COMMUNICATION:
Translocation of Autophosphorylated Calcium/Calmodulin-dependent Protein Kinase II to the Postsynaptic Density*

(Received for publication, February 12, 1997, and in revised form, March 27, 1997)

Stefan Strack , Sukwoo Choi , David M. Lovinger and Roger J. Colbran Dagger

From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0615

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Calcium/calmodulin-dependent protein kinase II (CaMKII) undergoes calcium-dependent autophosphorylation, generating a calcium-independent form that may serve as a molecular substrate for memory. Here we show that calcium-independent CaMKII specifically binds to isolated postsynaptic densities (PSDs), leading to enhanced phosphorylation of many PSD proteins including the alpha -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptor. Furthermore, binding to PSDs changes CaMKII from a substrate for protein phosphatase 2A to a protein phosphatase 1 substrate. Translocation of CaMKII to PSDs occurs in hippocampal slices following treatments that induce CaMKII autophosphorylation and a form of long term potentiation. Thus, synaptic activation leads to accumulation of autophosphorylated, activated CaMKII in the PSD. This increases substrate phosphorylation and affects regulation of the kinase by protein phosphatases, which may contribute to enhancement of synaptic strength.


INTRODUCTION

CaMKII1 isoforms comprise a family of broad specificity, calcium-activated kinases (1, 2). The alpha  and beta  isoforms are abundantly expressed in the brain, with alpha  making up as much as 2% of total protein in certain brain regions (3). CaMKII is particularly enriched in PSDs (4, 5), cytoskeletal specializations apposed to the postsynaptic membrane of excitatory synapses that are thought to be scaffolds for neurotransmitter receptors, ion channels, and their postsynaptic modulators and effectors (reviewed in Refs. 6 and 7). Earlier reports suggested that CaMKIIalpha constitutes as much as 50% of total PSD protein (8-10), but PSDs prepared from rapidly homogenized brains are only 2-3-fold enriched in CaMKIIalpha compared with whole forebrain extracts (3, 11). CaMKIIalpha knockout mice show impaired hippocampal long term potentiation, a cellular model for learning and memory (12). Conversely, introduction of CaMKIIalpha into neurons augments postsynaptic responses and occludes further electrically induced long term potentiation (13, 14).

CaMKIIalpha undergoes calcium/calmodulin-dependent autophosphorylation on Thr286 in its regulatory domain, rendering the kinase partially calcium-independent (1, 2). This reaction has been proposed as a "molecular switch," translating transient calcium elevation into prolonged kinase activity (15, 16), which becomes subject to regulation by protein phosphatases. In addition, Thr286 autophosphorylation promotes binding of CaMKIIalpha to a 190-kDa PSD protein by gel overlay (17). The present results extend these findings, demonstrating that Thr286 autophosphorylation controls subcellular targeting of CaMKII in neurons with important functional consequences.


EXPERIMENTAL PROCEDURES

Purification and Labeling of Recombinant CaMKIIalpha

CaMKIIalpha was expressed in Sf9 cells and purified (17). [35S]CaMKIIalpha (approx 1200 cpm/pmol) was purified from cells metabolically labeled with 35 µCi/ml [35S]methionine for 58 h prior to harvesting. Kinase was autophosphorylated in the presence (Thr286) or the absence (Thr305/Thr306) of calcium/calmodulin (0.2-0.6 and 0.6-1.2 mol 32P/mol kinase subunit, respectively) (17). Appropriate autophosphorylation of [35S]CaMKIIalpha with unlabeled ATP was confirmed by assaying calcium-dependent and -independent kinase activities (18) using specific CaMKII substrate autocamtide-2 (10 µM) (19).

Isolation of PSDs

PSDs were prepared from adult rat forebrains flash frozen within 45 s of euthanasia by detergent lysis of synaptosomes (20) except that 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 µg/ml leupeptin were included in all buffers. Synaptosomes were lysed in 1% (v/v) Triton X-100 and 150 mM KCl, and a second subsequent sucrose gradient was omitted because it yielded no further purification. PSDs displayed typical "donut" morphology by video-enhanced differential-interference contrast microscopy (21). PSDs prepared in the absence or the presence of the protein phosphatase inhibitor microcystin-LR (1 µM) contained similar amounts of CaMKII protein (5-10% by quantitative immunoblotting (17)) and calcium/calmodulin-dependent CaMKII activity (100-150 nmol/min/mg with 10 µM autocamtide-2). Calcium-independent kinase activity was <2% and approx 10% of calcium/calmodulin-dependent kinase activity when PSDs were isolated in the absence or the presence of microcystin-LR, respectively. Therefore, CaMKII (Thr286) was almost completely dephosphorylated during normal PSD isolation (minus microcystin-LR).

In Vitro Binding of CaMKIIalpha

PSDs and 35S- or 32P-labeled CaMKIIalpha were incubated at the indicated concentrations, temperatures, and times in Buffer A (20 mM HEPES, pH 7.5, 10 mM dithiothreitol, 0.1% (v/v) Triton X-100, 1 mM benzamidine, 20 µg/ml leupeptin) containing 1 mg/ml bovine serum albumin, 0.1 M NaCl, 1 µM microcystin-LR, and EDTA and EGTA at 2.5-fold molar excess over Mg2+ and Ca2+, respectively, carried over from the autophosphorylation. Binding was terminated by centrifugation of 90 µl through 0.5 ml of HEPES-buffered 0.5 M sucrose cushion into 20 µl of 10% glutaraldehyde in 0.6 M sucrose in a horizontal rotor (5000 × g, 45-60 s). The sucrose cushions were aspirated (excess ligand), and the tube bottoms (PSD pellets) were cut off and counted.

Protein Phosphatase Assays

Soluble [T286-32P]CaMKIIalpha (0.1-0.2 µM subunit) was incubated with 25-50 µg/ml isolated PSDs, whereas resuspended PSD·[T286-32P]CaMKIIalpha complexes (2-5 µg kinase/mg PSD) were incubated at a final concentration of 70 µg/ml PSD protein. Incubations were conducted for 30 min at 30 °C in Buffer A containing 1 mM EGTA, 1 mg/ml bovine serum albumin, and 0.1 M NaCl, plus specific inhibitors or activators indicated below. Phosphatase activity was quantitated as trichloroacetic acid-soluble (20%, w/v) [32P]phosphate by scintillation counting. Blanks, with no PSDs in soluble substrate assays or 2.5 µM microcystin-LR in "PSD complex" substrate assays, were subtracted from all assays to control for [32P]phosphate present at the beginning of the incubation. Microcystin-LR completely blocked dephosphorylation in both assays. PP1 activity was calculated by subtraction of dephosphorylation measured in the presence of 0.2 µM Inhibitor-2 (Upstate Biotechnology, Inc.) from total "constitutive" dephosphorylation. PP2A activity was defined as activity inhibited by 2.5 nM okadaic acid (LC Laboratories); activity measured in the presence of 2.5 µM okadaic acid was assigned to unknown okadaic acid-resistant phosphatase(s); PP2C activity was defined as that stimulated by 10 mM magnesium acetate in the presence of 2.5 µM okadaic acid. Each protein phosphatase activity was expressed as a percentage of constitutive activity. Activities do not add up to 100% because inhibitor concentrations were chosen for optimum selectivity, not maximal efficacy.

Phosphorylation of PSD Proteins and Immunoprecipitation

After incubation without or with 1 µM [T286-P]CaMKIIalpha , PSDs (0.3 mg/ml) were washed by pelleting twice and suspended in Buffer A containing 10 mM magnesium acetate, 0.5 mg/ml bovine serum albumin, 1 µM microcystin-LR, and either 0.5 mM CaCl2, 6 µM calmodulin, or 1 mM EGTA. Phosphorylations were started by adding 0.5 µM [gamma -32P]ATP (100 cpm/nmol) and stopped with 25 mM EDTA after 1 min on ice. GluR1 was immunoprecipitated (22) using antibody from Chemicon. No phosphorylated band co-migrating with GluR1 was detected in control precipitations with normal serum (not shown).

Translocation of CaMKII in Hippocampal Slices

Preparation and electrophysiological recordings from hippocampal slice were described previously (23). Four to six slices (400 µm, 3-6 week old rats) were divided into control/experimental hemislices and maintained in oxygenated, artificial cerebrospinal fluid for 60 min at 32 °C. After treatment with or without calyculin A (1 µM for 60 min) or tetraethylammonium (TEA) (25 mM for 10 min, 15-min wash) slices were flash frozen in liquid N2. Slices were homogenized in 50 mM Tris, pH 7.5, 1% (v/v) Triton X-100, 0.1 M KCl, 20 mM sodium pyrophosphate, 50 mM NaF, 50 mM sodium beta -glycerophosphate, 1 µM microcystin-LR, 1 mM benzamidine, 20 µg/ml leupeptin, and the PSD-enriched particulate fraction was isolated by centrifugation (30 min at 14,000 × g). Crude PSD fractions were immunoblotted in triplicate for the indicated proteins using mixed alkaline phosphatase- and 125I-labeled secondary antibodies. Gamma counts for CaMKIIalpha /beta and GluR1 in each lane were normalized to neurofilament heavy subunit (NF-H) immunoreactivity. Normalized values for experimental slices were expressed as a percentage of control slice values. Quantifications were performed in the linear range of the assay, which was determined separately for each protein by probing serial extract dilutions.


RESULTS AND DISCUSSION

Thr286 autophosphorylated CaMKIIalpha has been shown previously to bind a 190-kDa PSD protein on gel overlays (17). One criticism of gel overlays is that denatured proteins are partially renatured prior to binding, potentially exposing binding domain(s) that are cryptic in native protein or PSDs. To examine possible interactions of CaMKII under native conditions, we investigated binding of [35S]CaMKIIalpha to isolated PSDs (Fig. 1A). Calcium/calmodulin-dependent autophosphorylation at Thr306, generating the calcium-independent form of the kinase ([T286-P]CaMKII), enhanced in vitro binding about 5-fold. Calcium/calmodulin-independent autophosphorylation at Thr305/Thr306, inactivating the enzyme ([T306-P]CaMKII), reduced binding somewhat below nonphosphorylated kinase. Binding of [T286-P]CaMKII by PSDs was independent of calcium/calmodulin or magnesium (not shown). The apparent binding was not due to exchange of [35S]CaMKII for endogenous enzyme but represented true accumulation of CaMKIIalpha in the PSD (Fig. 1A, inset). [T286-P]CaMKIIalpha binding to PSDs was specific and may be functionally relevant, because the low levels of binding to mitochondria and membranes were unaffected by autophosphorylation (Fig. 1A).


Fig. 1. Characterization of CaMKIIalpha binding to PSDs in vitro. A, comparison of CaMKIIalpha binding to different cellular structures. Nonphosphorylated or Thr286- or Thr306-autophosphorylated (non-P, T286-P, and T306-P, respectively) [35S]CaMKII (1 µM each) was incubated with PSDs, liver mitochondria (mito.), or NIH 3T3 fibroblast membranes (0.25 mg/ml each) for 10 min at 25 °C. Inset, PSDs were incubated with each form of CaMKIIalpha (2.5 µM), washed twice, and analyzed by quantitative CaMKII immunoblot. The amounts of endogenous CaMKIIbeta does not change following binding. Data (mean ± S.D., n = 2) representative of two (mitochondria and membranes) or four (PSDs) experiments are shown. B, time course of binding. Incubations at 4 °C containing 50 nM [T286-32P]CaMKIIalpha and 0.1 mg/ml PSDs were stopped at the indicated times. C, time course of dissociation. PSDs (0.25 mg/ml) were incubated (25 °C, 15 min) with [T286-P] (bullet , open circle ) or nonphosphorylated (square ) [35S]CaMKIIalpha (1 µM) and unbound kinase was removed by centrifugation. PSD·CaMKIIalpha complexes (16.8 µg [T286-P]CaMKIIalpha per mg, 1 µg/mg nonphosphorylated) were resuspended in fresh buffer with (bullet , square ) or without (open circle ) microcystin-LR (1 µM), and [35S]CaMKII released into the supernatant was assayed at the indicated times. Data (means ± S.D., n = 2) representative of three experiments are shown in B and C. D, determination of binding affinity. Initial rates of binding to PSDs were determined by incubating PSDs (0.1 mg/ml) with [T286-32P]CaMKIIalpha (50 nM) for 5 min at 4 °C in the absence or the presence of nonradioactive CaMKIIalpha in different autophosphorylation states. Mean ± S.D. (n = 2) data from two experiments are plotted. The apparent IC50 for [T286-P]CaMKIIalpha was 1.06 µM (95% confidence interval: 0.87-1.31 µM).
[View Larger Version of this Image (37K GIF file)]

[T286-P]CaMKIIalpha binding to PSDs was rapid, reaching saturation after 15 min at 4 °C (Fig. 1B) or 5 min at 25 °C (not shown). Whereas nonphosphorylated kinase bound reversibly (approx 25% dissociated in 3 h), binding of [T286-P]CaMKIIalpha appeared essentially irreversible under these conditions (<3% dissociated in 3 h) (Fig. 1C). Interestingly, although binding to PSDs was enhanced by autophosphorylation, continued phosphorylation was not required to maintain the interaction because dissociation was not accelerated when dephosphorylation of bound kinase by endogenous protein phosphatases was allowed (calcium-independent CaMKII activity 17 versus 10% ± microcystin-LR at 60 min) (Fig. 1C, open circle). This may explain why during PSD isolation CaMKII remains PSD-associated even though it is mostly dephosphorylated (see "Experimental Procedures"). In fact, [T286-P]CaMKIIalpha dissociated from PSDs as slowly as endogenous CaMKIIalpha measured by immunoblotting (not shown), suggesting that in vitro and in vivo association of CaMKII with the PSD are mechanistically similar. The reason for this very slow reversibility is not known, but mechanisms such as proteolysis (24, 25) may be required to dissociate CaMKII from the PSD in vivo.

To assess the affinity of binding to PSDs, initial rates of [T286-32P]CaMKIIalpha binding were measured in the absence or the presence of nonradioactive kinase. Whereas non-P CaMKIIalpha and [T306-P]CaMKIIalpha were poor competitors, [T286-P]CaMKIIalpha was an effective competitor (apparent IC50 approx 1.1 µM) (Fig. 1D). Because this IC50 is approximately 10 times lower than the average concentration of CaMKII in forebrain (3, 17), sufficient CaMKII exists for binding to be regulated in vivo. Significantly, this IC50 is very similar to the affinity of [T286-P]CaMKIIalpha for p190 estimated by gel overlay (17); p190 is therefore a candidate for targeting the calcium-independent form of CaMKII to PSDs.

It seemed important to determine which protein phosphatases (PPs) influence the lifetime of calcium-independent CaMKII in the PSD. CaMKII can be dephosphorylated by purified PP1 (26), PP2A (27), and PP2C (28), but not by PP2B. Each of these major phosphatases is present in PSDs, although only PP1 is enriched in this fraction (29). Their activities can be distinguished based on requirements for divalent cations and sensitivity to specific inhibitors (30). With soluble exogenous [T286-32P]CaMKIIalpha as the substrate, the PP2A:PP1 activity ratio was 3:1 (Fig. 2), in agreement with previous data (29). However, under these conditions, 5-10% of the substrate binds to PSDs. When assays were repeated using [T286-32P]-CaMKIIalpha previously bound to the PSD as a substrate, PP1 appeared to be mostly responsible for dephosphorylation (PP2A:PP1 activity ratio, 1:6; Fig. 2).2 This is consistent with previous reports that dephosphorylation of CaMKII endogenous to PSDs is catalyzed by PP1 (29, 31, 32); however, CaMKII endogenous to isolated PSDs (i.e. in vivo translocated CaMKII) may represent a modified form of the kinase (46), possibly due to post-mortem ischemic conditions (11, 47). The present data demonstrate that interaction of the calcium-independent form of CaMKII with PSDs directly regulates its inactivation, in that binding to the PSD in vitro converts CaMKII from a PP2A substrate to a substrate for PSD-bound PP1. The mechanism for this change in protein phosphatases responsible for dephosphorylating Thr286 is unknown. CaMKII may undergo a conformational change after binding to PSDs that favors dephosphorylation by PP1. Alternatively, dephosphorylation by PP1 may be enhanced by physical proximity in the PSD.


Fig. 2. Dephosphorylation of [T286-P]CaMKIIalpha . Protein phosphatases endogenous to isolated PSDs were allowed to dephosphorylate soluble and [T286-32P]CaMKIIalpha previously bound to PSDs. Specific inhibitors and activators were used to define contributions of PP1, PP2A, PP2C, and an okadaic acid-resistant activity (see "Experimental Procedures"). Binding of CaMKIIalpha to PSDs had no effect on the dephosphorylation of glycogen phosphorylase a, a selective PP1 substrate, or casein, a substrate for PP2A and PP2C (not shown), demonstrating that CaMKIIalpha binding to PSDs does not activate PP1 or inactivate PP2A. Similar data were obtained in four or five independent experiments.
[View Larger Version of this Image (28K GIF file)]

AKAP79 is a protein that is thought to anchor inactive forms of protein kinase A, protein kinase C, and PP2B to PSDs (33, 34). In contrast, CaMKII bound to PSDs remains active, because binding of [T286-P]CaMKIIalpha to PSDs increased calcium-independent activity toward autocamtide-2 peptide substrate by an amount corresponding to binding of [T286-32P]CaMKIIalpha quantified in parallel reactions (not shown). More importantly, binding of [T286-P]CaMKIIalpha (approx 10 µg/mg PSD protein) led to a approx 2-fold increase of calcium-dependent phosphorylation and a approx 13-fold increase of calcium-independent phosphorylation of many proteins in PSDs (Fig. 3A).


Fig. 3. Activity of CaMKIIalpha bound to PSDs. A, phosphorylation of PSD proteins. PSDs or PSDs with bound [T286-P]CaMKIIalpha (PSD:CaMKII(P)) phosphorylated in the absence (-) or the presence (+) of calcium/calmodulin were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The relative radioactivities of each lane (duplicate determinations by scintillation counting) are indicated. Arrows mark autophosphorylated alpha  and beta  subunits of endogenous CaMKII. B, identification of the glutamate receptor GluR1 subunit as a substrate. GluR1 immunoprecipitations were carried out from samples of PSD and PSD·CaMKII(P) phosphorylated in the absence of calcium/calmodulin (lanes 2 and 4 of panel A). Similar data were obtained in four (A) and two (B) independent experiments.
[View Larger Version of this Image (38K GIF file)]

In the presence of calcium/calmodulin, CaMKII phosphorylates GluR1 subunits of AMPA-type glutamate receptors in PSDs, increasing channel permeability and postsynaptic responses (22). GluR1 immunoprecipitation experiments were carried out with PSDs phosphorylated in the absence of calcium, with or without bound [T286-P]CaMKIIalpha . Phosphorylated GluR1 was immunoprecipitated only from PSDs to which [T286-P]CaMKIIalpha had been bound (Fig. 3B), identifying GluR1 as a substrate for the PSD-bound, autophosphorylated, calcium-independent form of CaMKII.

Brain injuries involving increased intracellular calcium such as ischemia/hypoxia (11, 35), hypoglycemia (36), and excitotoxic insults (37) cause CaMKII translocation to the cytoskeleton, including PSDs. Ischemia also transiently increases calcium-independent CaMKII activity (38). We therefore tested the hypothesis that Thr286 autophosphorylation is sufficient for translocation of CaMKII to occur in neurons. Calyculin A, a cell-permeant protein phosphatase inhibitor, was shown previously to increase Thr286 phosphorylation in hippocampal slices (39) without affecting general excitability or viability (40). Here, calyculin A increased calcium-independent CaMKII activity in hippocampal slice extracts from 25.6 ± 1.2% to 43.4 ± 3.7% (n = 6, p < 0.01), accompanied by a >2-fold increase in CaMKII associated with PSD-enriched fractions (Fig. 4, A and C). We next induced a form of long term potentiation with the K+-channel blocker TEA (41). There was a 83.6 ± 21.7% (n = 4, p < 0.05) enhancement of transmission at CA3-CA1 synapses (Fig. 4B) 15 min after TEA removal, accompanied by increased calcium-independent CaMKII activity (20.3 ± 1.9% to 26.7 ± 4.2%, n = 5, p = 0.07) and a 70-80% increase of CaMKII protein in the PSD-enriched fraction (Fig. 4C). Thus, CaMKII translocation occurs in intact neurons in response to treatments that induce CaMKII autophosphorylation and potentiate synaptic transmission.


Fig. 4. Translocation of CaMKII in hippocampal slices. A, effect of calyculin A. Representative immunoblot of PSD-enriched fraction from hippocampal slices of the same rat treated with or without 1 µM calyculin A for 60 min. B, TEA potentiates synaptic transmission. Slices were treated with 25 mM TEA for 10 min while recording field potentials in the CA1 region in response to test stimuli applied to the Schaffer collateral. Averages of 10 traces obtained before treatment and 15 min after treatment are shown. C, summary of translocation experiments. Calyculin A (1 µM for 60 min) and TEA (25 mM for 10 min, 15 min wash) treatment resulted in a significant increase of both alpha  and beta  isoforms of CaMKII (* p < 0.05; ** p < 0.01; n = 6) in PSD-enriched fractions, whereas GluR1 (n = 3) remained unaffected.
[View Larger Version of this Image (32K GIF file)]

In conclusion, the present data suggest a molecular mechanism for association of CaMKII with the PSD that is likely to be important in physiological and pathological states. Stimulation of CaMKII autophosphorylation by calcium influx into dendritic spines may increase the amount of active CaMKII in the PSD resulting in enhanced phosphorylation of GluR1 and other key substrates, in turn leading to an enhanced postsynaptic response. In addition, translocation may sequester CaMKII away from the cytosolic phosphatase activity of PP2A, making it available for dephosphorylation by PSD-bound PP1 only. Interestingly, PP1 itself is highly regulated by phosphorylation and association with targeting and inhibitory subunits (42) and is also involved in synaptic plasticity (43). Finally, because CaMKII has been proposed to play a structural role in the PSD (5), translocation of substantial amounts of cytosolic CaMKII to the PSD in response to calcium signals may result in long term changes in synapse morphology (44, 45).


FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM47973 (to R. J. C.) and NS30470 (to D. M. L.) and an American Heart Association Established Investigator Award (to R. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232-0615. Tel.: 615-936-1630; Fax: 615-322-7236; E-mail: Roger.Colbran{at}mcmail.vanderbilt.edu.
1   The abbreviations used are: CaMKII, calcium/calmodulin-dependent protein kinase II; CaMKIIalpha or beta , alpha  or beta  isoform of CaMKII; [T286-P]CaMKIIalpha , CaMKIIalpha autophosphorylated on threonine 286; [T306-P]CaMKIIalpha , CaMKIIalpha autophosphorylated on threonine 305 and/or threonine 306; PSD, postsynaptic density; PP, protein serine/threonine phosphatase; TEA, tetraethylammonium; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid.
2   Total activities in soluble and PSD-bound CaMKII phosphatase assays were 7.8 and 0.6 pmol/min/mg, respectively. It is difficult to interpret this difference because both substrate and enzyme are immobilized on the PSD in the latter assay. The apparent decrease in phosphatase activity after binding may reflect a preferential loss of the PP2A component or may simply be due to restricted mobility of PSD-bound CaMKII.

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