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Originally published In Press as doi:10.1074/jbc.M502191200 on August 24, 2005

J. Biol. Chem., Vol. 280, Issue 42, 35329-35336, October 21, 2005
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Multivalent Interactions of Calcium/Calmodulin-dependent Protein Kinase II with the Postsynaptic Density Proteins NR2B, Densin-180, and {alpha}-Actinin-2*

A. J. Robison, Martha A. Bass, Yuxia Jiao, Leigh B. MacMillan, Leigh C. Carmody, Ryan K. Bartlett, and Roger J. Colbran1

From the Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Vanderbilt-Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee 37232-0615

Received for publication, February 25, 2005 , and in revised form, August 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKII{alpha} is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and {alpha}-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of {alpha}-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKII{alpha} holoenzyme [P-T286]CaMKII bind to {alpha}-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, {alpha}-actinin-2 binds to representative splice variants of each CaMKII gene ({alpha}, {beta}, {gamma}, and {delta}), whereas densin-180 binds selectively to CaMKII{alpha}. In addition, C-terminal truncated CaMKII{alpha} monomers can interact with NR2B and {alpha}-actinin-2, but not with densin-180. Soluble {alpha}-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized {alpha}-actinin-2 by {approx}10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or {alpha}-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged {alpha}-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKII{alpha} can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CaMKII2 plays a ubiquitous and central role in calcium signaling. Alternative splicing of the four CaMKII genes ({alpha}, {beta}, {gamma}, and {delta}) gives rise to ~30 known mRNA/protein products. CaMKII{alpha} and CaMKII{beta} predominate in the brain and are involved in normal regulation of synaptic transmission (1-4). Calcium/calmodulin-dependent autophosphorylation at Thr286 (numbered as in CaMKII{alpha}) enhances the affinity for calcium/calmodulin and confers autonomous (calcium-independent) activity after calmodulin dissociates. Consequently, CaMKII is capable of integrating information conveyed by the amplitude, frequency, and duration of local calcium transients to which it is exposed. Autophosphorylation at Thr305/306 occurs only in the absence of bound calcium/calmodulin and blocks subsequent calmodulin binding (reviewed in Ref. 5).

Knock-in mutation of the Thr286 or Thr305/306 autophosphorylation sites in murine CaMKII{alpha} to Ala or Asp drastically alters some forms of hippocampal synaptic plasticity and disrupts spatial learning behaviors (reviewed in Ref. 6). The specificity of changes in synaptic responses to their dedicated inputs implies that postsynaptic actions of CaMKII, such as modulation of the trafficking and activity of AMPA-type glutamate receptors, are exquisitely regulated in a spatial and temporal manner (7-9).

CaMKII interacts with other proteins that we refer to collectively as CaMKII-associated proteins (CaMKAPs). At postsynaptic sites, these include multiple subunits of the N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) (10-13), densin-180 (14, 15), {alpha}-actinin (15, 16), cyclin-dependent protein kinase 5 (16), synGAP{beta} (17), and filamentous actin (18, 19). However, specific contributions of each of these interactions to neuronal signaling and the regulation of synaptic plasticity remain unclear (reviewed in Refs. 7-9). To specifically address this issue, it is critical to have a thorough understanding of the molecular bases for these interactions and the factors regulating complex formation.

Prior studies have identified dissimilar high affinity CaMKII-binding domains in the NR2B subunit of the NMDA receptor and in densin-180. These interactions are differentially regulated by calcium/calmodulin-binding, autophosphorylation at Thr286, and phosphorylation of the binding proteins (10, 11, 13-15, 20, 21). Moreover, densin-180 and NR2B do not compete with each other for binding to CaMKII (14). Interestingly, densin-180 makes an additional direct interaction with {alpha}-actinin (15), and {alpha}-actinin can bind to the NR1 and NR2B subunits of the NMDA receptor (21-24). However, little is known about the molecular determinants for CaMKII binding to {alpha}-actinin. Here, we report an initial molecular dissection of the CaMKII-binding domain in {alpha}-actinin-2 and explore the relationship of this interaction to CaMKII binding with densin-180 and NR2B. The data indicate that CaMKII itself may serve as a structural scaffold for the assembly of a postsynaptic signalosome.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Western blotting was performed with mouse anti-CaMKII{alpha} (Affinity Bioreagents), mouse anti-{alpha}-actinin (Sigma) antibodies, or mouse anti-NR2B (BD Transduction Laboratories). The goat anti-CaMKII antiserum was described previously (25). Rabbit anti-densin-180 serum was raised against a GST fusion protein including residues 1247-1290 and 1378-1412 of densin-180. Goat anti-densin-180 serum was generated by co-injection of peptides corresponding residues 451-470 and 652-671 of densin-180. A detailed characterization of the densin antibodies will be published separately.3

Coimmunoprecipitation—Adult male Sprague-Dawley rats were sacrificed by decapitation. Forebrains were quickly removed and homogenized on ice in 0.32 M sucrose containing 4 mM HEPES, pH 7.4 (Buffer 1, 8 ml per forebrain) using a Potter-Elvehjem Teflon-glass device. All remaining steps were performed at 4 °C. Following centrifugation (800 x g, 10 min), the supernatant was removed and re-centrifuged (9,200 x g, 15 min). The 9,200 x g pellet was resuspended in Buffer 1 (8 ml per forebrain) and centrifuged again (10,200 x g, 15 min). The new pellet (crude synaptosomes) was resuspended in Buffer 2 (20 mM HEPES/NaOH, pH 8.0, 0.1 M NaCl, 5 mM EDTA, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 20 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, 1 µM microcystin; 4 ml per forebrain), incubated on ice for 30 min, and then centrifuged (100,000 x g, 30 min). The supernatant was diluted in Buffer 2 to 2 mg/ml protein and used for immunoprecipitations using anti-CaMKII, anti-densin, or preimmune antibodies (5 µl of goat serum per 2 mg of protein) (see above). Protein G-Sepharose beads (Pierce, 25 µl) were added after 2 h, and the incubation continued overnight. Beads were collected by centrifugation and washed five times with Buffer 2 (1 ml each). Proteins were eluted in 2x SDS-PAGE sample buffer and analyzed by Western blot.

GST- and His6-tagged Fusion Proteins—The {alpha}-actinin-2 cDNA was a generous gift from Dr. Alan Beggs (Harvard). Desired fragments of {alpha}-actinin-2, NR2B, or densin-180 cDNAs were amplified by PCR using oligonucleotide primers containing BamHI (5') and EcoRI (3') restriction enzyme sites as described (10, 14). PCR products were ligated into the pGEX-2T (Amersham Biosciences) or pRSET-A (Qiagen) vectors and transformed into BL21-DE3 Gold Escherichia coli bacteria. After induction of protein expression, glutathione S-transferase (GST) or His6 fusion proteins were purified using glutathione-agarose (Sigma) or His-Select Nickel Affinity Gel (Sigma), according to the manufacturer's protocol. His6-NR2B was solubilized and purified in the presence of 8 M urea; the urea was then removed by dialysis. GST-densin-(1247-1495) represents the entire C-terminal tail of the A splice variant; GST-densin-(1247-1405) represents the C-terminal tail of the A variant without the PDZ domain; and GST-densin-(1247-1542) represents the entire C-terminal tail of the D variant (14). GST-NR2B includes amino acids 1260-1339, with the point mutation S1303A (20). His6 fusion proteins were densin-(1247-1542), actinin-(547-894), and NR2B-(965-1339). Purified protein concentrations were determined using Bradford (Bio-Rad) or bicinchoninic acid (Pierce) assays.

CaMKII Isoforms and Truncation Mutants—Murine CaMKII{alpha}, Xenopus CaMKII{beta}, porcine CaMKII{gamma}B, and rat CaMKII{delta}2 were purified from baculovirus-infected Sf9 cells as described (25, 26). The CaMKII{gamma}B and CaMKII{delta}2 cDNAs were generous gifts from Dr. C. M. Schworer in the laboratory of Dr. H. Singer (Albany Medical College, NY). The C-terminal truncation mutants of CaMKII{alpha}-(1-420) and CaMKII{alpha}-(1-380) were generated as described (20) and were monomeric as determined by size-exclusion chromatography on a Superdex 200 column (Amersham Biosciences).

CaMKII Autophosphorylation—CaMKII isoforms were selectively autophosphorylated at Thr286/287, as described (25). Briefly, kinase (5 µM subunit) was incubated on ice with 50 mM HEPES, pH 7.5, 10 mM magnesium acetate, 1.5 mM CaCl2, 10 µM calmodulin, and 40 µM ATP. After 90 s, the reaction was stopped with EDTA (12.5 mM final). CaMKII{alpha}-(1-420) and CaMKII{alpha}-(1-380) were autophosphorylated using identical conditions, except that ATP was increased to 200 µM, and incubations were performed at 30 °C for 5 min.

GST Cosedimentation Assays—Purified GST fusion proteins or GST alone ({approx}250 nM full-length protein) were incubated for 2 h at 4 °C with [P-T286]CaMKII or Thr287-autophosphorylated CaMKII{beta}/{gamma}/{delta} ({approx}250 nM) and glutathione-agarose beads (Sigma, 25 µl of packed resin) in pull-down (PD) buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.5% Triton X-100; 500-µl final volume). Where indicated, His6-tagged fusion proteins ({approx}250 nM each) were included with the kinase. Beads were sedimented by centrifugation and washed in 500 µl of PD Buffer 6 times for 5 min each. Beads were transferred to new microcentrifuge tubes, and sedimented proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were stained using Ponceau S and digitally scanned to quantify protein loading using ImageJ (rsb.info.nih.gov/ij/). Pilot studies showed that pixel densities of individual protein bands are linearly related to the amount of protein loaded on the gel in the range of 0.04-2.5 µg per lane for both GST and CaMKII{alpha} (0.7-42 pmol). Amounts of CaMKII{alpha} sedimented on the beads were normalized to the recovered GST fusion protein, and background binding to GST alone was subtracted. Statistical comparisons (one-way analysis of variance with Tukey post-hoc tests) were performed using Prism (GraphPad Software). To confirm the identity of Ponceau-stained proteins, membranes were immunoblotted for CaMKII or His6-tagged proteins using affinity purified polyclonal goat anti-CaMKII primary antibodies (25), or monoclonal mouse anti-His6 antibodies (Amersham Biosciences), respectively, and alkaline-phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch).

CaMKII Overlay Assays—Nitrocellulose membranes containing immobilized GST fusion proteins (1-5 µg) were incubated with [32P-T286]CaMKII (50 nM subunit), washed and autoradiographed, essentially as described (25). Where indicated, [32P-T286]CaMKII was incubated on ice for 30 min with soluble GST fusion proteins before addition to the membrane. Films exposed within the linear range were digitally scanned, and band intensities (determined using ImageJ) were normalized to GST protein loading (see above).

Biotinylated Densin-180 Overlay—GST-densin (200 µg) was incubated at a 1:20 molar ratio with EZ-Link Sulfo-NHS-SS-Biotin (Pierce). Unconjugated biotin was removed by dialysis, and biotinylated protein was stored in 10 mM HEPES, pH 7.5, with 0.1% Triton X-100 at -80 °C. Nitrocellulose membranes containing GST-actinin (5 µg) were blocked for 1 h at room temperature in 5% milk powder in TTBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Triton X-100), and then incubated with 2 µg/ml alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch) with or without biotinylated GST-densin-(1247-1542) (65 nM) in the same buffer. After washing in TTBS (5 times, 5 min each), bound alkaline phosphatase was detected colorimetrically.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CaMKII Is Associated with Multiple CaMKII-binding Proteins in PSD-enriched Fractions—PSD-enriched synaptosomal fractions isolated from adult rat forebrain were enriched in CaMKII and three CaMKII-binding proteins, {alpha}-actinin, NR2B, and densin-180. About 50% of the CaMKII{alpha}, but less than 20% of the CaMKAPs, were solubilized from the PSD-enriched fraction using Triton X-100. CaMKII immunoprecipitates from this solubilized extract also specifically contained {alpha}-actinin, densin-180, and occasionally NR2B (Fig. 1). Densin-180 immunoprecipitates also contained CaMKII and NR2B, but no detectable {alpha}-actinin (Fig. 1). These data confirm previous observations that CaMKII, {alpha}-actinin, NR2B, and densin-180 are enriched in PSDs. The effects of solubilization conditions and the differential regulation of each interaction4 preclude definitive conclusions about the nature of specific protein-protein interactions in PSDs. However, the data are consistent with the existence of multiprotein complexes containing CaMKII in vivo.

The CaMKII-binding Region of {alpha}-Actinin-2—The {alpha}-actinin isoforms are inverted dimers in the native state, containing N-terminal actin-binding domains, four spectrin repeats, and two putative C-terminal EF hand domains (Fig. 2A) (27). To investigate whether {alpha}-actinin-2 binds directly to CaMKII and to delineate the CaMKII-binding domain, GST fusion proteins containing various portions of the C-terminal domain of {alpha}-actinin-2 were used in co-sedimentation assays. GST-actinin-(547-894) effectively bound Thr286-autophosphorylated CaMKII{alpha} ([P-T286]CaMKII) with similar efficacy to the GST-densin-(1247-1542) control (Fig. 2B and also see Fig. 3). Similar amounts of non-phosphorylated CaMKII{alpha} and [P-T286]CaMKII associated with GST-actinin-(547-894) under these conditions.4 Truncation of 80 amino acids from the C terminus (GST-actinin-(547-814)) reduced CaMKII binding to the very low nonspecific levels detected with GST alone. Moreover, C-terminal fragments (GST-actinin-(809-894) and GST-actinin-(819-894)) bound similar amounts of [P-T286]CaMKII as did GST-actinin-(547-894). Interestingly, the CaMKII-binding C-terminal domain (75 amino acids) displays 70% amino acid sequence identity (84% similarity) across three {alpha}-actinin isoforms that interact with CaMKII (15, 16) (Fig. 2D). Deletion of the more highly conserved extreme N- or C-terminal regions within this domain to yield GST-actinin-(839-894) or GST-actinin-(819-885) abrogates CaMKII binding (Fig. 2, B and C). Thus, determinants for [P-T286]CaMKII interactions with {alpha}-actinin-2 appear to be distributed within a minimal functional binding domain of residues 819-894.



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  		FIGURE 1.
CaMKII associates with multiple proteins in PSD-enriched fractions. A Triton X-100-solubilized PSD-enriched fraction was immunoprecipitated using anti-CaMKII serum (CaMKII), anti-densin-180 serum (densin), or preimmune serum (PIS) and immune complexes were immunoblotted (IB) as indicated. The anti-CaMKII immune complex also contained {alpha}-actinin and densin-180, whereas the anti-densin-180 complex also contained CaMKII and NR2B. These data are representative of three similar experiments.

 



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  		FIGURE 2.
CaMKII directly binds residues 819-894 {alpha}-actinin-2. A, schematic domain structure of {alpha}-actinin dimers. ABD, actin-binding domain; R1-4, spectrin repeat domains; EF, putative EF hand domains. B, glutathione-agarose cosedimentation assays using [P-T286]CaMKII and the indicated GST-actinin fusion proteins or GST-densin-(1247-1542). Sedimented proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and visualized by Ponceau S for total protein (top) or by immunoblotting for CaMKII (bottom). Asterisks on the Ponceau-stained membrane mark full-length (intact) GST fusion proteins, and the arrowhead indicates the CaMKII{alpha} bands. Data are representative of three to eight experiments. C, summary of glutathione-agarose cosedimentation studies mapping the CaMKII binding domain. D, amino acid sequence alignment of the minimal CaMKII binding domain of {alpha}-actinin-2 with corresponding regions of {alpha}-actinin-1 and {alpha}-actinin-4. Identical residues are indicated by black boxes, with conservative changes in gray boxes.

 
It is well established that [P-T286]CaMKII interacts with residues 1290-1309 of NR2B and with residues 1355-1382 of densin-180 (10, 11, 13, 14, 21). Comparable amounts of [P-T286]CaMKII bound to GST-actinin-(547-894), GST-NR2B, or GST-densin-(1247-1542) (Fig. 3, A and C). The ratio of binding of CaMKII to GST-densin-(1247-1542) and to GST-actinin-(547-894) was 0.9 ± 0.1 and 0.8 ± 0.2, respectively, when normalized to NR2B (Fig. 3C). However, in gel overlay assays, [32P-T286]CaMKII bound to GST-actinin-(547-894) very weakly when compared with the strong binding to GST-NR2B and GST-densin-(1247-1542) (Fig. 3). In fact, binding of [32P-T286]CaMKII to immobilized GST-actinin-(547-894) was not consistently above the very weak nonspecific interaction with GST alone (Fig. 3, B and C). These data suggest that, unlike NR2B and densin-180, the CaMKII-binding domain of {alpha}-actinin-2 is unable to refold into a binding-competent conformation on the membrane.



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  		FIGURE 3.
CaMKII isoform binding to {alpha}-actinin-2, NR2B, and densin-180: role of the C-terminal association domain. A, Thr286/287-autophosphorylated CaMKII{alpha} (wild type (WT) or C-terminal truncated), CaMKII{beta}, CaMKII{gamma}B, or CaMKII{delta}2 were incubated with GST, GST-densin-(1247-1495), GST-NR2B, or GST-actinin-(819-894). The top panel shows a Ponceau stained gel of the GST fusion protein preparations used in these experiments. The lower panels show CaMKII immunoblots of proteins isolated on glutathione-agarose (P: 40% of total pellet) and the soluble proteins remaining in the supernatant (S: 2% of total). B, CaMKII overlay assay. GST fusion proteins were immobilized on nitrocellulose membranes and incubated with wild type [32P-T286]CaMKII. Membranes were washed and autoradiographed. C, quantification of wild type (WT) CaMKII{alpha} binding to the three GST fusion proteins in pull-down (top) and overlay (bottom) assays. Binding to GST-actinin and GST-densin was normalized to binding to GST-NR2B in each experiment and averaged across three to eight experiments; data are shown as mean ± S.E. D and E, quantitative comparison of CaMKII isoform (D) and CaMKII{alpha} truncation mutant (E) binding to densin, NR2B, and {alpha}-actinin-2 in glutathione-agarose cosedimentation assays. Binding of each isoform or truncation mutant was normalized to wild type CaMKII{alpha} for each GST fusion protein, and then averaged across three to eight experiments. Data are expressed as mean ± S.E. *, p < 0.001; {diamondsuit}, p < 0.01 versus binding to wild type CaMKII{alpha}.

 
CaMKII Isoform Selectivity—Proteins encoded by the four CaMKII genes are highly similar within their N-terminal catalytic and regulatory domains (89-93% amino acid sequence identity) but are more divergent in the C-terminal association domains (53-89% amino acid sequence identity) (28). Representative splice variants of each gene were autophosphorylated at Thr286 (CaMKII{alpha}) or at Thr287 (CaMKII{beta}/{gamma}B/{delta}2) and then used in glutathione-agarose cosedimentation assays. The four CaMKII isoforms bound with similar efficacy to GST-NR2B and GST-actinin-(819-894). In contrast, GST-densin-(1247-1495) selectively interacted with CaMKII{alpha} (Fig. 3); binding of CaMKII{beta} and CaMKII{gamma}B was reduced ~13-fold relative to binding of CaMKII{alpha}. Although GST-densin-(1247-1495) appeared similarly ineffective in binding CaMKII{delta}2 by immunoblotting (Fig. 3A), this could not be quantitated from the Ponceau-stained membrane, because CaMKII{delta}2 and GST-densin-(1247-1495) co-migrate on the gel.

Role of the C-terminal Association Domain of CaMKII{alpha}—The role of the C-terminal domain in binding to each of the CaMKAPs was investigated using CaMKII{alpha} truncations at either residue 420 or residue 380. Both proteins are monomeric but can be Thr286-autophosphorylated to a similar extent as the wild type kinase using modified reaction conditions, as assessed by immunoblotting using phospho-Thr286-specific antibodies and by the generation of autonomous kinase activity (data not shown). We previously showed that CaMKII-(1-420) exhibited reduced binding to GST-NR2B (20), but under the conditions used here, both GST-NR2B and GST-actinin-(819-894) bound with comparable efficacy to the monomeric kinases and the wild type holoenzyme. However, there was no statistically significant binding of either truncation mutant to GST-densin-(1247-1495) (Fig. 3, A and E). These data show that the NR2B and {alpha}-actinin-2 binding sites lie within the first 380 amino acids of CaMKII and that formation of a CaMKII holoenzyme is not essential for these interactions. However, CaMKII{alpha} binding to densin-180 appears to require an intact association domain.

Non-competitive Binding of Densin-180, NR2B, and {alpha}-Actinin-2 to CaMKII—Residues 819-894 of {alpha}-actinin-2 share no obvious amino acid sequence similarity to the CaMKII-binding domains of NR2B or densin-180. To compare mechanisms for interactions of CaMKII with {alpha}-actinin-2, NR2B, and densin-180, competition overlay assays were performed. Nitrocellulose membranes containing immobilized GST fusion proteins were incubated with [32P-T286]CaMKII (50 nM) in the absence or presence of each of the soluble GST fusion proteins (up to 1.25 µM). Binding of kinase to immobilized proteins was determined by autoradiography of the washed membranes.

With immobilized GST-densin-(1247-1495), soluble GST-densin-(1247-1495) decreased [32P-T286]CaMKII binding in a concentration-dependent manner ({approx}55% of control at 1.25 µM), but similar concentrations of soluble GST-actinin-(819-894) or soluble GST-NR2B had no significant effect (Fig. 4). Similarly, with immobilized GST-NR2B, soluble GST-NR2B competed for binding of [P-T286]CaMKII ({approx}15% binding at 1.25 µM), but soluble GST-densin-(1247-1495) or soluble GST-actinin-(819-894) had no significant effect. Control experiments showed that soluble GST had no effect on CaMKII binding to any immobilized protein (data not shown). It was not possible to test for potential effects of higher competitor concentrations because of the limited solubility of these GST fusion proteins.

As shown above, [32P-T286]CaMKII binds very weakly to immobilized {alpha}-actinin-2 in overlay assays. However, soluble GST-densin-(1247-1495) increased {approx}8-fold the apparent interaction of CaMKII with immobilized GST-actinin-(819-894), whereas soluble GST-NR2B and soluble GST-actinin-(819-894) had no effect (Fig. 4). In combination, these data demonstrate that interactions of NR2B, densin-180, and {alpha}-actinin-2 with [P-T286]CaMKII are non-competitive and suggest that densin-180 may facilitate binding of CaMKII to {alpha}-actinin-2.



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  		FIGURE 4.
Effects of soluble {alpha}-actinin-2, densin, or NR2B on CaMKII{alpha} binding to immobilized GST fusion proteins in overlay assays. A, GST-densin-(1247-1495) (top), GST-NR2B (middle), or GST-actinin-(819-894) (bottom) were immobilized on nitrocellulose membrane. Membranes were incubated with [32P-T286]CaMKII (50 nM) in the presence of the indicated concentrations of each soluble GST fusion protein (columns). Data are representative of more than three experiments. B, quantification of the effects of soluble GST fusion proteins (1250 nM each) on CaMKII binding to each immobilized fusion protein. CaMKII binding was normalized to binding observed in the presence of soluble GST in each experiment; the mean ± S.E. from three to five experiments is shown.

 
Mechanism for Potentiation of CaMKII Binding to {alpha}-Actinin-2 by Densin-180—Facilitation of CaMKII binding to immobilized {alpha}-actinin-2 by soluble densin-180 in the overlay assay (above) may be influenced by interactions of CaMKII with both densin-180 and {alpha}-actinin-2, as well as by direct interaction of densin-180 with {alpha}-actinin-2 (15). To determine whether the densin-180 PDZ domain interacts with the C terminus of {alpha}-actinin under our conditions, biotinylated GST-densin-(1247-1495) was incubated with immobilized {alpha}-actinin-2 constructs in overlay assays. Densin interacted with GST-actinin-(819-894) and GST-actinin-(839-894), but not with GST-actinin-(819-885), which lacks the C-terminal 9 amino acids (Fig. 5A). Thus, densin-180 interacts with the consensus PDZ-binding motif in {alpha}-actinin-2 under our overlay conditions.

We next assessed the role of direct CaMKII interaction with {alpha}-actinin-2 in densin-enhanced binding to immobilized {alpha}-actinin-2. Soluble GST-densin enhanced by 16 ± 3-fold the binding of [32P-T286]CaMKII to GST-actinin-(839-894) (Fig. 5B), which cannot bind CaMKII directly in cosedimentation assays (Fig. 2), but retains an intact C terminus. These data suggest that CaMKII interactions with actinin are not involved in the densin-enhanced immobilization of [32P-T286]CaMKII by denatured GST-actinin-(819-894).

We next investigated contributions of the densin-180 PDZ domain and the C terminus of {alpha}-actinin-2 to the densin-enhanced binding of [32P-T286]CaMKII to {alpha}-actinin-2. The potentiation was essentially abrogated if the PDZ domain was deleted from soluble densin (GST-densin-(1247-1405)), or if nine amino acids were deleted from the C terminus of the immobilized {alpha}-actinin-2 (GST-actinin-(819-885)) (Fig. 5B). Moreover, preincubation of immobilized GST-actinin-(819-894) with soluble GST-densin-(1247-1542) potentiated ({approx}5-fold) the apparent interaction of [32P-T286]CaMKII (added only after washing to remove excess soluble densin) (data not shown). These data are consistent with a model in which densin-180 simultaneously interacts with the C terminus of {alpha}-actinin via its PDZ domain (residues 1405-1492), and with CaMKII via the independent CaMKII-binding domain (residues 1355-1382).

CaMKII Simultaneously Binds to Multiple CaMKAPs—Because at least three CaMKAPs interact non-competitively with CaMKII, we hypothesized that CaMKII could simultaneously bind multiple CaMKAPs. To test this idea, GST, GST-NR2B, GST-densin-(1247-1542), or GST-actinin-(819-894) were incubated with either [P-T286]CaMKII, a mixture of His6-tagged versions of all three CaMKAPs, or all four proteins. Complexes harvested on glutathione-agarose were analyzed for bound CaMKII and His6-tagged proteins. GST alone did not interact with CaMKII or any of the His6-CaMKAPs (Fig. 6A).



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  		FIGURE 5.
Densin-180 simultaneously interacts with {alpha}-actinin-2 and CaMKII. A, overlay of GST-actinin proteins with biotinylated GST-densin-(1247-1495). A Ponceau-stained membrane containing the indicated immobilized GST-actinin proteins is shown (left), along with the binding of biotinylated GST-densin (middle), and a control membrane incubated with streptavidin-AP in the absence of biotinylated GST-densin (right). Data are representative of three independent experiments. B, GST-actinins (indicated at the top) were analyzed by [32P-T286]CaMKII overlay in the presence of soluble GST or GST-densins indicated below (1250 nM each). Densin: GST-densin-(1247-1495). Densin{Delta}PDZ: GST-densin-(1247-1405) (lacking the PDZ domain). Data from three to four experiments were quantitated as in Fig. 3 and the mean ± S.E. is shown. *, p < 0.001 compared with GST and GST-Den{Delta}PDZ.

 
Similar, approximately stoichiometric amounts of [P-T286]CaMKII were isolated using GST-NR2B in the absence and presence of the mixture of His6-densin-(1247-1542), His6-actinin-(547-894), and His6-NR2B(965-1339). The His6-tagged CaMKAPs cosedimented with GST-NR2B in the presence of [P-T286]CaMKII but did not directly associate with GST-NR2B (Fig. 6A). Thus, CaMKII holoenzymes appear capable of simultaneously binding to GST-NR2B and to His6-NR2B, His6-actinin, or His6-densin.

GST-actinin-(819-894) isolated comparable amounts of CaMKII in the presence and absence of the mixture of His6-tagged CaMKAPs. In addition, His6-densin associated with GST-actinin-(819-894) in the presence and absence of [P-T286]CaMKII, presumably due to CaMKII-independent interaction of the PDZ domain of densin-180 with the C terminus of actinin. However, His6-NR2B and His6-actinin were isolated using GST-actinin-(819-894) only in the presence of CaMKII, showing that the holoenzyme simultaneously bound to GST-actinin-(819-894) and either His6-NR2B or His6-actinin.

Similarly, His6-actinin associated with GST-densin-(1247-1542) in both the absence and presence of CaMKII. However, His6-NR2B and His6-densin associated with GST-densin-(1247-1542) only when CaMKII was present (Fig. 6A). Moreover, similar stoichiometric amounts of CaMKII associated with GST-densin-(1247-1542) in the presence or absence of mixed His6-tagged CaMKAPs.



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  		FIGURE 6.
Simultaneous binding of CaMKII to NR2B, densin-180, and {alpha}-actinin-2. GST, GST-NR2B, GST-actinin-(819-894), or GST-densin-(1247-1542) (A, left column to right column, as indicated) or a GST-densin construct without a PDZ domain (1247-1405) (B, right column) were incubated with [P-T286]CaMKII alone, with a mixture of His6-NR2B-(965-1339), His6-actinin-(547-894), and His6-densin-(1247-1542), or with all four proteins, as indicated at the bottom of each column. Complexes isolated on glutathione-agarose (P: 40% of the total), as well as the remaining soluble proteins (S: 2% of the total), were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and then sequentially stained with Ponceau S to detect GST fusion proteins and CaMKII{alpha} (top two rows) and immunoblotted for His6-tagged proteins (bottom three rows). Data are representative of at least three independent experiments.

 
To determine the role of the interaction between the PDZ domain of densin-180 and the C terminus of {alpha}-actinin in CaMKII-mediated assembly of this multiprotein complex, we performed similar assays using GST-densin-(1247-1405), which lacks the PDZ domain. GST-densin-(1247-1405) bound CaMKII equally well in the presence or absence of the His6-tagged CaMKAPs, but bound the three His6-tagged CaMKAPs in the presence, but not the absence, of CaMKII (Fig. 6B). Therefore, a PDZ-mediated interaction of densin and {alpha}-actinin is not necessary for their simultaneous binding to CaMKII.

The role of CaMKII holoenzyme structure in assembly of this multiprotein complex was examined using the monomeric truncation mutant CaMKII{alpha}-(1-420). Although CaMKII{alpha}-(1-420) cannot bind densin-180, it is capable of interacting with both NR2B and {alpha}-actinin-(Fig. 3). CaMKII{alpha}-(1-420) facilitated the association of His6-actinin with GST-NR2B on glutathione-agarose (Fig. 7). The amount of His6-actinin isolated was somewhat less than that recovered with the wild type holoenzyme in side-by-side experiments (not shown), but this might be expected, because there is only one available binding site for {alpha}-actinin when a CaMKII monomer binds to a GST-NR2B molecule, whereas binding of a CaMKII holoenzyme to GST-NR2B immobilizes 12 binding sites for {alpha}-actinin. Thus, single CaMKII subunits are capable of binding both NR2B and {alpha}-actinin simultaneously.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CaMKII partially colocalizes with NMDA receptor NR2B subunits, densin-180, and {alpha}-actinin in dendritic spines of cultured neurons (10, 29, 30). Moreover, several studies have shown that CaMKII and NR2B, as well as CaMKII and densin-180, can be coimmunoprecipitated from different types of brain extract (10, 11, 15, 31). Although CaMKII{alpha} was detected in {alpha}-actinin-1 immunoprecipitates from whole rat brain lysates, {alpha}-actinin was not detected in CaMKII immunoprecipitates (16). Here we extend these findings by showing that {alpha}-actinin is present in CaMKII immunoprecipitates from partially solubilized PSD-enriched extracts (Fig. 1), suggesting that CaMKII interacts with PSD-associated {alpha}-actinin, presumably {alpha}-actinin-2 (32, 33). Densin-180 was also detected in these samples. Moreover, densin-180 immunoprecipitates from these solubilized PSD extracts contained NR2B and CaMKII (Fig. 1). It is unclear how the solubilization conditions affect specific protein-protein interactions. Moreover, Ca2+/calmodulin binding and autophosphorylation at Thr286 or Thr305/306 differentially regulate CaMKII interactions with NR2B and {alpha}-actinin (10, 11, 13, 20, 21).4 Nevertheless, these data support the idea that CaMKII can interact with several CaMKAPs found in dendritic spines, including NR2B, densin-180, and {alpha}-actinin. The experiments described here dissect the interactions of CaMKII with these three CaMKAPs.



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  		FIGURE 7.
Simultaneous binding of NR2B and {alpha}-actinin-2 to monomeric CaMKII subunits. GST or GST-NR2B were incubated with [P-T286]CaMKII-(1-420) alone, with His6-actinin-(547-894) alone, or with a mixture of both proteins, as indicated below. Complexes isolated on glutathione agarose (P: 40% of the total), as well as the remaining soluble proteins (S: 2% of the total), were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and then sequentially stained with Ponceau S to detect GST fusion proteins and CaMKII{alpha} (top two rows) and immunoblotted for His6-tagged proteins (bottom row). Data are representative of three independent experiments.

 
CaMKII was previously reported to bind {alpha}-actinin-1 and {alpha}-actinin-4 via a poorly defined C-terminal domain (15). Here, we show that CaMKII directly binds to {alpha}-actinin-2 in vitro and provide an initial characterization of the molecular determinants for interaction. Together, our data show that the interactions of CaMKII with NR2B, densin-180, and {alpha}-actinin are mechanistically distinct in multiple ways. First, the minimal CaMKII-binding domain identified in {alpha}-actinin-2 (residues 819-894, Fig. 2) is substantially larger than, and shares no amino acid sequence similarity with, the minimal CaMKII-binding domains of NR2B and densin-180 (14, 20). However, deletion of sequences toward the extreme N and C termini of the CaMKII-binding domain that are highly conserved between three {alpha}-actinin isoforms abrogates CaMKII binding (Fig. 2). Second, in overlay assays, CaMKII binding to {alpha}-actinin-2 did not exceed background levels (presumably because {alpha}-actinin-2 failed to refold correctly on the membrane), whereas CaMKII bound effectively to NR2B and densin-180 (Fig. 3). This observation likely explains why initial overlay analyses of brain subcellular fractions failed to detect significant CaMKII-binding proteins with similar electrophoretic mobility to native {alpha}-actinin (25). Third, NR2B and {alpha}-actinin-2 interacted effectively with all four CaMKII isoforms, whereas densin-180 was selective for CaMKII{alpha} holoenzymes (Fig. 3). Fourth, activation of CaMKII by calcium/calmodulin-binding or autophosphorylation at Thr286 is essential for the interaction with NR2B and potentiates interaction with densin-180 (10, 11, 13-15, 20, 21), whereas binding of CaMKII to {alpha}-actinin-2 is independent of Thr286 autophosphorylation.4 Last, these CaMKAPs do not compete for binding to CaMKII (Figs. 4 and 6). In combination, the data demonstrate that in contrast to AKAPs, which use a conserved molecular determinant (an amphipathic {alpha}-helix) to bind cAMP-dependent protein kinase regulatory subunits (34), CaMKAPs use divergent mechanisms to interact with CaMKII, with the potential for unique functional effects beyond simply dictating the subcellular localization of CaMKII.

We also explored the role of CaMKII holoenzyme structure in the interactions with {alpha}-actinin-2, densin-180, and NR2B. NR2B and {alpha}-actinin-2 bind monomeric truncation mutants of CaMKII{alpha}. Thus, binding determinants for NR2B and {alpha}-actinin lie within the first 380 amino acids (Fig. 3), which contain the catalytic and regulatory domains. Consistent with these findings, Bayer et al. (13) have shown that catalytic domain mutations affect binding of CaMKII to NR2B, and yeast two-hybrid studies have shown that {alpha}-actinin interacts with residues 1-316 of CaMKII{alpha} (15). In contrast, densin-180 does not bind to the monomeric CaMKII{alpha}-(1-420) (Fig. 3). Previous studies using yeast two-hybrid approaches have shown that residues 1-316 of CaMKII{alpha} are incapable of binding to densin-180, and that residues 317-478 are sufficient for binding (15). These data suggest two hypotheses: 1) essential components of the densin-180 interaction domain lie within residues 421-478 of CaMKII; 2) holoenzyme structure is required for association of densin-180 with a domain within residues 317-420. Additional experiments will be required to resolve this issue.

Interactions of CaMKII with individual CaMKAPs have been suggested to play a role in targeting CaMKII within dendritic spines (reviewed in Refs. 4, 7, and 9). In particular, the dynamic regulation of CaMKII interactions with NR2B seems to parallel the transient translocation of CaMKII to PSDs in neurons stimulated electrically, or by glutamate (35, 36). However, under some conditions CaMKII translocation to synapses is associated with the loss of synaptic NMDA receptors (37). Interactions with {alpha}-actinin-2 and/or densin-180 may further modulate CaMKII targeting to facilitate appropriate downstream signaling to substrates, such as AMPA receptor GluR1 subunits (38) and possibly NMDA receptors themselves (39). The CaMKAPs also may functionally modulate CaMKII in different ways (40).4

It was previously proposed that CaMKII clusters synaptic proteins via diverse protein-protein interactions (1). The present data provide the first direct biochemical demonstration of this potential in that [P-T286]CaMKII was shown to simultaneously interact with NR2B, densin-180, and {alpha}-actinin in vitro. GST-NR2B interacts with all three His6-tagged CaMKAPs in a CaMKII-dependent manner (Fig. 6). Similarly, GST-densin and GST-actinin isolated complexes containing CaMKII and the other CaMKAPs. Although close to stoichiometric amounts of CaMKII bound to each GST fusion protein in these experiments, it was difficult to detect His6-tagged proteins by protein staining of the membranes (not shown), indicating that sub-stoichiometric amounts were recovered. This may result from difficulties in maintaining multiple interactions throughout our rigorous washing procedure and is thus unlikely to reflect the actual stoichiometries of the complexes. Although NR2B and {alpha}-actinin can bind simultaneously to monomeric CaMKII subunits (Fig. 7), the ability of CaMKII to assemble the complex may in part depend on the holoenzyme structure, permitting independent interactions of individual subunits with CaMKAPs as well as physiological substrate proteins such as AMPA-type glutamate receptor GluR1 subunits (38). Taken together, our data suggest that CaMKII holoenzymes can nucleate multiprotein complexes in dendritic spines.

It is well established that the NMDA receptor is part of a large multiprotein complex (41-44). Both NR2B and densin-180 interact with MAGUK proteins (45, 46). Moreover, the spectrin-repeat domain of {alpha}-actinin-2 interacts with the "C0" cassette of the NMDA receptor NR1 subunit and with residues 1361-1482 of NR2B (22-24). Our data showing that the C terminus of {alpha}-actinin-2 binds to the PDZ domain of densin-180 (Fig. 5) confirm previous studies showing a similar interaction with {alpha}-actinin-4 (15, 16). Because soluble densin-180 facilitates CaMKII binding to immobilized {alpha}-actinin-2 in overlay assays (Figs. 4 and 5B), densin-180 is clearly capable of simultaneously interacting with CaMKII and {alpha}-actinin-2. However, His6-densin did not enhance CaMKII association with GST-actinin in glutathione-agarose cosedimentation assays (Fig. 6). Thus, it is not clear whether a similar potentiation will occur in cells.

CaMKII{alpha} is highly abundant in neurons (e.g. 1-2% of total hippocampal protein), greater than might be expected for a strictly enzymatic role (47, 48). Thus, nucleation of protein complexes by CaMKII holoenzymes may play one or more structural roles in dendritic spines. A variety of scaffolding proteins such as PSD-95 and CASK participate in multivalent interactions with cytoskeletal, receptor, and signaling proteins that are essential for proper targeting of receptor and signal transduction complexes (reviewed in Ref. 49). Recent work shows that isolated PSDs contain similar numbers of PSD-95 molecules and CaMKII holoenzymes (50). Interestingly, CASK contains an inactive kinase domain homologous to the catalytic domain of CaMKII that may have evolved from an ancestral calmodulin-dependent protein kinase to lose catalytic function while retaining the ability participate in protein-protein interactions (51, 52). Because CaMKII interactions with CaMKAPs are dynamically modulated,4 we propose that CaMKII may function as an autoregulated structural component, contributing to the reorganization of postsynaptic protein complexes depending on its activation state. These dynamic processes may play a role in the morphological changes in postsynaptic densities and dendritic spines following induction of synaptic plasticity (43, 53, 54), the ordered clustering of CaMKII in "towers" within the PSD (55), and/or the synaptic insertion of AMPA receptors (1). Further investigation of these structural roles will require selective manipulation of these interactions while preserving the catalytic function of the kinase.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants F32-MH068129, 5T32-DK07563, RO1-MH63232, and RO1-NS44282 and by the American Heart Association. 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

1 To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, TN 37232-0615. Tel.: 615-936-1630; Fax: 615-322-7236; E-mail: roger.colbran{at}vanderbilt.edu.

2 The abbreviations used are: CaMKII, calcium/calmodulin protein kinase II; [P-T286]CaMKII, Thr286-autophosphorylated CaMKII{alpha}; NMDA, N-methyl-D-aspartate; NR2B, NMDA receptor 2B subunit; GST, glutathione-S-transferase; PSD, postsynaptic density; AMPA, aminomethylphosphonic acid; ATP, adenosine triphosphate; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; CaMKAP, CaMKII associated protein; PDZ, postsynaptic density-95/disc large/zona occludens-1. Back

3 Y. Jiao, A. J. Robison, Y, Nikandrova, and R. J. Colbran, manuscript in preparation. Back

4 A. J. Robison, R. K. Bartlett, M. A. Bass, and R. J. Colbran, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Brian Wadzinski and Michelle Mazei-Robison for their assistance in manuscript preparation.



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 DISCUSSION
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