Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. A protein-protein interaction domain, called the L27 domain, has been identified which is present twice in mLin-2/CASK. This study reports the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identifies the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. A novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, contains a region highly related to the SAP97 N terminus and binds Camguk, a Drosophila orthologue of mLin-2/CASK. These data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice (Lee, 2002; full text of article).
A schematic portrayal of the recognized protein interaction domains in mLin-2/CASK shows the two L27 domains between the CKII domain and PDZ domain; an alignment of L27 domains is also portrayed. Included are the deduced amino acid sequences of MAGUKs, including mLin-2/CASK, Drosophila Camguk, PALS2/VAM-1, and mLin-7. This alignment revealed two adjacent regions of high homology, which were named the L27N and L27C domains and has been previously reported as L27A and L27B (Doerks, 2000), respectively. After examining its sequence similarity to the adjacent L27C domain, which mediates interaction with mLin-7, it was postulated that the L27N domain was also a novel protein-protein interaction domain that mediates homo- or heterodimerization. To test this hypothesis, several proteins consisting of fusions of GST and the L27N and L27C domains of mLin-2/CASK, mLin-7, and PALS2 were constructed and an in vitro affinity binding assay was performed to examine each domain's ability to bind either Myc-tagged mLin-2/CASK or mLin-7. GST fusion proteins containing both L27N and L27C were used as positive controls. The GST fusion protein containing the minimal L27C domain of mLin-2/CASK interacted with mLin-7 while the L27N domain, previously considered to be part of the mLin-7 interaction domain, did not. In addition, the minimal L27 domain of mLin-7 interacted with mLin-2/CASK. The assay also revealed that the L27N domain of mLin-2/CASK does not function as a homodimerization domain. Next, tests were performed to see whether the L27N domain of mLin-2/CASK interacts with another protein. The GST-mLin-2/CASK L27N domain bound to a protein doublet with a molecular mass of 120 to 130 kDa, identified as SAP97 (Lee, 2002).
It was of interest to determine whether or not the interaction between the N terminus of SAP97 and the L27N domain of mLin-2/CASK was conserved in other organisms. It was postulated that, if the L27N domain-mediated interaction between MAGUK proteins is one of the mechanisms that govern the targeting of membrane proteins, it should be evolutionarily conserved. To test the hypothesis, other proteins containing sequences that are similar to the first 75 aa residues of SAP97 were sought using BLAST analysis. As a result, a small Drosophila gene product, called CG1730 (accession no. AAF48037) was identified, which contained N-terminal sequences that are highly homologous to the N-terminal domain of SAP97. After analyzing the genomic sequence encoding CG1730 and its chromosomal location, it was realized that the sequence was located near the beginning of the gene encoding Drosophila Dlg (CG1725) . Subsequently the full-length CG1730 protein, containing 198 aa, was cloned by PCR using an EST (LP07807) as a template. The protein CPD, component protein of Dlg, was named for its potential role as a missing N-terminal domain of the Drosophila Dlg protein, which is also present in other orthologues such as C. elegans DLG-1. A sequence alignment of different SAP97 homologues reveals the high degree of sequence similarity within the first 65 aa (Lee, 2002).
The possibility was examined that a splice variant of the Drosophila Dlg gene might contain regions of the CPD gene due to their close genomic location. To identify the transcript, RNA from two different mixed stages of Drosophila embryos (0 to 8 h and 8 to 16 h) was isolated and reverse transcribed. The CPD gene-Dlg gene hybrid 5' end was amplified by reverse transcription-PCR from cDNA using primers specific for CPD gene sequences, and the 3' end was amplified by using primers specific for the Dlg (aa 1 to 493) coding sequence. When primers specific for sequences generating full-length CPD (aa 1 to 198) and full-length DLG (aa 1 to 978) were used as internal controls, all three fragments with the predicted sizes were amplified. Subsequent sequencing of those DNA fragments revealed the existence of a unique CPD-Dlg hybrid transcript consisting of the first 146 aa of CPD fused to aa 30 of Dlg, resulting in a protein of 1,094 amino acids (Lee, 2002).
Since the SAP97 N terminus mediates interaction with mLin-2 via the L27N domain, whether regions of CPD interacted with the L27N domain of mLin2/CASK as well as with Drosophila Camguk, the orthologue of mLin-2/CASK, was tested by using a GST fusion protein affinity binding assay. Bacterially expressed GST fusion proteins were incubated with lysate from MDCK cells stably expressing either Myc-mLin-2/CASK or HEK 293 cell lysate transiently expressing Myc-Camguk proteins (aa 1 to 500). Full-length CPD interacted with both mammalian and Drosophila proteins. The binding region was narrowed to the first 65 aa of CPD in agreement with the alignment. It was also determined that the first 26 aa residues were necessary for the interaction. Upon deletion of these residues, the interaction between CPD and either Camguk or mLin-2/CASK was significantly reduced or abolished, respectively. It was also found that the L27N domain of mLin-2/CASK is conserved in Camguk by demonstrating that the L27N domain of Camguk can bind CPD (Lee, 2002).
This study identified a novel interaction between the L27N domain of mLin-2/CASK and SAP97, a mammalian homologue of Drosophila Dlg tumor suppressor protein and another MAGUK family member. Recently, the SH3 domain of SAP97 was reported to associate with the GUK domain of mLin-2/CASK (Nix, 2000). However, these experiments showed that association between the L27N domain of mLin-2/CASK and the N terminus of SAP97 was stronger than the interaction between the GUK and SH3 domains of these proteins. It is possible that there is more than one point of attachment between these proteins. Furthermore, their association site may vary upon binding to other interaction partners since, like other MAGUK proteins, SAP97 is also a multidomain scaffolding protein that recruits transmembrane proteins and signaling molecules to the plasma membrane sites (Lee, 2002).
SAP97 contains three PDZ domains as well as one SH3 domain, one Hook domain, and one GUK domain. In neuronal cells, SAP97 is expressed in presynaptic membranes and mediates the clustering of receptor molecules, such as the voltage-gated Shaker K+ channels and the NR2 subunits of the N-methyl D-aspartate receptor, by interacting with PDZ domains. It is also expressed at the basolateral membrane of epithelial cells and at the lymphocyte immune synapse, but its precise role in epithelia remains to be characterized. The PDZ domains of SAP97 interact with adenomatous polyposis coli, PTEN tumor suppressor protein (1), and several viral oncoproteins, while the GUK domain interacts with the GKAP/SAPAP1/DAP1 family. There have been reports suggesting the possibility of oligomerization of SAP97 through the N-terminal protein sequences (aa 106 to 120), which are also conserved in other SAP proteins such as PSD-95 and SAP102. In addition, the functional significance of the N termini of these MAGUK proteins in targeting and receptor clustering has been previously demonstrated. The first 65 aa residues of SAP97 have been reported to direct selective subcellular localization and the attachment to the cytoskeleton assembly at sites of cell-cell contact. However, the protein that interacts with the N terminus of SAP97 and that is responsible for its targeting to the lateral membrane was unknown. This report using dominant-negative mLin-2/CASK revealed that the membrane localization of SAP97 was dependent on its association with mLin-2/CASK. When dominant-negative protein mLin-2/CASK(1-612) was overexpressed, SAP97 failed to properly localize to the lateral membrane, while the dominant-negative effect was removed upon deletion of the interacting L27N domain. The hypothesis that SAP97 basolateral membrane localization was mediated through its interaction with the mLin-2/CASK protein was further supported by demonstrating that expression of a dominant-negative mLin-2/CASK missing the Hook domain also results in mislocalization of SAP97 in the epithelia. However, it was difficult to show complete mislocalization of all SAP97 proteins due to high expression of the endogenous full-length mLin-2/CASK protein present in cells (Lee, 2002).
CaMKII is critical for structural and functional plasticity. Camguk (Cmg, also known as Caki, the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca2+/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca2+/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca2+/CaM, can provide a localized source of active kinase. When Ca2+/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses (Lu, 2003).
Cmg, mammalian CASK, and C. elegans Lin-2 form a MAGUK subfamily that is defined by an N-terminal CaMKII-like domain in addition to the SH3, PDZ, and guanylate kinase domains typical of the MAGUK family. In mammalian neurons, CASK has been shown to be localized to synapses both pre- and postsynaptically where it associates with cell surface molecules like neurexins, calcium channels, and syndecans, the cytoskeletal protein 4.1, PSD95, and other adaptor molecules. CASK may also participate in signaling to the nucleus by directly interacting with a transcription factor (Lu, 2003 and references therein).
Cmg null animals have impaired locomotion, but their ability to learn had not been tested. Modification of courtship behavior in Drosophila has been used to test both associative and nonassociative memory formation; CaMKII is required for both. Exposure of a male to a mated female leads to decreased courtship of a subsequently presented virgin. This suppression reflects formation of an associative memory that requires mushroom body and central complex circuits. Courtship suppression also occurs during the training period via a non-mushroom body central circuit. Habituation to a courtship stimulating cue can be demonstrated after training with immature males. In these behavioral assays, the locomotor defects of the Cmg null flies could only cause an underestimate of behavioral defects since 'failure' in the assay is signified by an increase in courtship activity. Wild-type, Cmg null, and cmg deficiency heterozygotes were tested for mated female conditioning. All genotypes were normal for associative memory formation when compared to males that had been sham conditioned in an empty chamber. Behavior during the training period, however, was abnormal; Cmg null males fail to decrease courtship during training. Deficiency heterozygotes are intermediate. To determine if the Cmg null has a habituation defect, the response to immature males, who emit courtship promoting pheromones distinct from the female type, was examined. Cmg null animals fail to habituate. Courting Cmg null males have normal initial courtship levels and the ability to discriminate between virgin and mated females, indicating that peripheral sensory pathways are intact (Lu, 2003).
Association of Cmg with CaMKII is stimulated by ATP bound to the CaMKII catalytic domain. At physiological levels of ATP, Cmg and CaMKII are likely to be associated. ATP hydrolysis in the presence of Ca2+/CaM leads to autophosphorylation of CaMKII on T287 and production of a Cmg-localized constitutively active kinase. Cmg can also promote ATP hydrolysis by CaMKII in the absence of Ca2+/CaM. Under these conditions and without prior T287 autophosphorylation, CaMKII preferentially autophosphorylates its own CaM binding domain. This reaction leads to the fast release of the CaMKII in a form that cannot be activated by Ca2+/CaM. The ability of genetic manipulation of Cmg, PP2A, and synaptic activity levels in vivo to alter the amount of postsynaptic CaMKII that is in the inactivated state argues strongly that these reactions are physiologically relevant and define a new mechanism for the regulation of CaMKII activity (Lu, 2003).
What is the function of this interaction in vivo? One possible role is to provide a basis for maintaining differences between active and inactive synapses. The effect of cmg on courtship conditioning, a behavior known to require CaMKII, is suggestive of a role for this interaction in behavioral plasticity. Differences in synaptic strength can fine tune networks and are crucial to most memory models. Local CaMKII inactivation by Cmg could serve to decrease the gain on CaMKII-mediated calcium signaling at synapses that have been inactive. At low Ca2+, T306 autophosphorylation would occur, and inactive kinase would be released from Cmg. This provides a 'use it or lose it' mechanism for preserving synapse-specific strength differentials, since CaMKII tethered to Cmg at synapses that see higher Ca2+ levels would be protected from inactivating autophosphorylation by bound CaM. This complexed kinase can undergo T287 autophosphorylation, resulting in constitutive kinase activity and a slowed CaM dissociation rate that would further prolong the lifetime of the complex. Whether the Cmg-mediated inactivation of CaMKII operates locally or globally remains to be determined and likely depends on the ability of the inactivated kinase to escape from complexes with its other binding partners in the postsynaptic density (Lu, 2003).
Establishment of a pool of autophosphorylated CaMKII that cannot be directly stimulated by Ca2+/CaM also provides a mechanism for phosphatase activity to regulate CaMKII. In vitro, both PP1 and PP2A are able to dephosphorylate the CaM binding domain of CaMKII. In Drosophila, PP2A is the relevant in vivo phosphatase for postsynaptic pT306. The opposing actions of Cmg and PP2A provide machinery for dynamic regulation of the maximum level of calcium-activatable CaMKII at synapses (Lu, 2003).
In mouse, knockin of a CaMKII T305D mutation blocks LTP and learning and decreases postsynaptic localization of the kinase, suggesting that this mutant is unable to interact with one or more of its normal synaptic binding partners. Inability to undergo inactivating autophosphorylation (T305VT306A knockin) is associated with a lower threshold for LTP and loss of behavioral fine tuning. These data argue that, in mammals, autophosphorylation of the CaM binding domain of CaMKII is also an important regulatory mechanism for both function and localization of CaMKII. Whether in mammals CASK (the Cmg homolog) is the critical synaptic binding partner and regulator of synaptic T305 autophosphorylation is unknown (Lu, 2003).
This study also demonstrates a novel binding mechanism for CaMKII on a MAGUK scaffolding protein. It is proposed that this interaction consists of two discrete steps. First, binding of ATP to the catalytic domain of CaMKII reveals an interaction surface on the kinase that forms an initial bond to the Cmg protein. This initial interaction can occur either in the presence or absence of Ca2+/CaM, implying that it does not involve the C-terminal CaM binding sequences of the autoinhibitory domain. The ability of a peptide corresponding to the N terminus of the autoinhibitory domain to partially block complex formation suggests that this region, which is known to be involved in regulation of ATP binding or the catalytic domain surface that the N terminus of the autoinhibitory domain binds to, is the site of this initial interaction (Lu, 2003).
Once an initial interaction is established, bound ATP is no longer required. This could be due to a stabilization of the ATP-bound conformation by Cmg binding. Stabilization of the open form of the kinase autoregulatory domain is postulated to occur after Ca2+/CaM-dependent NR2B binding, since this interaction is insensitive to CaM stripping. Another possibility for the loss of ATP requirement is that a secondary interaction occurs that abrogates the need for the initial interaction. Such a secondary interaction does occur, but this does not rule out a concurrent stabilization of the ATP-bound conformation (Lu, 2003).
The secondary interaction that forms after ATP-dependent binding of Cmg involves the C-terminal region of the autoinhibitory domain, including the CaM binding sequences. Three lines of evidence suggest that this secondary interaction provides the most stable form of the complex. (1) N-terminal peptides only partially block complex formation, while peptides that contain the CaM binding domain completely block complex formation. (2) CaMKIIs with mutations of T306/7 do not bind to Cmg at all. These mutants are fully capable of binding ATP, but the exposure of the initial binding domain is insufficient to maintain a salt-stable interaction in the absence of a normal CaM binding domain. The failure of the relatively subtle threonine to serine mutant to bind suggests a direct interaction of Cmg with T306/7. (3) The natural release mechanism of this complex is via autophosphorylation of the CaM binding domain. Protection of this region by Ca2+/CaM can prevent both the autophosphorylation and dissociation of CaMKII (Lu, 2003).
The two-step association mechanism and the autophosphorylation-regulated dissociation of CaMKII from Cmg provide a new way in which CaMKII can be localized and regulated using its autoinhibitory domain. As defined for catalytic activation, the regulatory region of CaMKII is a small but complicated protein interaction domain. It is becoming clear that this region can support multiple intra- and intermolecular interactions that have profound effects on local CaMKII activity and synaptic plasticity (Lu, 2003).
Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. This study demonstrates that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-a-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium- and calmodulin-dependent protein kinase II. CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. These findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel (Marble, 2005; full text of article).
Mutations have been generated in the single Drosophila Calmodulin gene and the effects of these mutations then examined on behavior,
synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam3c1, a mutation in the first Ca2+-binding site, exhibit behavioral,
neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight.
The effects on motor neuron function and transmitter release of many behavioral mutations, particularly those affecting ion channels, are enhanced by application of the K+ channel-blocking drug quinidine.
This drug, when applied at a concentration of 0.1 mM, completely and specifically blocks the delayed rectifier K+ current in the Drosophila larval muscle. Application of quinidine enhances the effects of ion channel mutations, such as Shaker and Hyperkinetic, on the duration of
motor nerve terminal depolarization and transmitter release. The phenotypes of
other excitability mutants, such as inebriated and pushover, are also enhanced by quinidine application. Application
of quinidine has no significant effect on excitatory junctional current (EJC) amplitude in the wild-type control larvae or in larvae
heterozygous for Cam mutations. In contrast, quinidine application to Cam3c1/Camnull at the three lowest external [Ca2+] levels tested causes an approximately threefold increase
in EJC amplitude. The muscle responsiveness to neurotransmitter is shown to be normal, and that the increased amplitude EJCs observed in
Cam3c1/Camnull larvae reflect increased transmitter release. One might imagine that impaired activation of a K+ channel in Cam3c1/Camnull might have no observable phenotypic consequences under otherwise normal
conditions; however, in combination with quinidine, which could block a distinct, functionally redundant K+ channel, this effect could lead to increased nerve terminal depolarization and increased Ca2+ influx
into the nerve terminal. The observation that the effects of Cam3c1/Camnull occur only at low external
[Ca2+] is consistent with this view: the effects of most excitability mutations, including Shaker, Hyperkinetic, and inebriated, are also revealed only at low external [Ca2+]. This observation has been
proposed to result from the activation of a Ca2+-activated K+ current at higher external [Ca2+]. Alternatively, Cam3c1/Camnull could be defective in Ca2+ buffering. In this view,
a broadening of the action potential conferred by quinidine application combined with reduced Ca2+ buffering
as a consequence of Cam3c1/Camnull could lead to increased transmitter release (Arredondo, 1998).
Synaptic bouton structure at motor nerve terminals is altered.
Structural abnormalities are present in the nerve
terminals of Cam3c1/Camnull larvae in muscle 13 of abdominal segments 3 - 5. In
particular, rather than cascading into a string of distinct type I and II boutons as in the control larvae,
the terminal arbor of the Cam3c1/Camnull larvae forms a thickened, or large, misshapen structure with
few distinct boutons. The large structure results in a reduced number of boutons and a nearly complete
lack of terminal branching in muscle 13. In comparison to control larvae, no abnormalities in the structure of nerve terminals on muscles 6, 7, or 12 have been observed. Despite this altered synapse morphology, muscle 13 synaptic transmission in Cam3c1/Camnull larvae resembles the muscle 6 properties. This observation raises the possibility that this defective bouton might still be functional. Although mutations in several neuronal signaling genes confer defects in the pattern of motor neuron
innervation of the target muscle, the morphological defects of Cam3c1/Camnull at the nerve terminal
differ from any that have been reported previously and do not appear to result from the same mechanisms as the "activity-dependent" increases in synaptic bouton number or axonal branching Rather, the phenotype observed appears to
result from defects in the formation of distinct boutons at the proper locations along the muscle surface. It is unclear why this defect in bouton formation is observed only in muscle 13. Proper synapse formation on muscle 13 may be more sensitive to altered CaM function, or perhaps distinct mechanisms control bouton formation in muscle 13 vs. other muscles. Analysis of the genes required for proper bouton formation is less characterized than for axon pathfinding or growth cone guidance. The results presented here suggest a role for CaM in this process (Arredondo, 1998).
These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and
CaM-activated adenylyl cyclase (CaMAC or Rutabaga). Mutations
in rutabaga reduce facilitation and post-tetanic potentiation at the larval neuromuscular junction, whereas activation of an inhibitory domain of
CaMKII confers a number of behavioral, electrophysiological, and anatomical defects, including defects in courtship conditioning, an abnormal spontaneous firing of motor axon action potentials, increases in axon branching and transmitter release, and a reduction in facilitation and augmentation. Cam3c1/Camnull larvae do not show these phenotypes; e.g., Cam3c1/Camnull larvae possess a normal number of axon branches and display normal EJC amplitude (in the absence of quinidine) and normal paired pulse facilitation. Similarly, whereas application of quinidine to Cam3c1/Camnull substantially increases evoked transmitter release, quinidine application
has little or no effect on CaMKII-inhibited larvae. Thus, it is
unlikely that the Cam3c1 mutation is exerting its effects via either CaMAC or CaMKII, but rather via
an alternative target. One alternative target, the Drosophila CaM-activated protein kinase Caki, is expressed in the central nervous system; mutants defective in caki exhibit behavioral defects
related to those described here (Martin, 1996). The catalytic domain of Caki shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is
enzymatically active. 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'. Thus, the
Cam3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target, distinct from CaMKII or CaMAC (Arredondo, 1998).
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