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Originally published In Press as doi:10.1074/jbc.M310728200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 11, 10206-10214, March 12, 2004
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The eag Potassium Channel Binds and Locally Activates Calcium/Calmodulin-dependent Protein Kinase II*

Xiu Xia Sun{ddagger}, James J. L. Hodge{ddagger}, Yi Zhou§, Maidung Nguyen, and Leslie C. Griffith{ddagger}||

From the {ddagger}Department of Biology and Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454-9110 and the §Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074

Received for publication, September 29, 2003 , and in revised form, December 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of neuronal excitability in many systems. Recent studies suggest that local regulation of membrane potential can have important computational consequences for neuronal function. In Drosophila, CaMKII regulates the eag potassium channel, but if and how this regulation was spatially restricted was unknown. Using coimmunoprecipitation from head extracts and in vitro binding assays, we show that CaMKII and Eag form a stable complex and that association with Eag activates CaMKII independently of CaM and autophosphorylation. Ca2+/CaM is necessary to initiate binding of CaMKII to Eag but not to sustain association because binding persists when CaM is removed. The Eag CaMKII-binding domain has homology to the CaMKII autoregulatory region, and the constitutively active CaMKII mutant, T287D, binds Eag Ca2+-independently in vitro and in vivo. These results favor a model in which the CaMKII-binding domain of Eag displaces the CaMKII autoinhibitory region. Displacement results in autophosphorylation-independent activation of CaMKII which persists even when Ca2+ levels have gone down. Activity-dependent binding to this potassium channel substrate allows CaMKII to remain locally active even when Ca2+ levels have dropped, providing a novel mechanism by which CaMKII can regulate excitability locally over long time scales.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium/calmodulin-dependent protein kinase II (CaMKII)1 has been shown to regulate ion channels and neuronal excitability in both vertebrates (1–6) and invertebrates (4, 5, 7–11). Regulation of excitability has been proposed as a mechanism by which neurons can globally modify their firing to keep spike rates in a scalable range in the presence of synapse-specific plasticity (12–14). Recently, it has become clear that regulation of excitability can also occur as a local phenomenon (15–19). Regional changes in excitability mediated by differing levels of A-type potassium currents were shown to be important for gating Hebbian plasticity in CA1 pyramidal cell dendrites and limiting propagation of action potentials (19). These effects have important implications for the computational abilities of neurons that integrate over many inputs. Both slow, expression level changes and fast, enzymatic modifications could underlie local phenomena, and in CA1, local regulation of excitability was associated with both regional differences in current density (19) and region-specific post-translational modifications of ion channels (18). Fast modulation offers the advantage of dynamic retooling of the computational capability of the neuron.

For signal transduction pathways to effect fast local changes in excitability, the activity of effector enzymes has to be spatially restricted. The ability of protein kinases with many substrates to act with specificity to regulate cellular functions is increasingly being ascribed to interactions with scaffolding molecules that bring kinases and their substrates into close proximity. Signaling platforms range from small complexes containing only a few proteins to very complex cellular specializations like the postsynaptic density of the mammalian central nervous system (20). The advantages of scaffolding enzymes and substrates include enhancement of reaction rates because of elimination of diffusional barriers and increased local concentration of substrates and segregation of the enzyme to prevent reaction with other substrates. Localization of effector enzymes to sources of small molecule regulators can also establish very restricted signaling domains. Scaffolding may be particularly important for regulation of membrane-bound proteins in neurons; diffusion in the membrane bilayer is relatively slow, and the ability of soluble enzymes to access substrates can be influenced by the complex geometry of neurons.

The regulation of firing in Drosophila motor axons involves the ether-à-go-go (eag) potassium channel. eag is the founding member of an evolutionarily conserved superfamily of voltage-gated potassium channels with homology to cyclic nucleotide-gated channels (21). In adult flies, mutation of eag causes ether-induced shaking (22) and memory formation defects (7) and in larvae, spontaneous axonally generated excitatory junctional potentials in the body wall muscle and supernumerary responses to nerve stimulation (23). Genetic interactions between eag and the CaMKII ala inhibitor peptide transgene suggested that CaMKII may be a regulator of Eag (7). Biochemical and electrophysiological studies demonstrated that current amplitude is regulated by phosphorylation of Thr-787 by the kinase (11). In this study, we investigate whether CaMKII can locally regulate Eag function. We find that, in addition to providing a scaffold for CaMKII, Eag is also an activator of the kinase. The interaction of these two proteins provides a localized source of CaMKII that remains active, in the absence of calcium, for the lifetime of the complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Anti-CaMKII rabbit polyclonal antibodies were raised against the C-terminal 13 amino acids of the Drosophila CaMKII (24) and against a GST fusion protein containing the CaMKII catalytic domain (25). Anti-CaMKII mouse monoclonal antibody (26) was a gift of Yoshiki Takamatsu (Tokyo Metropolitan Institute for Neuroscience). Anti-Eag rabbit polyclonal antibodies were raised against the last 143 amino acids of Eag (amino acids 1032–1174) fused to GST. Eag antibodies were affinity purified on a maltose affinity column loaded with a fusion of maltose-binding protein and Eag(1032–1174).

Other reagents used include 0.5 µg/ml biotinylated CaM (biotin-CaM, generated using NHS-biotin (Pierce) according to the manufacturer's instructions), horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, anti-rabbit and anti-mouse), streptavidin-horseradish peroxidase (Jackson ImmunoResearch Laboratories), fluorescein isothiocyanate- and Cy5-conjugated anti-rabbit Ig (Jackson ImmunoResearch Laboratories).

Plasmids and Construction—PCR products of Eag fragments, with the exception of Eag-C2 (11), were cloned between the BamHI and EcoRI sites of pGEX-2T (Amersham Biosciences) to make GST fusion proteins. PCR-based site-directed mutagenesis was done using the QuikChange kit (Stratagene) to produce point mutations in GST-Eag-C1. Bac-to-Bac pFastBac plasmids containing wild type CaMKII were a gift from Neal Waxham, University of Texas at Houston School of Medicine. Recombinant virus used for infection was produced using the pFastBac plasmid in monolayers of Sf21 cells as described by the manufacturer. All mutations were verified by sequencing (Brandeis Core Sequencing Facility).

Fly Strains—Flies were grown on standard medium at 25 °C. Canton S was used as the wild type strain. Panneural expression was achieved by crossing the C155-GAL4 driver line (27) with either UAS-T287A, UAS-T287D (28) or UAS-GFP-nSyb (29). eagSC29 is a null allele and was a gift of Barry Ganetzky (University of Wisconsin, Madison).

Coimmunoprecipitation from Fly Head Extracts—Flies were frozen in liquid N2 and decapitated by vortexing. Heads were separated from bodies with a sizing sieve. 90 µl of fly heads was homogenized in 500 µl of solubilization buffer (50 mM Tris, pH 8.0, 0.15 M NaCl, 0.1% SDS, and 0.1% Triton X-100 with Complete protease inhibitor mixture (Roche Applied Science), and either 4 mM CaCl2 or 1 mM EDTA and 4 mM EGTA). After centrifugation for 5 min at 13,000 rpm, pellets were homogenized one more time. Combined supernatants and pellets were rotated at 4 °C for 1 h and centrifuged again. The supernatant was then incubated with 5 µl anti-Eag antibody at 4 °C for 1 h, and the immunocomplex was precipitated by incubating for another 1 h with 40 µl of 50% protein A/G-agarose slurry (Santa Cruz). After three washes with the solubilization buffer, proteins bound to beads were resolved by 7.5% SDS-PAGE. The gel was cut into two halves; the upper half with proteins above 60 kDa was transferred electrophoretically to nitrocellulose at 230 mA for 40 min and probed with anti-Eag and the lower half transferred at 230 mA for 30 min and probed with biotin-CaM or mouse anti-CaMKII. Bands were visualized with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit-horseradish peroxidase for anti-Eag and streptavidin-horseradish peroxidase for biotin-CaM) and enhanced chemiluminescence reagents (ECL, Amersham Biosciences).

Expression and Purification of GST Fusion Proteins—Overnight bacterial cultures of Escherichia coli BL21 transformed with the appropriate plasmid were diluted 1:100 in LB broth with 100 µg/ml ampicillin and grown at 37 °C until A600 = 0.6. Protein expression was induced with 0.2 mM isopropyl-1-thio-{beta}-D-galactopyranoside at 37 °C for 4 h. Cells were harvested by centrifugation at 6,000 x g for 10 min.

To purify proteins, 50-ml cell pellets were resuspended in 5 ml of cold lysis buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride) with 0.5 mg/ml lysozyme and incubated on ice for 15 min. 1% Triton X-100 was added, and the mixture was sonicated for 5 x 10 s. After spinning at 10,000 x g for 10 min, the pellet was resuspended in 3 ml of lysis buffer including 0.6% sarcosyl and incubated at 4 °C until clear; then 180 µl of 20% Triton X-100 was added. Glutathione-Sepharose 4B beads equilibrated with lysis buffer were added and incubated at 4 °C for 2 h or overnight. Beads were washed with 10 volumes of buffer (0.2 M Tris, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 0.25% Nonidet P-40, and 1 mM Na3VO4) three times. Beads were then either washed twice with 20 mM sodium orthophosphate, pH 7.2, 0.1% Triton X-100, and 0.15 M NaCl for binding assay or eluted with freshly made elution buffer (50 mM Tris, pH 8.5, 0.1 M NaCl and 20 mM reduced glutathione) followed by dialysis against 1 x phosphate-buffered saline with 10% glycerol to obtain soluble proteins.

Expression and Purification of CaMKII—CaMKII was purified from cells utilizing a protocol adapted from Putkey and Waxham (30). Sf21 cells (a gift of Neal Waxham) at a density {approx} 1.5 x 106 were infected by baculovirus at a multiplicity between 2 and 10. Cell pellets collected after growing for 2–3 days were washed with cold phosphate-buffered saline. Cells were either frozen at –80 °C or resuspended immediately in 10 volumes of 40 mM HEPES, pH 7.5, 5% betaine, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 5 mg/liter soybean trypsin inhibitor, and 20 mg/liter leupeptin with 10 strokes of a glass/Teflon homogenizer and clarified by centrifugation at 100,000 x g at 4 °C for 1 h. The proteins were precipitated from the supernatant with ammonium sulfate (50% final), resuspended in starting buffer (40 mM HEPES, pH 7.5, containing 2 mM CaCl2, 0.15 M NaCl, and 10% glycerol and protease inhibitors), and applied to a CaM-Sepharose column. The column was washed with starting buffer, then starting buffer with 2 M NaCl instead of 0.15 M and washed then again with starting buffer. The bound proteins were eluted with 40 mM HEPES, pH 7.5, 2 mM EGTA, 0.5 M NaCl, 10% glycerol and dialyzed into storage buffer (10 mM HEPES, pH 7.5, 0.1 mM EGTA, 100 mM KCl, 50% glycerol) then frozen as aliquots at –80 °C.

Binding Assays with Immobilized GST Fusion Proteins—20-µl beads conjugated to GST-Eag fusion protein were incubated with 100 µl of binding mix (200 nM kinase, 50 mM PIPES, 15 mM MgCl2, 20 mM sodium orthophosphate, pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 0.5 mg/ml bovine serum albumin, and either 1 mM CaCl2 and 3 µM CaM or 1 mM EGTA) at 4 °C for 1–2 h. Beads were then washed with 10 volumes of buffer (50 mM PIPES, 15 mM MgCl2, 20 mM sodium phosphate, pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 0.5 mg/ml bovine serum albumin, and either 1 mM CaCl2 or 1 mM EGTA or 1 mM CaCl2 and 3 µM CaM as indicated) three or four times. Eag-bound CaMKII was eluted by boiling in 1x SDS-PAGE loading buffer followed by separation using SDS-PAGE and immunoblotting with anti-CaMKII antibodies.

Kinase Activity and Protein Phosphorylation Assay—Wild type CaMKII or the T287A mutant was bound to immobilized GST-Eag-C2, and CaM was removed with EGTA washes. The activity of soluble or Eag-bound kinase was assayed as described previously (31). CaMKII, GST-Eag, or CaMKII bound to Eag was phosphorylated in an assay reaction of 50 µl containing 50 mM PIPES, pH 7.0, 15 mM MgCl2, 1 mM CaCl2, 10 µg/ml calmodulin, and 50 µM [{gamma}-32P]ATP (1 Ci/mmol). Reactions were started by the addition of hot ATP and run at 30 °C for 1 min and stopped by adding 50 µl of 2x SDS sample loading buffer. Samples were heated at 100 °C for 3 min before separation by SDS-PAGE. Gels were dried and subjected to autoradiography.

Immunohistochemistry—Third instar larval brains were dissected and stained as described previously (32). Images were acquired using the 20x objective of a Leica TCS SP2 confocal scanning microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Eag and CaMKII Coimmunoprecipitate in a Calcium-dependent Manner from Drosophila Heads—Eag is a substrate of CaMKII in vitro and in vivo (7, 11). To determine whether these two proteins were part of a stable signaling complex, we immunoprecipitated Eag from extracts of adult Drosophila heads, separated the precipitated proteins by SDS-PAGE, and probed for CaMKII using either mouse anti-CaMKII (Fig. 1A, top panel) or biotin-CaM overlay (Fig. 1A, bottom panel). CaMKII is coimmunoprecipitated with Eag from wild type flies, whereas immunoprecipitates from eagsc29 flies, which do not express any Eag protein or mRNA (33), do not contain CaMKII. The ability to bring down CaMKII was enhanced by homogenizing tissue in a buffer containing calcium. In the top panel of Fig. 1A, immunoprecipitations were done in the presence of calcium. In the bottom panel immunoprecipitation from calcium-containing extracts was compared with pull-down from extracts made in the presence of EGTA and EDTA. The multiple bands detected in the 50-kDa range by mouse anti-CaMKII and biotin-CaM overlay likely correspond to different alternative splice isoforms of CaMKII (24).



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  		FIG. 1.
CaMKII and Eag interact in vivo. A, coimmunoprecipitation of CaMKII and Eag from fly head extracts is enhanced by calcium. Fly head extracts from adult wild type Canton S and Eag-null eagsc29 flies were immunoprecipitated (IP) with rabbit anti-Eag and protein A/G-Sepharose beads in buffer including either 4 mM CaCl2 or 1 mM EDTA and 4 mM EGTA. Bound proteins were resolved by 7.5% SDS-PAGE and transferred to nitrocellulose. Eag was detected by immunoblotting with rabbit anti-Eag (1:2,000). Top panel, immunoprecipitations were done in the presence of calcium and CaMKII detected by immunoblotting (1:30) with an anti-CaMKII mouse monoclonal antibody (26). Bottom panel, immunoprecipitations were done both with and without calcium. CaMKII was detected by biotin-CaM overlay and streptavidin-horseradish peroxidase detection. Calcium enhanced CaMKII coimmunoprecipitation. In both panels, CaMKII was coimmunoprecipitated only in wild type (WT) extracts. In biotin-CaM overlays a background band of approximately 50 kDa which comigrates with the lowest molecular mass CaMKII isoform is seen in both wild type and Eag-null immunoprecipitations. Increased intensity of this band in the eag-null lanes results from a longer exposure time. n >= 3 for all experiments, and representative data are shown. B, Eag is localized to axons and synapses in the Drosophila central nervous system. Left panels, wild type Canton S and Eag-null eagsc29 larvae were stained with anti-Eag (1:1,000). Staining was visualized with fluorescein isothiocyanate-conjugated anti-rabbit Ig (1:150). No staining is seen in mutant animals. Right panels, C155-GAL4; UAS-GFP-nSyb/+ third instar larvae were stained with anti-Eag (1:1,000), and staining was visualized using Cy5-conjugated anti-rabbit Ig (1:150). GFP and antibody images were acquired using the 20x objective of a Leica TCS SP2 confocal scanning microscope. Scale bars = 40 µm.

 
Eag Is a Synaptic Protein in Drosophila Central Neurons—To determine whether Eag is present in neurons and at synapses, we stained third instar larval brains with antibodies directed against Eag. Although Eag has been studied in Drosophila for many years, its distribution has not been described previously. Fig. 1B (left panel) shows wild type and eagsc29 brains stained for Eag. Staining is seen primarily in the neuropil (synaptic area) and axon tracts in the wild type animal, with no staining in eagsc29 brains, which do not express Eag. Colocalization of Eag staining with the synaptic marker GFP-nSyb (29), which is expressed under control of the panneural C155-GAL4, demonstrates that Eag is present at neuronal synapses (Fig. 1B, right panel). The synaptic localization of Eag puts it in the same cellular compartment as CaMKII, which has been shown to be a synaptic protein in both vertebrates (34) and invertebrates (26).

CaMKII Requires Ca2+/CaM to Initiate, but Not Sustain, Binding to Eag—The calcium-dependent interaction of CaMKII with Eag was investigated further using an in vitro binding assay. Purified CaMKII (R3 isoform) was mixed with glutathione-Sepharose beads that had been conjugated to a GST fusion protein containing the entire cytoplasmic C terminus, amino acids 556–1174, of Eag (GST-Eag-C2) in the presence of Ca2+/CaM or EGTA, and washed with buffers containing Ca2+/CaM, Ca2+, or EGTA as indicated. Beads conjugated to GST were used as a control for nonspecific binding. In other experiments, the cytoplasmic N terminus of Eag was found not to interact with CaMKII (data not shown). Bound proteins were separated by SDS-PAGE and CaMKII and CaM detected by immunoblotting. In Fig. 2 it can be seen that CaMKII cannot associate with GST-Eag-C2 when there is no Ca2+/CaM in the binding buffer. If allowed to interact initially in Ca2+/CaM-containing buffer, washes with Ca2+ alone allow both CaMKII and CaM to remain bound. Once formed, the CaMKII·Eag complex is stable even after EGTA washes, which effectively strip CaM from the complex. GST alone does not bind CaMKII even in the presence of Ca2+/CaM.



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  		FIG. 2.
CaMKII and Eag interact in a Ca2+/CaM-dependent manner in vitro. Binding of CaMKII to the full-length Eag C terminus is Ca2+/CaM-dependent. CaMKII was incubated with glutathione-Sepharose beads conjugated to GST-Eag-C2 (a GST fusion protein containing amino acids 556–1174 of Eag) or GST alone in the presence of EGTA or Ca2+/CaM and washed with buffers containing EGTA, CaM, and/or Ca2+. Bound protein was tested for CaMKII and CaM by immunoblotting. Ca2+/CaM is necessary to initiate binding of CaMKII to GST-Eag-C2; however, it is not necessary to sustain the complex because binding persists after even washes with EGTA which effectively strip CaM from the complex. During washes with Ca2+ but without CaM, CaM remained bound to the complex. GST alone does not bind CaMKII under any conditions tested. Residual CaM in the GST control (fourth lane) is the result of the carryover from CaM present in the last wash; CaM does not bind specifically to GST (data not shown). n >= 3, representative data are shown.

 
Consistent with the in vivo coimmunoprecipitation result that multiple isoforms of CaMKII appeared to associate with Eag, in vitro binding assays also showed that all tested CaMKII isoforms (R1, R2, R3, R3A, R4, R5, and R6; see Ref. 24) can interact with GST-Eag-C2 in the presence of Ca2+/CaM but not when bound in an EGTA buffer (data not shown).

Deletion Mapping Defines the CaMKII-binding site on Eag— Deletion analysis was used to define the minimal region of GST-Eag-C2 necessary to bind to CaMKII (Fig. 3). CaMKII was bound in the presence of Ca2+/CaM and beads washed with a Ca2+-containing buffer. Bound CaMKII was detected by immunoblotting. Binding ability mapped to amino acids 772–803 of the C terminus (Fig. 3, B and C). A further truncation to 794 (data not shown) was also found to bind CaMKII. Sequence analysis revealed a high degree of homology to the CaMKII autoinhibitory domain over the amino acids 773–794 region (see Fig. 6A). In CaMKII, this domain interacts directly with the catalytic domain of the kinase to suppress activity (35). Binding of CaM to the kinase disrupts this interaction and allows substrate access and autophosphorylation. This suggests that Eag may bind to the CaMKII catalytic domain on the interaction face used by the autoregulatory domain of the kinase itself. This mechanism of kinase binding is similar to that demonstrated for the N-methyl-D-aspartate receptor NR2B subunit (36).



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  		FIG. 3.
Localization of the CaMKII binding site in the C terminus of Eag. A, transmembrane topology of Eag. The CaMKII-binding domain (residues 773–794) is highlighted. B, GST fusion constructs encoding truncated portions of the C-terminal region of Eag were tested for their ability to bind to CaMKII. The amino acids included in each construct are as indicated. The ability to bind with CaMKII is indicated by a plus or minus sign. C, purified CaMKII was mixed with glutathione-Sepharose beads conjugated to the GST fusion constructs of Eag indicated in the presence of Ca2+/CaM for 2 h, and then beads were washed with buffer containing Ca2+. CaMKII was detected by immunoblotting with anti-CaMKII (1:1,000). n >= 2 for all constructs, and representative data are shown.

 



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  		FIG. 6.
Mapping of residues critical for the interaction of CaMKII with Eag. A, interaction of Eag point mutants with wild type CaMKII. Top panel, homology between Eag CaMKII-binding domain and the autoinhibitory domain of CaMKII is shown by boxed residues. Dots indicate residues of CaMKII which have been shown to be important for the interaction of the autoinhibitory domain and catalytic domains (42). Amino acids in GST-Eag-C1 were mutated individually (*) or in pairs (*). Middle panel, CaMKII binding was performed as in Fig. 3C. Bottom panel, Coomassie staining is shown to demonstrate equivalent loading of Eag fusion proteins onto glutathione-Sepharose beads. B, interaction of CaMKII catalytic domain point mutants with GST-Eag-C2. CaMKII was incubated with glutathione-Sepharose beads conjugated to GST-Eag-C2 in the presence of Ca2+/CaM or EGTA and washed with buffer containing either Ca2+ or EGTA corresponding to the initial condition. Bound kinase was detected by immunoblotting with anti-CaMKII (1:1,000). WT, wild type.

 
Direct Binding of CaM to Eag Is Not Required for CaMKII Binding or Phosphorylation—Ca2+/CaM could exert its positive effects on the CaMKII/Eag interaction either by binding to CaMKII or to Eag. Human hEAG1 has been shown to be inhibited by direct Ca2+/CaM binding (37). Drosophila Eag has very high homology to hEAG1 over this region (amino acids 725–744), which is just proximal to the CaMKII-binding site (Fig. 4A). Association of CaM with GST-Eag-C1 can be completely blocked by mutations of critical phenylalanines in this motif to serine (Fig. 4A, bottom panel).



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  		FIG. 4.
Direct binding of CaM to Eag does not affect Thr-787 phosphorylation. A, top panel, Drosophila Eag shows high homology to hEAG1 in the region demonstrated to bind CaM in the human protein (37) (boxed residues). Residues mutated in Schonherr et al. (37) to block CaM binding are labeled with an asterisk (*). Bottom panel, GST-Eag-C1(F732S/ F735S) does not bind CaM. CaM bound to wild type (WT) and mutant GST-Eag-C1-coupled Sepharose beads was detected by immunoblotting with anti-CaM. B, CaM binding to Eag does not affect its phosphorylation. Soluble wild type CaMKII or constitutively active T287D CaMKII was incubated with GST-Eag-C1 or GST-Eag-C1(F732S/F735S)-bound beads and 50 µM [{gamma}-32P]ATP for 1 min in the presence of Ca2+/CaM or EGTA. Proteins were separated by SDS-PAGE and visualized by autoradiography.

 
Three lines of evidence indicate that direct binding of CaM to the Eag C terminus is not required for binding of CaMKII. First, deletion analysis (Fig. 3) indicates that this CaM-binding site is not involved in Ca2+/CaM-dependent CaMKII binding. If the CaM-binding region of Eag is disrupted (GST-Eag 731–803; data not shown) or entirely removed (GST-Eag 761–803) CaMKII still binds normally. Second, wild type CaMKII binds to F732S/F735S GST-Eag-C1 (data not shown), even though this Eag mutant does not bind CaM. Third, mutant CaMKIIs that are constitutively in the activated state (T287D mutation) bind to Eag in the absence of Ca2+/CaM (see Fig. 5A below).



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  		FIG. 5.
Activation of CaMKII is required for Eag binding. A, interaction of CaMKII autoregulatory domain point mutants with GST-Eag-C2. CaMKII was incubated with glutathione-Sepharose beads conjugated to GST-Eag-C2 in the presence of Ca2+/CaM or EGTA and washed with buffer containing either Ca2+ or EGTA corresponding to the initial condition. Bound kinase was detected by immunoblotting with anti-CaMKII (1:1,000). WT, wild type. B, T287D and Eag coimmunoprecipitate Ca2+-independently from fly head extracts. Extracts made from transgenic flies expressing either T287D or T287A CaMKII under control of a panneural promoter (C155-GAL4; UAS-T287D/+ and C155-GAL4; UAS-T287A/+) were immunoprecipitated with an antibody against Eag. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed for CaMKII by biotin-CaM overlay and streptavidin-horseradish peroxidase detection or for Eag by immunoblotting (1:2,000). n >= 3 for all experiments, and representative data are shown.

 
Although not required for kinase binding, it was still possible that interaction of the CaM- and CaMKII-binding sites could regulate phosphorylation of Thr-787, the major CaMKII site on Eag (11). CaM binding to a substrate protein has been shown to regulate site availability both positively (38) and negatively (39). We measured radioactive phosphate transfer from [{gamma}-32P]ATP into wild type GST-Eag-C1 and F732S/F735S GST-Eag-C1. These mutations have been shown in hEAG1 to block CaM binding (37), and they also block CaM binding to the Drosophila Eag C terminus (Fig. 4A). Wild type CaMKII was able to phosphorylate both wild type and mutant Eag to similar levels in the presence of Ca2+/CaM, but not in the presence of EGTA (Fig. 4B, top panels). This demonstrates that CaM binding to Eag is not necessary for Thr-787 phosphorylation. T287D CaMKII, which is constitutively active, was able to phosphorylate Thr-787 to the same level in both the presence and absence of CaM (Fig. 4B, bottom panels), demonstrating that direct binding of CaM to Eag does not inhibit Thr-787 phosphorylation. We conclude that the binding of CaM to Eag neither enhances nor suppresses phosphorylation or CaMKII binding.

Activation of CaMKII Is Necessary for Eag Binding—Ruling out a role for direct CaM binding to Eag suggests that CaM is enhancing association of Eag and CaMKII by binding to the kinase. This stimulation of CaMKII·Eag complex formation could be caused by either specific binding of Eag to autophosphorylated CaMKII or to CaMKII that has adopted an "active" conformation. CaMKII binds CaM at the distal end of its autoinhibitory domain. To determine which properties of CaMKII were important for Ca2+/CaM-dependent Eag binding, we examined the ability of CaMKII mutations that altered catalytic activity, autophosphorylation, or CaM binding to associate with beads conjugated to GST-Eag-C2 in the presence of Ca2+/CaM or EGTA. Beads were washed with the same buffer (but without CaM in the Ca2+-containing buffer) and bound kinase detected by immunoblotting. Under these conditions, wild type kinase associates with Eag only when Ca2+/CaM is present in the binding buffer (Fig. 5A).

A mutant that is incapable of binding CaM, T306D/T307D (31), was unable to bind Eag under any conditions tested (Fig. 5A). This failure was the result of the defective CaM binding of this mutant because another mutant that alters these residues but still binds CaM, T306A/T307A, associates in a Ca2+/CaM-dependent manner (data not shown) and the fact that binding of T306D/T307D can be rescued by addition of a T287D mutation (see below). These results support the conclusion of the deletion studies (Fig. 3) that CaM is required to be bound to CaMKII rather than directly to Eag for complex formation.

The binding of CaM to CaMKII is believed to initiate a dissociation of autoinhibitory sequences from the catalytic domain, opening the active site and allowing autophosphorylation and substrate phosphorylation. Autophosphorylation of Thr-287 maintains the open catalytic domain and allows the kinase to remain active even after CaM has dissociated (40). Mutation of Thr-287 to aspartate (T287D) produces a constitutively active kinase (31, 41) which was able to associate with Eag even when there was no CaM present during binding, and this mutation "rescued" the T306D/T307D mutant and allow it to bind constitutively (Fig. 5A). These results suggest that autophosphorylation of Thr-287 promotes Eag binding even in the absence of CaM. The ability of T287D to bind constitutively can also be demonstrated in vivo. Fig. 5B shows CaMKII binding to Eag immunoprecipitates from fly heads overexpressing either T287D or T287A CaMKII under control of C155-GAL4, a panneural driver. Immunoprecipitations were done either in the presence or absence of Ca2+. A significant increase in bound CaMKII, as detected by biotin-CaM binding, is seen in EGTA-containing buffers when the constitutively active kinase is expressed.

The effects of CaM binding and Thr-287 phosphorylation on Eag association could be the result of exposure of a binding surface on the catalytic domain or generation of a new autophosphorylation-dependent binding site. To distinguish between these possibilities, we examined the binding of T287A, which cannot autophosphorylate to become CaM-independent, and K43M, which cannot autophosphorylate at all because of an inability to hydrolyze ATP. Both mutants showed normal Ca2+/CaM-dependent binding in vitro (Fig. 5A), indicating that autophosphorylation and activity are not required for binding. These data are consistent with the Ca2+/CaM-dependent association of Eag with CaMKII occurring on a catalytic domain binding surface. In the case of wild type kinase Ca2+/CaM is required to expose the site, whereas in the case of kinase autophosphorylated at Thr-287, the enzyme is in a conformational state in which the catalytic domain is always open. Thus, neither CaM nor autophosphorylation is required per se, but both can serve to put the kinase in an active, open conformation that allows Eag to bind.

Point Mutations Define Critical Residues in the CaMKII-binding Domain of Eag—The homology between the CaMKII-binding domain of Eag and that of the CaMKII autoinhibitory domain suggests that Eag may interact with the catalytic domain of the kinase. A number of amino acids in the CaMKII-binding domain (marked by a dot below the CaMKII sequence in Fig. 6A) correspond to amino acids that were found, in rat {alpha}CaMKII, to be important for the functional interaction of the autoregulatory and catalytic domains (42). To identify amino acids within the CaMKII-binding domain of Eag which are critical for interaction with the kinase, point mutations were made in GST-Eag-C1. Residues, or pairs of residues, that were mutated are indicated by an asterisk in the alignment in the top panel of Fig. 6A. The mutated proteins were tested for their ability to bind to wild type CaMKII in the presence of Ca2+/CaM followed by a Ca2+ wash. Fig. 6A (middle panel) shows CaMKII immunoreactivity associated with beads. Coomassie staining of the fusion protein recovered from the beads is shown in the lower panel to demonstrate that equal amounts of each mutant fusion protein were present.

Only four of the Eag mutants had reduced CaMKII binding. Two of them (R784K/Q785K and D789K/E790K) contain amino acids conserved between Eag and CaMKII. These residues (Arg-784 and Asp-789 in Eag) are homologous to residues in the CaMKII autoinhibitory domain which were shown by charge-reversal mutagenesis to make direct contact with amino acids in the catalytic domain (42). The other two mutants (L782K/A783K and G794K/E795K) are in nonconserved residues, and for one of them (G794K/E795K), the mutation actually makes the Eag region more similar to the CaMKII autoinhibitory domain. Mutation of other residues that are in positions that were shown for the CaMKII autoinhibitory domain to have contacts with the catalytic domain (K774E, A783K, T787A, T787D, V794E) had no effect. The lack of effect of mutations in Thr-787, the major CaMKII phosphorylation site of Eag, also suggests that phosphorylation of Eag does not modulate its binding to CaMKII. These results imply that, despite its homology to the autoinhibitory domain, the Eag CaMKII-binding domain is not interacting with the CaMKII catalytic domain with all the same molecular contacts.

Eag Interacts with the Catalytic Domain of CaMKII—Although we found that association of the Eag CaMKII-binding domain with wild type CaMKII only uses a subset of the residues that are homologous to the autoinhibitory domain of the kinase, the catalytic domain residues defined in the charge reversal study were still good candidates for defining the interaction surface that binds to Eag. We therefore looked at the ability of three mutations in CaMKII catalytic domain residues known to interact with its autoinhibitory domain to suppress binding to wild type Eag. D239K and F99K both bound GST-Eag-C2 in a Ca2+/CaM-dependent manner (Fig. 6B). Only I206K was able to block Eag association. A homologous mutation in rat {alpha}CaMKII, I205K, was found to block the Ca2+/CaM-dependent interaction of the N-methyl-D-aspartate receptor with CaMKII (36). No other catalytic domain mutants were examined in that study. These results support the idea that Eag is interacting with the catalytic domain of CaMKII but that its contacts differ from those of the autoinhibitory domain of the kinase itself. These differences in the details of binding could be important for determining the effect of Eag binding on activation of the kinase.

Binding to Eag Generates Ca2+/CaM-independent CaMKII Activity—The interaction of the CaMKII catalytic domain with its autoinhibitory domain keeps the kinase in an inactive state. Eag interacts with the catalytic domain of the kinase as well, but it has a different set of molecular contacts. To determine whether association with Eag had an affect on CaMKII activity, we measured transfer of radioactive phosphate from [{gamma}-32P]ATP by CaMKII, GST-Eag-C2, and CaMKII bound to GST-Eag-C2. Soluble CaMKII was able to autophosphorylate, but only in the presence of Ca2+/CaM (Fig. 7A). GST-Eag-C2 by itself had no kinase activity. CaMKII that had been bound to GST-Eag-C2 in the presence of Ca2+/CaM, then washed in EGTA to remove CaM, was able to both autophosphorylate and to phosphorylate Eag in the presence of EGTA. Ca2+/CaM stimulated both reactions but was not required. These results indicate that Eag is both a scaffold and an activator for CaMKII.



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  		FIG. 7.
Interaction with Eag activates CaMKII independently of CaM and autophosphorylation. CaMKII was bound to immobilized GST-Eag-C2, and CaM was removed by EGTA wash. A, bound wild type CaMKII can autophosphorylate and phosphorylate Eag in a Ca2+/CaM-independent manner. Soluble CaMKII or GST-Eag-C2 or beads with CaMKII bound to GST-Eag-C2 were incubated with 50 µM [{gamma}-32P]ATP for 1 min in the presence of Ca2+/CaM or EGTA. Proteins were separated by SDS-PAGE and visualized by autoradiography. The positions of Eag and CaMKII are indicated by arrows on the left. B, bound CaMKII can phosphorylate exogenous substrate in a Ca2+/CaM- and autophosphorylation-independent manner. Kinase activity of bound wild type (WT) or T287A CaMKII was compared with soluble enzyme in the presence of either Ca2+/CaM or EGTA. Activity was measured as described under "Experimental Procedures" using a synthetic peptide substrate. Maximum Ca2+/CaM-stimulated activity was set as 100% and autonomous activity expressed as a percentage of this value. Data are presented as the mean ± S.E. of six experiments. The autonomous activity of both wild type and T287A CaMKII bound to Eag was increased significantly compared with soluble kinase (indicated by *, p < 0.005, Student's t test).

 
Binding to Eag can cause CaMKII to autophosphorylate and phosphorylate its scaffold. To determine whether soluble substrates were able to access the CaMKII catalytic site when it is bound to Eag, we assayed kinase activity using a peptide substrate. Both soluble and bound CaMKII were able to phosphorylate the peptide, but their levels of Ca2+/CaM-independent activity were significantly different (Fig. 7B). Kinase bound to Eag and washed with EGTA to remove CaM showed a 3.9-fold increase in Ca2+/CaM-independent activity compared with soluble kinase (p < 0.005, Student's t test). This increase in activity was not caused by autophosphorylation of Thr-287 because T287A CaMKII had a similar (6.5-fold, p < 0.005 compared with soluble T287A, Student's t test) stimulation of autonomous activity. These data demonstrate that the interaction of CaMKII and Eag allows the kinase to adopt an active conformation that does not require autophosphorylation or CaM to maintain. The ability to remain active in the channel-bound state would allow CaMKII in a neuron to continue to phosphorylate Eag and other accessible proteins even after intracellular Ca2+ returns to basal levels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In Drosophila, inhibition of CaMKII and mutation of the gene for the eag potassium channel both cause hyperexcitability at the larval neuromuscular junction and adult memory formation defects. Eag is a CaMKII substrate, and phosphorylation of its single CaMKII site, Thr-787, enhances current amplitude, helping to repolarize the neuron after an action potential. CaMKII is an abundant enzyme in both vertebrate and invertebrate nervous systems. How CaMKII achieves specificity of action in a sea of potential substrates is an open question. In this study we demonstrate that the Drosophila eag potassium channel is both a scaffold for, and an activator of, CaMKII. This close and stable interaction allows activation of CaMKII to bring the kinase to the plasma membrane and tether it there.

The mechanism by which Eag binds CaMKII is likely to prove to be a general one. The activation of a protein kinase like CaMKII requires conformational changes that alter the relative orientations of the catalytic and regulatory domains of the protein to allow the active site to have access to substrates. These conformational changes can expose residues that are normally hidden and create new binding surfaces. Proteins such as Eag and the N-methyl-D-aspartate receptor NR2B subunit (36) can take advantage of this and bind to the kinase in an activation-dependent manner. In both of these cases, binding requires either CaM or Thr-287 (Thr-286 in rat) autophosphorylation to initiate the interaction by exposing the binding surface, but once bound, the complex is stable to removal of the activator.

This mode of binding suggests that Eag and NR2B interact directly with the catalytic domain of CaMKII in a manner analogous to the kinase regulatory region. The interaction of the CaMKII catalytic and autoregulatory domains has been studied by a number of groups. In the absence of a crystal structure, the most detailed information has come from a charge reversal study in which the contacts of autoinhibitory domain residues were identified by looking for mutations in the catalytic domain which would compensate for disruptive regulatory domain mutations (42). This study defined a number of residues in the catalytic domain which interact with the autoinhibitory domain, most likely directly.

We sought to determine the molecular details of how Eag interacts with CaMKII by investigating the effects of mutations in its CaMKII-binding domain on binding. This allowed us to identify residues that make critical contacts with CaMKII. We found that some of these residues, but not all, were analogous to autoinhibitory domain amino acids that interacted with the catalytic domain. We also looked at a subset of CaMKII catalytic domain mutations known to affect autoregulation. Only one of these, I206K, blocked Eag binding, whereas other residues used in the autoinhibitory domain interaction did not.

The differences in the details of autoinhibitory domain and Eag binding to CaMKII catalytic domain are not surprising. The displacement of the autoinhibitory domain of CaMKII from its catalytic domain is a prerequisite for its activation. Accordingly, interactions with proteins that are able to effect or prolong this displacement will modulate kinase activity. Binding of a peptide or protein to the catalytic domain of CaMKII could have several different outcomes for kinase activity: the kinase could be inhibited (as in the case of the autoregulatory domain), the kinase could be activated (as in the case of Eag and NR2B), or the kinase could remain Ca2+/CaM-dependent (as may be the case for binding to L-type calcium channels (43)). Because the functional outcome differs, it is likely that the exact contacts that are made between the kinase and the ligand also differ. As more binding partners of each class are characterized, it will be interesting to see whether interaction patterns emerge which can predict the effect of binding on activity.

The mapping of interaction contacts for proteins that activate CaMKII will also allow a better understanding of how the autoinhibitory domain of the kinase functions. For example, the catalytic domain residue Ile-205 (Ile-206 in Drosophila) is important for autoinhibitory domain function and binding of both NR2B and Eag. This suggests that this residue may function as a part of a general binding surface instead of directly participating in inhibition of kinase activity. The failure of Eag to utilize Phe-99 or Asp-239 may indicate that binding to these residues is important for inhibition of kinase activity.

The functional importance of activation-dependent tethering may lie in its ability to change the subcellular address of the kinase. The range of substrates and their local concentration seen by a bound kinase are not the same as that sampled by a soluble enzyme. When bound to an ion channel, the kinase is close to the plasma membrane and to other transmembrane proteins and their binding partners. This arrangement can define subcellular signaling domains that may function in response to local, instead of global, cues. The stability of these complexes would be regulated by the off rates of kinase binding and could persist well after the activating stimulus has dissipated. The utility of local signaling complexes for the neuron is that they allow independent regulation of membrane and synaptic properties based on inputs specific to that region. Processing of information locally may be important in integration of synaptic activity that contributes to neuronal plasticity.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R01 GM54408 (to L. C. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by National Institutes of Health Grant R01 NS17910 (to Irwin B. Levitan). Present address: Dept. of Neurobiology, University of Alabama at Birmingham, 1719 6th Ave. South, SRC 543, Birmingham, AL 35294-0021. Back

|| To whom correspondence should be addressed: Dept. of Biology, MS008, Brandeis University, 415 South St., Waltham, MA 02454-9110. Tel.: 781-736-3125; Fax: 781-736-3107; E-mail: griffith{at}brandeis.edu.

1 The abbreviations used are: CaMKII, calcium/calmodulin-dependent protein kinase II; GFP, green fluorescent protein; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Neal Waxham (University of Texas at Houston) for providing viral constructs and advice on kinase purification. Ed Dougherty of the Brandeis Confocal Facility (supported by shared instrumentation Grant S10 RR16780) provided help with imaging, and Gisela Wilson (University of Michigan) provided helpful discussion. Yoshiki Takamatsu provided mouse monoclonal anti-CaMKII.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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