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).
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
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
Irie M., et al. (1999). Isolation and characterization of mammalian homologues of Caenorhabditis elegans lin-7: localization at cell-cell junctions. Oncogene 18(18): 2811-7. Medline abstract: 99289097
Laverty, H. G. and Wilson, J. B. (1998). Murine CASK is disrupted in a sex-linked cleft palate mouse mutant. Genomics 53(1): 29-41. Medline abstract: 9787075
Lee, S., Fan, S., Makarova, O., Straight, S. and Margolis, B. (2002). A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia. Mol. Cell Biol. 22(6): 1778-91. Medline abstract: 11865057
Lehtonen, S., et al. (2005). Cell junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and alpha-actinin are components of the nephrin multiprotein complex. Proc. Natl. Acad. Sci. 102(28): 9814-9. Medline abstract: 15994232
Lopes, C., Gassanova, S., Delabar, J. M. and Rachidi, M. (2001). The CASK/Lin-2 Drosophila homologue, Camguk, could play a role in epithelial patterning and in neuronal targeting. Biochem. Biophys. Res. Commun. 284(4): 1004-10. Medline abstract: 11409895
Lu, C. S., Hodge, J. J., Mehren, J., Sun, X. X. and Griffith, L. C. (2003). Regulation of the Ca2+/CaM-responsive pool of CaMKII by scaffold-dependent autophosphorylation. Neuron 40: 1185-1197. Medline abstract: 8602221
Marble, D. D., et al. (2005). Camguk/CASK enhances Ether-a-go-go potassium current by a phosphorylation-dependent mechanism. J. Neurosci. 25(20): 4898-907. Medline abstract: 15901771
Martin, J. R. and Ollo, R. (1996). A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed. EMBO J. 15: 1865-1876. Medline abstract: 8617233
Mburu, P., et al. (2006). Whirlin complexes with p55 at the stereocilia tip during hair cell development. Proc. Natl. Acad. Sci. 103(29): 10973-8. Medline abstract: 16829577
Mehren, J. E. and Griffith, L. C. (2004). Calcium-independent calcium/calmodulin-dependent protein kinase II in the adult Drosophila CNS enhances the training of pheromonal cues. J. Neurosci. 24: 10584-10593. Medline abstract: 15564574
Merrill et al., et al. (2005). Activity-driven postsynaptic translocation of CaMKII. Trends Pharmacol. Sci. 26: 645-653. Medline abstract: 16253351
Nix, S. L., et al. (2000). hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions. J. Biol. Chem. 275: 41192-41200. Medline abstract: 10993877
Petrosky, K. Y., Ou, H. D., Lohr, F., Dotsch, V. and Lim, W. A. (2005). A general model for preferential hetero-oligomerization of LIN-2/7 domains: mechanism underlying directed assembly of supramolecular signaling complexes. J. Biol Chem. 280(46): 38528-36. Medline abstract: 16147993
Samuels, B. A., et al. (2007). Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK. Neuron 56(5): 823-37. PubMed citation: 18054859
Stetak, A., et al. (2006). Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development. EMBO J. 25(11): 2347-57. Medline abstract: 16688213
Tabuchi, K., Biederer, T., Butz, S. and Sudhof, T. C. (2002). CASK participates in alternative tripartite complexes in which Mint 1 competes for binding with caskin 1, a novel CASK-binding protein. J. Neurosci. 22(11): 4264-73. Medline abstract: 12040031
Wang, G. S., et al. (2004). Transcriptional modification by a CASK-interacting nucleosome assembly protein. Neuron 42(1): 113-28. Medline abstract: 15066269
Zordan, M. A., et al. (2005). Drosophila CAKI/CMG protein, a homolog of human CASK, is essential for regulation of neurotransmitter vesicle release. J. Neurophysiol. 94(2): 1074-83. Medline abstract: 15872064
date revised: 15 July 2008
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.