of this site would have important consequences for plasticity (Hodge, 2006).

This study presents evidence that dCASK can also act as an activity-dependent modulator of the levels of constitutively active CaMKII in vivo. CaMKII pT287 autophosphorylation occurs within the dodecameric holoenzyme and requires Ca²⁺/CaM to be bound to both the subunit doing the catalysis and the subunit that contains the substrate threonine. This cooperativity implies that modifications that alter the ability of Ca²⁺/CaM to bind would affect the ability of the kinase to become Ca²⁺ independent. Because it promotes phosphorylation of T306, interaction with dCASK regulates T287 phosphorylation by altering the occupancy of CaM binding sites in the holoenzyme. In vivo this postsynaptic interaction provides a synapse-specific mechanism to alter the probability of generating autonomous activity that is controlled by the activity history of the synapse. This makes dCASK an important gain controller for the CaMKII molecular switch (Hodge, 2006).

dCASK, the Drosophila homolog of the mammalian MAGUK scaffold protein CASK and C. elegans Lin-2, is a synapse-specific regulator of the ability of CaMKII to become Ca²⁺ independent via T287 autophosphorylation. The transition to Ca²⁺ independence has been shown to be a critical part of CaMKII's role in neural plasticity. The plasticity-inducing effects of constitutively active CaMKII have been documented at the cellular and behavioral levels in both vertebrates and invertebrates. This feature of CaMKII's activity is fundamental to its role in the neuron, and both too much and too little constitutive activity have deleterious effects on learning. Regulation of both the transition to the Ca²⁺-independent state and the maximum achievable level of constitutive activity is therefore likely to be important for brain function. dCASK is the first protein other than CaM to be shown to modulate this process (Hodge, 2006).

The mechanism underlying dCASK's ability to impose gain control on CaMKII T287 autophosphorylation is based on its ability to stimulate autophosphorylation of the kinase's CaM binding domain at T306 in low-calcium/low-activity conditions. Phosphorylation of T306 (T305 in mammalian αCaMKII) was first seen in vitro with purified kinase and only occurs when the kinase is first rendered constitutively active by T287 phosphorylation and then has Ca²⁺/CaM stripped by addition of EGTA. This results in a doubly phosphorylated (pT287 + pT306) enzyme that is active but cannot bind CaM. In contrast, dCASK-stimulated T306 phosphorylation does not require previous T287 phosphorylation and can therefore produce a monophosphorylated (pT306) enzyme that is inactive and cannot bind CaM (Lu, 2003; Hodge, 2006).

Monophosphorylation of T306 would have two major consequences for CaMKII activity. The first is that individual pT306 subunits within a holoenzyme cannot be activated by Ca²⁺/CaM. In a neuron, this will produce a linear decrease in the level of CaMKII activity elicited by a calcium pulse since each subunit is an independent kinase. The second consequence is more subtle, but perhaps more important because of the special role of autonomously active CaMKII in plasticity. T306 monophosphorylation decreases T287 phosphorylation, because T287 phosphorylation obligatorily occurs between subunits within a holoenzyme and requires CaM binding to both catalytic and substrate subunits. The cooperativity of T287 phosphorylation with respect to CaM binding means that there is a greater than stoichiometric disruption of T287 phosphorylation with increasing pT306. Thus, T306 phosphorylation has a greater impact on T287 autophosphorylation than on Ca²⁺-stimulable activity for intermediate levels of pT306 within a holoenzyme (Hodge, 2006).

In Drosophila, there is evidence for behavioral defects that may be a result of alterations in CaMKII activity. Long-term memory of odor-shock conditioning requires CaMKII and is associated with large increases in the amount of total protein in specific brain areas (Ashraf, 2006). Associative courtship suppression has been shown to require antennal lobe CaMKII activity and to be enhanced by overexpression of constitutively active CaMKII in the antennal lobes. These studies suggest that both the total amount of CaMKII and the level of Ca²⁺-independent activity can be altered by plasticity-inducing events (Hodge, 2006).

These two modifiable parameters operate on different time scales and may have different roles. Autophosphorylation is very fast while new protein synthesis can take minutes to hours and is likely to be more important in long-term memory. In the retina, changes in total CaMKII levels were evident with the chronic exposure to a light:dark cycle, but changes in CaMKII autophosphorylation that were independent of kinase level also occurred. In the deeper reaches of the optic system, changes in autophosphorylation appeared to be the dominant effect. The insignificant change in CaMKII levels that were observed between mated and virgin males may be due to the very fast processing of the males after copulation or to the fact that pT287 was averaged over the entire antennal lobe. It is possible that there are glomerulus-specific changes in pT287 or total CaMKII that were not detected (Hodge, 2006).

There may be some short-term behaviors that are driven primarily by changes in autophosphorylation, such as suppression of courtship during training with a mated female. In this behavior, overexpression of a Ca²⁺-dependent form of the kinase has no effect, implying that it is the Ca²⁺-independent kinase rather than total kinase activity that is important. The enhanced courtship suppression caused by constitutively active kinase is dose dependent and is manifested by a reduction in initial courtship index that parallels the level of autonomous kinase during the 1 hr training period. At extremely high doses, such as those achieved by the strong GAL4 driver GH146, courtship is completely suppressed and therefore no longer plastic. This indicates that there is a limited range over which CaMKII constitutive activity supports plasticity. Cellular mechanisms that regulate T287 phosphorylation are therefore critical to maintaining animals within that range (Hodge, 2006).

When activity-dependent autophosphorylation is examined in dCASK null animals, the in vivo role of the dCASK/CaMKII interaction can be dissected. Basal levels of pT306 in dCASK-deficient animals are decreased. Presumably, the residual pT306 in these animals is a result of autophosphorylation secondary to T287 phosphorylation, and most of the pT306 therefore would be expected to be found in doubly phosphorylated pT287 + pT306 kinase. The profound activity-dependent decrease in pT306 in null animals is likely due to this dependence on primary T287 phosphorylation (Hodge, 2006).

Is the dCASK/CaMKII interaction important for behavior? Basal levels of pT287 are increased in dCASK null animals, presumably as a secondary effect of the decrease in monophosphorylated pT306 subunits. For neurons in which the absolute level of constitutive CaMKII activity is important for behavioral or cellular plasticity, this basal elevation of pT287 would be expected to have consequences since neurons would be closer to their threshold for the plasticity process. Given the dependence of suppression on constitutive CaMKII activity, the elevation of pT287 in dCASK-deficient males would therefore be expected to reduce initial courtship. Consistent with this, a slight reduction weas observe in courtship of anesthetized mature virgin females by dCASK-deficient males compared to wild-type. Since dCASK has many roles in the cell, it is difficult to assign this reduction solely to the change in pT287, but it is consistent with the robust effects of dCASK on CaMKII and the role of CaMKII in courtship behavior. Genetic rescue with a UAS-linked transgene (which is unlikely to provide exactly the correct amount of dCASK) would be uninformative on this issue since there appears to be a directly dose-dependence of dCASK level with pT306, and both decreases and increases in CaMKII activity can affect behavior (Hodge, 2006).

When neuronal circuits are activated in the context of this higher basal level of pT287, the response to activity in primary cells of the circuit is blunted, as if there is a ceiling effect. The inability to increase the level of constitutive activity over this elevated baseline might also affect plastic processes in neurons where the biochemical processes subserving plasticity rely on the incremental change over baseline rather than on some absolute level of constitutive activity. There is evidence in Drosophila for such mechanisms in associative memory formation of courtship conditioning and in hippocampus where it has been shown that LTP is occluded by overexpression of active CaMKII. Changes in both the baseline and stimulable levels of T287 phosphorylation could disrupt the ability of neurons to respond to plasticity-inducing signals (Hodge, 2006).

Alterations in CaMKII autophosphorylation also appear to have consequences for circuit function. Mushroom bodies in Drosophila receive information about visual stimuli indirectly. In wild-type animals, light does not stimulate phosphorylation of T287 in this neuropil. In dCASK-deficient animals, there is inappropriate activation of the kinase in the mushroom body calyx. This suggests that dCASK also has a role in controlling spread of information within neuronal circuits, perhaps by dampening CaMKII activation (Hodge, 2006).

The results from biochemical assays and intact animal studies support a model in which the normal function of the dCASK/CaMKII interaction is to allow the activity history of the synapse to alter the probability with which CaMKII can become Ca²⁺ independent. At synapses where activity has been low, the dCASK/CaMKII interaction will decrease CaM binding in the holoenzyme, making it less able to initiate T287 phosphorylation even after a strong calcium pulse. In synapses that have been active, Ca²⁺/CaM protects CaMKII T306 from autophosphorylation, and the kinase can robustly respond to calcium influx. This makes dCASK an important regulator of plasticity. How this protein is localized to synapses and how its availability for interaction with CaMKII might be regulated will be important to determine (Hodge, 2006).

GENE STRUCTURE

cDNA clone length - 4139 (RA)

Bases in 5' UTR - 1415

Exons - 13 (RA)

Bases in 3' UTR - 921

PROTEIN STRUCTURE

Amino Acids - 591 (isoform A)

Structural Domains

Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. A new Drosophila CaM kinase gene has been cloned, named caki. The caki gene is extremely large; comparison of the genomic and cDNA sequences reveals that the caki transcription unit is at least 150 kb. The catalytic domain of this new CaM kinase protein shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active since it causes a 3-fold increase in the level of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter in a co-transfusion assay. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm' (Martin, 1996).

MAGUKs have been classified into two subfamilies: Dlg-like with three DHR/PDZ domains and p55-like with a single DHR/PDZ domain. There is now a new subfamily whose members have a novel domain structure: a calcium/calmodulin-dependent protein kinase domain in the N-terminus as well as the DHR/PDZ, SH3 and GUK domains in the C-terminus. These new MAGUKs may regulate transmembrane molecules that bind calcium, calmodulin, or nucleotides. camguk (cmg) is a Drosophila member of this novel MAGUK subfamily. The C-terminal domain of Camguk shows a strong resemblance to p55, with a single DHR domain, an SH3 domain, and a GUK domain with an intact ATP binding site. The DHR domain is 54% identical to that of human p55. At the N-terminus the sequence predicts a domain with striking similarity to calcium/calmodulin-dependent protein kinase (CaM kinase, including the entire putative catalytic domain (37% identity with that of Drosophila CaM kinase type IIbeta). The cmg locus is the same gene as caki previously thought to encode only a brain-specific CaM kinase type II. There are two reported homologs of Camguk: C. elegans lin-2 and rat CASK (Dimitratos, 1997).

date revised: 1 March 2007

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