Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. A new Drosophila CaM kinase gene named caki has been cloned. 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; full text of article).
Drosophila Camguk (Cmg) is a member of the CAMGUK subfamily of the MAGUK family of proteins which are localized at cell junction and other plasma membrane specialized regions, from worms to mammals. The protein structure of Cmg, as the other CAMGUK proteins, is characterized by only one PDZ domain and an additional CaM kinase domain, similar to CaMKII. While the mammalian ortholog CASKs play an important role in synaptic protein targeting and in synaptic plasticity, the Drosophila Cmg role is unknown. To study its potential role, a detailed analysis was performed of mRNA distribution of the Drosophila cmg gene at cellular and developmental level, during embryonic, larval, pupal and adult stages. The transient cmg transcription in midgut and Malpighian tubules may suggest a potential function in cell junction formation and in epithelial tissue patterning. Interestingly, cmg transcription increases substantially during embryonic neuroblast proliferation, becoming predominant in the developing central nervous system (CNS) during embryonic and postembryonic development stages and in the mature brain. In addition, a high transcriptional level was detected in the eye imaginal discs and in the adult retina, demonstrating a specific and continuous expression of cmg in neuroblasts and photoreceptor neurons, from the onset of cytodifferentiation. These findings suggest that Cmg could play a potential role in transmembrane protein targeting, particularly in synapses. These observations suggest the existence of a common highly conserved mechanism involved in forming and maintaining proper synaptic protein targeting, which are fundamental features of synaptic plasticity, learning and memory. Through its function, the CaM kinase domain-containing Cmg may be involved in signal transduction cascade. Its potential relation to Calmodulin and CaMKII is discussed (Lopes, 2001).
Assessment of NMJ autophosphorylation gave openned the opportunity to probe the CaMKII/dCASK interaction under very defined conditions at a well-characterized synapse to demonstrate that dCASK is a synapse-specific and activity-dependent modulator of T287 phosphorylation. Changes in dCASK function (Lu, 2003), CaMKII levels (Ashraf, 2006), and CaMKII autophosphorylation (Mehren, 2004) have been shown to be important for learning-related neuronal plasticity of the adult Drosophila brain. To determine if regulation of CaMKII autophosphorylation could be mediated by dCASK in adults, sections of wild-type male brains were double-stained with antibodies that recognized total CaMKII protein or were specific to CaMKII phosphorylated at either T287 or T306. CaMKII protein is found in all neuropil regions and at lower levels in somatic regions surrounding them (Takamatsu, 2003). Autophosphorylation of CaMKII at T287 and T306 occurred primarily in synaptic areas. dCASK has previously been shown to be expressed relatively uniformly (Lopes, 2001; Martin, 1996) in all synaptic regions in adult Drosophila brain (Hodge, 2006).
Although staining with the two phosphospecific antibodies occurred in all the regions containing CaMKII, the absolute intensity of staining did not directly parallel total kinase levels. Regions in which total CaMKII appeared similar could have very different levels of autophosphorylation. It was also clear that the patterns of staining seen with the anti-pT306 and anti-pT287 antibodies were distinct. Obvious examples of this are the retina and the lamina where the R1-6 photoreceptors synapse. In retina, pT306 is very high, but there is less pT287. In the laminar synaptic region, the opposite is true. In other regions such as the antennal lobe (AL) and mushroom body (MB), calyx phosphorylation levels were roughly equivalent. This is made clear by examination of the ratios of phosphospecific antibody CaMKII staining in various brain regions. While these numbers have no intrinsic meaning in terms of site occupancy for either phosphorylation, the ratio does reflect relative occupancy within a region. These data strongly suggest that autophosphorylation of T287 and T306 are regulated independently and that properties of particular circuits or subcellular locations can modulate autophosphorylation (Hodge, 2006).
dCASK, the Drosophila homolog of C. elegans Lin-2 and mammalian CASK, is a member of the MAGUK family of scaffolding proteins that contains an N-terminal CaMKII-like domain along with the canonical MAGUK PDZ, SH3, and GUK domains. dCASK has been shown to physically interact with CaMKII and regulate phosphorylation of CaMKII T306 postsynaptically in Drosophila (Lu, 2003). The levels of pT287 CaMKII were examined in wild-type and dCASK-deficient fly head extracts by Western blotting with a phosphospecific antibody to T287 to determine if dCASK affected autophosphorylation of this site in vivo. Animals carrying a deletion of the dCASK locus have very little pT306 compared to wild-type animals, but total CaMKII levels are unchanged (Lu, 2003). Phosphorylation of T287 is also sensitive to dCASK levels but is modulated in opposite direction from T306; deficiency animals with no dCASK protein have significantly higher pT287 levels than wild-type (Hodge, 2006).
Synaptic phosphorylation of T286 in mammals is activity regulated. To determine if CaMKII autophosphorylation is regulated by acute changes in activity in Drosophila and whether this is modulated by dCASK, the third-instar larval neuromuscular junction (NMJ) was examined. CaMKII pT306 levels are regulated by both dCASK and chronic changes in neuronal activity at this synapse (Lu, 2003). To find out if acute activity could regulate CaMKII autophosphorylation, a strong stimulus was applied to the motor axon on one side of the animal and staining was performed with an antibody that recognizes total kinase and an antibody that recognizes either pT287 or pT306. CaMKII and pT306 staining were examined of two muscle 12 NMJs from the same animal (the unstimulated side, and the stimulated side). A small decrease in pT306 is can be seen with stimulation, suggesting that synaptic activity may activate a pT306 phosphatase. NMJs from an animal stained with anti-CaMKII and anti-pT287 show a significant increase in pT287 on the stimulated side (Hodge, 2006).
To quantify these changes, immunoreactivity of phosphospecific antibodies was normalized to total synaptic kinase. Comparison of the stimulated side to the unstimulated side in wild-type animals demonstrates that acute activity slightly decreases pT306 in wild-type and dCASK-deficient animals. In animals that overexpress dCASK postsynaptically, activity causes a much more significant percent decrease in pT306. This may be due to the fact that these animals have elevated baseline levels of pT306 (Lu, 2003). Alternatively, dCASK might directly or indirectly regulate activity of a phosphatase in periods of high activity (Hodge, 2006).
The effect of acute activity on pT287 levels is qualitatively different than its effects on pT306. Wild-type flies have a modest but significant increase in pT287, and postsynaptic overexpression of dCASK blocks this effect. The NMJs of animals that lack dCASK have an exaggerated response to activity, which quadruples the pT287 content of CaMKII compared to wild-type. These data support the idea that dCASK acts as a gain control on activity-dependent T287 phosphorylation (Hodge, 2006).
The ability to independently regulate the strength of individual synapses on a neuron confers computational strength to the nervous system. CaMKII autophosphorylation within the postsynaptic apparatus is believed to be synapse specific in mammalian neurons and to contribute to synapse-specific plasticity (for review see Merrill, 2005). For dCASK modulation of CaMKII autophosphorylation to be useful for such processes it must also be synapse specific. To investigate this in Drosophila, electrical activity or dCASK levels were altered selectively in muscle 12 and the phosphorylation of CaMKII at synapses on muscles 12 and 13 that are made by a single type II motor neuron was examined (Hodge, 2006).
Phosphorylation of T306 is increased at muscle 12 synapses containing more postsynaptic dCASK relative to synapses made by the same neuron onto muscle 13. Decreasing electrical activity in the postsynaptic cell by expressing dORKΔC, a hyperpolarizing potassium channel, had little effect on T306. This may be due to the low levels of endogenous dCASK at type II synapses (Hodge, 2006).
Phosphorylation of T287 was decreased in manipulated synapses compared to synapses made by the same neuron onto a normal postsynaptic cell. Both electrical silencing with dORKΔC and postsynaptic overexpression of dCASK led to lower pT287 levels specifically in muscle 12 synapses. None of the manipulations changed total CaMKII levels (Hodge, 2006).
To determine if a natural stimulus that alters the level of activity within a neural pathway could affect CaMKII autophosphorylation specifically within that circuit, pT306 levels in sections of animals that had been dark reared were compared to those found in animals raised in a 12 hr:12 hr light:dark cycle. Levels of pT306 measured at boutons of adult muscles were unaffected by light conditions but were decreased by dCASK deficiency, consistent with previous work at the larval NMJ (Lu, 2003). In the CNS of wild-type animals, phosphorylation of T306 was decreased by light input in the retina and in the medulla where R8 photoreceptor axons terminate in neuropil layer M3. Light did not significantly affect the level of pT306 at R1–R6 or R7 photoreceptor synapses in the lamina and medulla of wild-type animals (Hodge, 2006).
In the retina of dark-reared dCASK mutant animals, pT306 levels were decreased in comparison to wild-type. In lamina and medulla, pT306 levels at the R1–R6 and R7 synapses in dark-reared animals were not significantly affected, but at medullar R8 photoreceptor synapses it was decreased. For dCASK-deficient animals, light profoundly decreased pT306, even below levels seen in wild-type. This may reflect a difference in the “set point” of multiple kinase autophosphorylation sites in these animals (Hodge, 2006).
Increases in neuronal activity are known to stimulate autophosphorylation of CaMKII at T287 and convert it into a Ca2+/CaM-independent enzyme. To test the ability of light to stimulate T287 phosphorylation in the adult optic system, head sections stained with anti-pT287 were examined. In wild-type adult fly heads, light significantly increased pT287 in the retina and at synaptic regions in the optic lobes corresponding to R7 and R8 photoreceptor termini. The light-dependent increase in pT287 in the axon terminal fields of R1–R6 photoreceptors was not statistically significant (Hodge, 2006).
In the optic system of dark-reared animals containing no dCASK protein, pT287 levels were in general higher than wild-type (for R8 and R7 synaptic terminals p < 0.001). Increased neuronal activity due to light stimulus was able to increase pT287 level to that found in wild-type animals, but no higher. Since basal pT287 levels are elevated in this genotype, this suggests that the dynamic range of T287 phosphorylation is blunted in dCASK-deficient animals (Hodge, 2006).
The retina, lamina, and the medulla are areas of the brain specialized to receive and process light information, and as such it makes sense that they show activity-induced changes in CaMKII autophosphorylation. To test the specificity of the light-induced increases in pT287, levels of phosphorylation were measured in nonoptic areas. Areas of the nervous system such as the NMJ or antennal lobes that have no optic inputs and areas in the CNS that only indirectly receive information about light such as the mushroom bodies might be expected to have a smaller response. Quantification of bouton pT287 at the adult NMJ shows that there is no difference between light- and dark-reared animals. Likewise, in wild-type animals, there is no enhancement of T287 autophosphorylation in the antennal lobes or the mushroom bodies (Hodge, 2006).
While light does not affect CaMKII autophosphorylation at nonoptic synapses, the level of dCASK protein does. Multifactoral ANOVA of the entire data set indicates that there is a significant genotype effect. Animals with no dCASK protein have significantly higher levels of pT287 in both light- and dark-reared conditions at the NMJ, a synapse which does not get either direct or indirect information about light levels. Similarly, levels of pT287 are slightly, but not significantly, higher in antennal lobes of dCASK-deficient animals compared to wild-type. In neither of these synaptic regions does light alter pT287 in dCASK-deficient animals (p > 0.05) (Hodge, 2006).
The situation in the mushroom bodies is more interesting. This brain region may receive inputs from optic centers via a polysynaptic pathway. In mushroom body calyx, light is able to evoke an increase in pT287 in animals that do not have dCASK even though in wild-type pT287 levels in this region are insensitive to light. This suggests that the lack of dCASK is allowing downstream neurons to become hypersensitive to light, supporting a role for dCASK in activity-dependent gain control of CaMKII T287 autophosphorylation at the circuit level as well as the subcellular level. It cannot be distinguish from these data if the circuit level effects are realized via the effects of CaMKII on neuronal activity or are a consequence of loss of other functions of dCASK (Hodge, 2006).
Activity-driven autophosphorylation is not the only mechanism for altering CaMKII activity in vivo. In mammals, the mRNA for CaMKII is locally translated in response to light stimulation, and this translation is required for some forms of plasticity. CaMKII protein can also translocate between subcellular compartments in the mammalian CNS. Activity-dependent control of CaMKII levels has also been demonstrated in the Drosophila CNS (Ashraf, 2006). At NMJ of muscles 12 and 13, synaptic levels of CaMKII were not altered significantly by manipulation of activity or dCASK, indicating that changes in autophosphorylation were the predominant effector of activity-dependent CaMKII modulation at these synapses. To address this issue in the CNS, CaMKII immunoreactivity was quantified in the optic system in dark-reared and light-reared wild-type males. In the retina of animals reared in a 12 hr:12 hr light:dark cycle, CaMKII levels were 122% ± 6% of dark-reared animals. CaMKII levels in the lamina at the R1–R6 synapses and in the medulla were not significantly higher. When the pT306 and pT287 changes in the retina are normalized to account for the change in CaMKII, there is still a significant increase in pT287 (light-reared pT287/total CaMKII is 149% of dark-reared) and an even greater decrease in pT306 as a percentage of total kinase (light-reared pT306/total CaMKII is 39% of dark-reared). These data suggest that modulation of both CaMKII levels and autophosphorylation occur in the sensory cells of the optic system, but that at synapses within the CNS, the primary activity-driven changes are in autophosphorylation (Hodge, 2006).
The data has indicated that light, a single channel sensory input, regulates CaMKII autophosphorylation in the optic system. It was also of interest to determine if more complex experiences could also alter T287 phosphorylation. Drosophila courtship is a stereotyped behavior that is driven by visual, olfactory, gustatory, and tactile cues. Males can learn to suppress courtship during training with a mated female, and this suppression is blocked by inhibition of CaMKII in antennal lobes and enhanced by expression of Ca2+-independent CaMKII in the same region. To determine if mating experience acutely altered pT287 levels, antennal lobe pT287 in head sections of males that had been raised in isolation was compared to males that were raised in isolation but allowed to copulate with a mature virgin female immediately before they were sectioned. In wild-type males, mating caused a significant increase in pT287. Total CaMKII levels were not significantly increased. Animals lacking dCASK had a higher basal level of pT287 but were not able to significantly increase it with mating. These results are consistent with dCASK functioning in this brain region to regulate the level and dynamics of CaMKII constitutive activity (Hodge, 2006).
Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, a caki null mutant was studied in Drosophila. By means of electrophysiological methods, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system were analyzed in adult flies. In caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ is increased, the response of the giant fiber pathway to continuous stimulation is impaired, and electroretinographic responses to single and continuous repetitive stimuli is altered and optomotor behavior is abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function (Zordan, 2005; full text of article).
Neurexins are neuron-specific cell surface molecules thought to localize to presynaptic membranes. Recent genetic studies using Drosophila have implicated an essential role for the single Drosophila Neurexin in the proper architecture, development and function of synapses in vivo. However, the precise mechanisms underlying these actions are not fully understood. To elucidate the molecular mechanism of Neurexin in vivo, dnrx and caki mutant flies, combined with various methods, were used to analyze locomotion, synaptic vesicle cycling and neurotransmission of neuromuscular junctions. Dneurexin (DNRX) was found to be important for locomotion through a genetic interaction with the scaffold protein, CAKI/CMG, the Drosophila homolog of vertebrate CASK. Similar to its mammalian counterparts, DNRX is essential for synaptic vesicle cycling, which plays critical roles in neurotransmission at neuromuscular junctions (NMJ). However, this interaction appears not to be required for the synaptic targeting of DNRX, but may instead be needed for proper synaptic function, possibly by regulating the synaptic vesicle cycling process (Sun, 2009).
Genetic causes for disturbances of locomotor behavior can be due to muscle, peripheral neuron, or central nervous system pathologies. The Drosophila homolog of human CASK (also known as Caki or Camguk) is a molecular scaffold that has been postulated to have roles in both locomotion and plasticity. These conclusions are based on studies using overlapping deficiencies that largely eliminate the entire CASK locus, but contain additional chromosomal aberrations as well. More importantly, analysis of the sequenced Drosophila genome suggests the existence of multiple protein variants from the CASK locus, further complicating the interpretation of experiments using deficiency strains. This study generated small deletions within the CASK gene that eliminate gene products containing the CaMK-like and L27 domains (CASK-β), but do not affect transcripts encoding the smaller forms (CASK-α), which are structurally homologous to vertebrate MPP1. These mutants have normal olfactory habituation, but exhibit a striking array of locomotor problems that includes both initiation and motor maintenance defects. Previous studies had suggested that presynaptic release defects at the neuromuscular junction in the multigene deficiency strain were the likely basis of its locomotor phenotype. The locomotor phenotype of the CASK-β mutant, however, cannot be rescued by expression of a CASK-β transgene in motor neurons. Expression in a subset of central neurons that does not include the ellipsoid body, a well-known pre-motor neuropil, provides complete rescue. Full length CASK-β, while widely expressed in the nervous system, appears to have a unique role within central circuits that control motor output (Slawson, 2011).
Previous work has implicated disruption of CASK in a suite of behavioral deficits. These studies, however, all suffered from the same limitation, as the null animals used in these experiments had lost both of the proteins encoded at the CASK locus, and also had disruptions of other third chromosome genes. To address this, a new set of isoform-specific mutants was generated, so as to better dissect the behavioral contribution of the CASK homolog in the fly. While these mutants shared similarities with the 307/313 flies used in previous studies, they were strikingly different in other ways (Slawson, 2011).
The CASK locus encodes two distinct MAGUKs: CASK proteins have been defined as a subfamily of MAGUK protein with a unique N-terminal CaMK-like domain in addition to the more typical L27, PDZ, SH3 and GUK domains. The CaMK-like domain has a constitutively active structure that grants it low levels of Ca2+/calmodulin-independent activity against complexed substrate. Unlike all other known kinases, this activity is inhibited by Mg2+. This domain also participates in regulation of CaMKII autophosphorylation. CASK-β would therefore be expected to have properties different from other MAGUK proteins, and it represents the true ortholog of vertebrate CASK. The Drosophila genome project annotation of the CASK locus predicts that in addition to canonical CASK proteins (CASK-β), this locus has separately initiated transcripts that encode shorter proteins with a unique N-terminal region that is followed by PDZ, SH3 and GUK domains (CASK-α). These proteins are, in structure, more like the p55/MPP1-type MAGUKs than a true CASK. Phylogenetically, the MPP1 MAGUK group in vertebrates appears to be an offshoot of the CASK branch of the tree which arose from a gene duplication with subsequent loss of the CaMK-like and L27 domains (De Mendoza, 2010). Interestingly, Drosophila has no known MPP1 homolog, and it appears that the niche of this type of MAGUK has been filled by the short CASK gene product. It would therefore not be surprising if CASK-β and CASK-α had quite different roles. Indeed the transcripts encoding these two proteins have different developmental profiles. Elucidation of the functions of the MPP1-like isoforms awaits the generation of CASK-α-specific mutants and antibodies, but it is tempting to speculate that the high expression in ovaries might indicate that loss of CASK-α underlies the sterility phenotype of 307/313 flies (Slawson, 2011).
Loss of the CASK isoform containing the CaMK-like and L27 domains underlies the CASK locomotor deficit: Mutants lacking CASK-β displayed an obvious motor defect, which was further dissected using a high-resolution video tracking system. This analysis revealed a very complex defect, with deficits in four major areas: motor initiation, motor maintenance, speed, and acceleration. Furthermore, this defect is clearly dose-dependent, as the severity of the phenotype appears to change in a correlated fashion with the amount of CASK-β protein present in the animal, with CASKP18/+ heterozygotes being more normal than CASK P18 homozygotes, and with equivalent locomotor behaviors observed between these homozygous null flies and CASKP18/Df for three independent deficiency lines. Along with this, expression of CASK-β in a null fly rescues the behavioral deficit, also in a dose-dependent fashion; Gal4 lines with stronger expression can even make animals hyperactive. Taken together, these data indicate that the locomotor defect seen in these flies results from loss of CASK-β in the nervous system, and not from extragenic mutations that arose as a result of the P element excision (Slawson, 2011).
The fact that mRNA encoding CASK-α, a CASK gene product that contains the PDZ, SH3 and GUK domains of CASK-β, is still expressed in the CASKP18 mutant, suggests that there may be unique functions for the CaMK-like and L27 domains of the CASK-β form. The CaMK-like domain has been shown to have both biochemical activity (see Mukherjee, 2008) and specific binding partners, such as MINT1/Lin10 and CaMKII. The L27 domains also have specific binding partners such as DLG/SAP97 and Veli/Lin7. The inability of residual CASK-α to take over CASK-β function might also reflect a difference in localization of the two proteins, as CASK-α has a conserved palmitylation site at its very N-terminus, whereas CASK-β does not have such a motif. This assumes, however, that both CASK-α and CASK-β are expressed in the same populations of neurons, which can not be known for certain until better visualization tools for these proteins are developed (Slawson, 2011).
CASK-β functions in a pre-motor circuit: Although CASK-β is expressed throughout much of the nervous system, its role in locomotor behavior is restricted to a limited number of cells. The C164-Gal4 driver, which rescues locomotor behavior beyond wild type levels, has strong expression in only a subset of central neurons, including the antennal lobes, mushroom bodies, subesophageal ganglion (SOG), pars intercerebralis, and parts of the central complex (fan shaped body), while the periphery is completely devoid of expression. Interestingly, the ellipsoid body, which is known primarily for its role in locomotion, is not a region where the Gal4 protein is expressed with this driver, suggesting that CASK is not acting in this population of cells to rescue behavior (Slawson, 2011).
Strong CASK-β expression in glutamatergic cells with the OK371-Gal4 driver did not rescue locomotor behavior. This is an important finding because insect motor neurons are primarily glutamatergic, implying that this subpopulation of cells within the central nervous system is also not the site of action for CASK-β in locomotion. This finding is at odds with the conclusions of recent work, which has suggested that alterations in the regulation of neurotransmitter release at the NMJ in 307/313 larvae and adults underlie the defective motor behavior of the null. These experiments suggest that these NMJ defects (if they are indeed even present in the CASK-β-specific mutant) are not the basis of the locomotor problems demonstrated by CASKP18/+ flies. Instead, the site of action is within a premotor population of neurons in the central nervous system that does not include ellipsoid body cells (Slawson, 2011).
Judging by the expression pattern of C164-Gal4, the groups of neurons relevant for CASK-β action in locomotor behavior could include cells from the pars intercerebralis, mushroom bodies, thoracic ganglion interneurons or central complex structures such as the fan-shaped body or protocerebrum, all of which have been previously implicated in regulating insect motor activity. These cells could also include populations of antennal lobe neurons involved in sensory processing, or smaller groups of neurons, but are difficult to identify based on morphology and location alone. Behavioral rescue experiments using Gal4 lines with more restricted expression patterns will be necessary to elucidate the cells relevant for CASK-β action in locomotion. Along with this, the mechanisms behind proper subcellular localization of CASK-β within these cell populations will be of interest, as this could help determine potential binding partners and signaling cascades that interact with CASK-β (Slawson, 2011).
Loss of CASK-β does not impair olfactory habituation: Mutants lacking CASK-β display a lower courtship index and a longer courtship latency than control flies. This indicates that CASK-β mutants are less adept at finding the target fly, which could be explained by a reduced sensitivity to pheromonal cues. Surprisingly, however, when CASKP18/+ were tested for courtship habituation, which is a task requiring non-associative memory formation and olfactory processing, these flies performed similarly to control flies. This was seen when male CASKP18/+ flies were trained with either a decapitated target immature male or direct exposure to immature male pheromone. This finding suggests that both olfactory processing and plasticity remain intact in this assay following the loss of CASK-β. It should be noted that these results are specific to male-male courtship, and that plasticity defects involving other pheromonal cues or sensory modalities remain to be examined. 307/313 has additional chromosomal aberrations that affect behavior and fertility: In all behavioral assays, 307/313 flies perform very differently from CASK-β mutants in addition to being sterile. This is not surprising since 307/313 flies are transheterozygous for two overlapping deficiencies. These deficiencies eliminate CASK-α as well as CASK-β, and also contain mutations in genes besides CASK, which could have an effect on the resulting behavior of the flies. The low level of basal courtship observed in CASKP18/+ flies, which is likely attributable to locomotor problems, is far less severe than the deficit seen in 307/313 flies. Along with this, unlike the CASKP18/+ mutants, 307/313 flies display an abnormally high and unusually variable habituation index, consistent with previous work (LU et al. 2003). These additional problems of the 307/313 flies could reflect a reduction in olfactory sensitivity, or a short-term plasticity defect, stemming from the loss of CASK-α or from heterozygosity at other genes (Slawson, 2011).
Alternatively, these differences could also stem from the more severe courtship initiation defect observed in 307/313 flies, as a difficulty initiating any kind of movement could affect the reliability of training and testing. This idea is supported by the finding that 307/313 flies display a qualitatively different locomotor profile compared with CASKP18/+ flies. Importantly, multivariate analysis demonstrates that the individual parameters contributing to the qualitative difference between CASKP18/+ and 307/313 are primarily initiation parameters. This suggests that the loss of the MPP1-like CASK-α (or potentially genetic interactions between haploinsufficient loci) in 307/313 flies may confer a unique locomotor deficit. For this reason, 307/313 is not a good model for loss of CASK-β, the CaMK-like/L27-containing MAGUK, as it pertains to behavior (Slawson, 2011).
CASK and motor dysfunction: This work with CASK-β mutants shows that there is a clear motor phenotype resulting from loss of the Drosophila CASK homolog. These flies appear to suffer from problems with motor initiation, motor maintenance, speed, and acceleration. Such a complex deficit stemming from a higher-level region within the central nervous system suggests that CASK-β may work to allow integration of multiple parameters of locomotion together into coordinated movement. Not surprisingly, this strong locomotor phenotype also appears to affect other behavioral tasks involving a motor response, such as courtship and habituation (Slawson, 2011).
Many diseases such as Parkinson’s Disease and Huntington’s Disease are characterized by motor dysfunction that disrupts multiple motor parameters. Fly models for both of these movement disorders, as well as many others, have been developed and characterized, and show deficits similar to those of CASKP18/+ flies. Furthermore, recent work has suggested that molecular scaffolds like MAGUK family proteins, of which CASK is a member, interact directly or indirectly with many proteins thought to be associated with these diseases. Determining the role that scaffolds such as CASK play in such interactions may lead to a deeper understanding of motor disease and potentially provide a basis for development of novel therapeutics (Slawson, 2011).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Caki
Arredondo, L., (1998). Increased transmitter release and aberrant synapse morphology in a Drosophila Calmodulin mutant. Genetics 150(1): 265-274. Medline abstract: 9725845
Ashraf, S. I., McLoon, A. L., Sclarsic, S. M. and Kunes, S. (2006). Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila. Cell 124: 191-205. Medline abstract: 16413491
Atasoy, D., et al. (2007). Deletion of CASK in mice is lethal and impairs synaptic function. Proc. Natl. Acad. Sci. 104(7): 2525-30. Medline abstract: 17287346
Biederer, T. and Sudhof, T. C. (2000). Mints as adaptors. Direct binding to neurexins and recruitment of munc18. J. Biol. Chem. 275: 39803-39806. Medline abstract: 11036064
De Mendoza, A., Suga, H. and Ruiz-Trillo, I. (2010). Evolution of the MAGUK protein gene family in premetazoan lineages. BMC Evol Biol 10: 93. PubMed Citation: 20359327
Dimitratos, S. D., Woods, D. F. and Bryant, P. J. (1997). Camguk, lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains. Mech. Dev. 63 (1): 127-130. Medline abstract: 9178262
Doerks, T., et al. (2000). L27, a novel heterodimerization domain in receptor targeting proteins Lin-2 and Lin-7. Trends Biochem. Sci. 25: 317-318. Medline abstract: 10871881
Feng, W., Long, J. F. and Zhang, M. (2005). A unified assembly mode revealed by the structures of tetrameric L27 domain complexes formed by mLin-2/mLin-7 and Patj/Pals1 scaffold proteins. Proc. Natl. Acad. Sci. 102(19): 6861-6. Medline abstract: 15863617
Gerke, P., et al. (2006). Neuronal expression and interaction with the synaptic protein CASK suggest a role for Neph1 and Neph2 in synaptogenesis. J. Comp. Neurol. 498(4): 466-75. Medline abstract: 16874800
Harris, B. Z., Venkatasubrahmanyam, S., Lim, W. A. (2002). Coordinated folding and association of the LIN-2, -7 (L27) domain. An obligate heterodimerization involved in assembly of signaling and cell polarity complexes. J. Biol. Chem. 277(38): 34902-8. Medline abstract: 12110687
Hata, Y., Butz, S. and Sudhof, T. C. (1996). CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J. Neurosci. 16: 2488-2494. Medline abstract: 8786425
Hodge, J. J., Mullasseril, P. and Griffith, L. C. (2006). Activity-dependent gating of CaMKII autonomous activity by Drosophila CASK. Neuron 51(3): 327-37. Medline abstract: 16880127
Hong, C. J. and Hsueh, Y. P. (2006). CASK associates with glutamate receptor interacting protein and signaling molecules. Biochem. Biophys. Res. Commun. 351(3): 771-6. Medline abstract: 17084383
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date revised: 15 December 2011
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