©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Autophosphorylated Ca/Calmodulin-dependent Protein Kinase II with Neuronal Cytoskeletal Proteins
CHARACTERIZATION OF BINDING TO A 190-kDa POSTSYNAPTIC DENSITY PROTEIN (*)

R. Blair McNeill (§) , Roger J. Colbran (¶)

From the (1) Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Subcellular localization of Ca/calmodulin-dependent protein kinase II (CaMKII) by interaction with specific anchoring proteins may be an important mechanism contributing to the regulation of CaMKII. Proteins capable of binding CaMKII were identified by the use of a gel overlay assay with recombinant mouse CaMKII (mCaMKII) or Xenopus CaMKII (xCaMKII) P-autophosphorylated at Thras a probe. Numerous [P]CaMKII-binding proteins were identified in various whole rat tissue extracts, but binding was most prominent to forebrain proteins of 190 kDa (p190) and 140 kDa (p140). Fractionation of forebrain extracts localized p190 and p140 to a crude particulate/cytoskeletal fraction and isolated postsynaptic densities. [P]mCaMKII-bound to p190 with an apparent Kof 609 nM (subunit concentration) and a Bof 7.0 pmol of mCaMKII subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM [P]mCaMKII to p190 was competed by nonradioactive mCaMKII autophosphorylated on Thr(EC= 200 nM), but to a much lesser extent by nonradioactive mCaMKII autophosphorylated on Thr(EC> 2000 nM). In addition, nonphosphorylated mCaMKII was a poor competitor for [P]mCaMKII binding to p190. The competition data indicate that Ca/CaM-dependent autophosphorylation at Thrpromotes binding to p190, whereas, Ca/CaM-independent autophosphorylation at Thrdoes not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Caconcentration.


INTRODUCTION

Increases in cytosolic calcium concentration, either by release from intracellular stores or by influx from the extracellular space, mediate the effects of many hormones and neurotransmitters on target tissues. The primary intracellular receptor for calcium is calmodulin (CaM),() which, when activated by calcium, interacts with many proteins (1) . CaM targets include several serine/threonine protein kinases such as myosin light chain kinase, phosphorylase kinase, and Ca/calmodulin-dependent protein kinases I-IV. Within the Ca/calmodulin-dependent protein kinase class, CaMKII is somewhat unique in that it exhibits broad substrate specificity and is found in most, if not all, tissues. CaMKII was originally isolated from rat brain and rabbit liver (reviewed in Ref. 2), and four isoforms (, , , ) have now been identified using molecular techniques (3, 4, 5, 6) . Alternatively spliced mRNAs encoding and are predominantly and exclusively expressed in the nervous system, whereas mRNAs encoding and isoforms are predominant in peripheral tissues (6, 7, 8, 9, 10, 11) . A combination of 8-12 of the subtypes make up a CaMKII holoenzyme. In brain, the isoform is highly expressed in forebrain while subtypes are universally expressed (4, 5, 6, 12) . Most, if not all, neurons contain CaMKII by immunocytochemistry (13, 14) . In some brain regions, such as hippocampus, the isoform alone accounts for 1% of total protein (15) . Among neuronal processes regulated by CaMKII are neurotransmitter release, catecholamine synthesis, cytoskeletal protein interactions, gene expression, and postsynaptic responses such as long term potentiation (reviewed in Refs. 16 and 17). Transgenic mice lacking the CaMKII gene are deficient in spatial learning (18) and also have an abnormal fear response and aggressive behavior (19) . In addition, it is much more difficult to induce long term potentiation in hippocampal slices from the transgenic animals (20) .

CaMKII activity is regulated by Ca/CaM binding and by two modes of autophosphorylation. Ca/CaM stimulates autophosphorylation at Thrresulting in a Ca/CaM-independent form (21, 22, 23, 24) . In the absence of Ca/CaM, CaMKII undergoes autophosphorylation at Thrand Ser, which blocks Ca/CaM binding, thereby inactivating the enzyme (25, 26, 27) . Presumably, protein phosphatases contribute to the regulation of CaMKII autophosphorylation in vivo by dephosphorylating these sites.

Consistent with its diverse roles, CaMKII exhibits broad yet localized distribution within neurons, as demonstrated by immunohistochemistry (13, 14) . It is concentrated at postsynaptic densities, where it is ideally located to regulate postsynaptic events, such as long term potentiation. Discrete localization of CaMKII may play an important role in its physiological function in at least three ways. 1) Substrate proteins may have to be colocalized with the kinase to be phosphorylated. 2) Localized modulation of the Caconcentration (31) may differentially regulate CaMKII in discrete subcellular compartments. 3) Colocalization with different protein phosphatases may affect its dephosphorylation. Recently, CaMKIIwas shown to contain a nuclear localization signal sequence which is inserted by alternative splicing just carboxyl-terminal of the calmodulin binding domain (32) . However, mechanism(s) that determine subcellular localization of other CaMKII isoforms are unknown. Protein kinase A is localized, at least in part, by association with specific AKAPs (A-kinase anchoring proteins) (33, 34, 35, 36, 37) . In addition, activated protein kinase C is thought to bind to proteins termed RACKs (receptors for activated C kinase) (38, 39) . Interaction of multifunctional kinases with specific anchoring proteins may allow broad specificity kinases to have localized and specific effects within cells. The present studies were designed to test the hypothesis that localization of CaMKII may also be determined, at least in part, by anchoring proteins.


EXPERIMENTAL PROCEDURES

Expression of CaMKII in Baculovirus (AcMNPV)

All procedures were performed as described (40) . Recombinant baculovirus expressing wild type mouse CaMKII (mCaMKII), a mutant mCaMKII (T286A), and wild type Xenopus CaMKII (xCaMKII) were gifts from Drs. D. Brickey and T. Soderling (Vollum Institute) (41, 42) . Suspension cultures were infected with virus at a multiplicity of infection of 20 plaque-forming units per cell and grown for 72 h. Cell pellets were stored at -70 °C and then thawed in homogenization buffer at 4 °C for purification of the kinase as described (41) .

Rat Forebrain Fractionation

Forebrains (1 g each) were rapidly removed from female Sprague-Dawley rats (>50 days of age) and homogenized using a Polytron (two 15-s bursts separated by 30 s on ice) in 10 ml/forebrain ice-cold homogenization buffer (10 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 20 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin). The homogenate was centrifuged at 100,000 g for 60 min, and the supernatant (S1; cytosolic proteins) was removed. The pellet was rehomogenized in 10 ml of homogenization buffer plus 1% Triton X-100 and centrifuged as before. The supernatant (S2; membrane proteins) was removed, and the pellet was resuspended in 10 ml of homogenization buffer (P2; cytoskeletal proteins). Aliquots of fractions were stored at -70 °C until needed. All other rat tissues were homogenized following the same protocol in order to generate whole tissue extracts.

Ca/Calmodulin-dependent Autophosphorylation

Reactions (typically 0.5 ml) were performed in HEPES (50 mM, pH 7.5), magnesium acetate (2 mM), CaCl(1.5 mM), DTT (2 mM), and [-P]ATP (10 µM; 20000 cpm/pmol). mCaMKII or xCaMKII (in 50 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, and 10% (v/v) ethylene glycol) was added to 3-10 µM (subunit concentration) so that 15% glycerol was also present in the incubations. Calmodulin was added to 2 the kinase subunit concentration. Following incubation on ice for 60 s, nonradioactive ATP (2 mM final) was added, followed by an additional 30-s incubation on ice. The reaction was stopped by the addition of EDTA (10 mM), NaF (25 mM), NaCl (500 mM), and Tween 20 (0.1% v/v), and incubation continued for 1 h on ice. Controls established that the autophosphorylation stoichiometry did not significantly increase during the second (30-s) phase. However, the second phase enhanced recovery from desalting columns (see below), ensuring that the eluted material was >95% insoluble in 10% trichloroacetic acid. Autophosphorylation stoichiometries (typically 0.2-0.6 mol of [P]phosphate per mol of mCaMKII subunit) were determined by spotting aliquots on P81 papers (Whatman) and washing in 75 mM phosphoric acid, followed by liquid scintillation counting (43) . [P]CaMKII was desalted on a Sephadex G-50 (fine) column (15 ml) equilibrated with Tris-HCl (50 mM, pH 7.4), NaCl (500 mM), EGTA (0.1 mM), DTT (1 mM), and Tween 20 (0.1% v/v), collecting 0.5-ml fractions. Peak radioactive fractions, as determined by Cerenkov counting, were pooled, and the concentration of [P]mCaMKII in the pool was determined by liquid scintillation counting of aliquots from the pool. Generally, 60-100% of applied [P]mCaMKII was recovered in the pool. Nonradioactive autophosphorylations of mCaMKII, T286A-mCaMKII, and xCaMKII were performed using identical conditions, except that [-P]ATP was replaced with ATP, and the reaction was terminated with EDTA (5 mM) alone. Nonphosphorylated samples were incubated under identical conditions, except ATP was replaced with water. Nonradioactive and nonphosphorylated samples were not desalted.

Ca/Calmodulin-independent (Basal) Autophosphorylation

Reactions (typically 0.5 ml) were performed in HEPES (50 mM, pH 7.5), magnesium acetate (2.5 mM), EGTA (2 mM), bovine serum albumin (1 mg/ml), DTT (2 mM), and ATP (1 mM). Typically, 5 µM mCaMKII subunit (in 50 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, and 10% (v/v) ethylene glycol) was added, and storage buffer was added to bring the final glycerol concentration to 15% (v/v). Samples were incubated for 60 min at 30 °C, and then reactions were stopped with EDTA (5 mM). Autophosphorylation stoichiometries were about 1.2 mol of [P]phosphate/mol mCaMKII subunit, determined by running similar reactions in the presence of [-P]ATP. Nonphosphorylated mCaMKII was incubated under the same conditions except ATP was omitted.

Casein Phosphorylation

Casein was incubated overnight at 30 °C in Tris-HCl (50 mM, pH 7.5), EDTA (0.1 mM), magnesium acetate (10 mM), casein (5 mg/ml), ATP (0.2 mM), C-subunit of protein kinase A (8.4 µg/ml), and -mercaptoethanol (0.1% v/v) in a final volume of 0.5 ml (44) . The reaction was stopped by the addition of EDTA and sodium pyrophosphate (10 mM each). Similar reactions in the presence of [-P]ATP indicated a typical stoichiometry of about 0.1 mol of phosphate per mol of casein.

Phosphorylase b Phosphorylation

Phosphorylase b was incubated for 60 min at 30 °C in Tris/sodium glycerol 1-phosphate (100 mM each, pH 8.2), magnesium acetate (10 mM), ATP (0.2 mM), CaCl(0.1 mM), phosphorylase b (10 mg/ml), and phosphorylase kinase (0.2 mg/ml) in a final volume of 0.5 ml (45) . NaF (50 mM) and EDTA (25 mM) were then added, and the incubation was continued for another 60 min. Similar reactions in the presence of [P]ATP indicated a typical stoichiometry of about 0.5 mol of phosphate per mol of phosphorylase b. Phosphorylation of CaMK-(281-309)-The peptide (50 µM) was incubated for 30 min at 30 °C in HEPES (50 mM, pH 7.5), magnesium acetate (2.5 mM), CaCl(1.5 mM), ATP (0.5 mM), calmodulin (150 µM), and mCaMKII (100 nM) in a final volume of 0.5 ml (46) , and the reaction was stopped by addition of EDTA (5 mM). Typically, 0.5 mol of phosphate was incorporated per mol of peptide.

Overlay Technique

An overlay procedure (47) was used to detect binding of P-labeled mCaMKII or xCaMKII to cellular proteins. Protein fractions were separated by SDS-PAGE (10%; 7-cm or 16-cm gels) and transferred overnight at 4 °C and 30 V to a PVDF membrane (MilliPore or Gelman) in Tris/glycine buffer (25 mM and 192 mM, respectively). Prestained standards were also run to permit estimation of approximate molecular weights of [P]CaMKII-binding proteins. Membranes were blocked for at least 60 min in Tris-HCl (50 mM)/NaCl (200 mM) (TBS) containing Tween 20 (3% v/v) and non-fat powdered milk (5% w/v) (blocking buffer). The membrane was washed for 30 min in TBS containing Tween 20 (0.1% v/v) and non-fat powdered milk (5% w/v) (rinsing buffer) prior to incubation with [P]CaMKII. Membranes were then incubated for 2 h at room temperature with constant rocking in rinse buffer containing the indicated concentration of P-labeled CaMKII, followed by extensive washing with at least 5 changes of rinse buffer and autoradiography. For affinity determinations and competition experiments, preparative SDS-PAGE minigels were loaded with 350-720 µg of P2 fraction protein; the resulting membranes were cut into 5-mm strips (containing 23-48 µg of total protein), and incubations were performed in Bio-Rad mini incubation trays (0.5-ml incubation and 1-ml wash per strip).

Quantitation of Specific Bands

Binding of P-labeled CaMKII to p190 was quantitated by excising the band from the PVDF membrane and counting in a liquid scintillation counter. Blanks for each strip were cut from an area of the membrane which appeared to have no bands. The amount of kinase bound to p190 in each strip (picomoles) was normalized for the loading of P2 protein on the strip (milligrams of protein).

CaMKII Western Blot

PVDF membranes were prepared as described for the overlay assay. Membranes were blocked for 60 min in blocking buffer (see above) and then incubated with affinity-purified goat anti-CaMKII antibody (0.1 µg/ml) in blocking buffer overnight at 4 °C. The antibody was raised to purified native rat forebrain CaMKII (48) by Bethyl Laboratories (Montgomery, TX) and then affinity-purified on a column of recombinant mCaMKII coupled to Affi-Gel 15 (Bio-Rad), prepared according to the manufacturer's directions. Membranes were then incubated with alkaline phosphatase-conjugated rabbit anti-goat secondary antibody (Vector Laboratories) (1:1000 dilution) for 60 min and developed with AP Substrate Kit II (Vector Laboratories) according to the directions.

Miscellaneous

All protein concentrations were determined by a Bradford (49) protein assay (Bio-Rad) using bovine serum albumin as standard. Phosphorylase kinase, phosphorylase b, casein, and prestained standards were from Sigma. The peptide CaMK-(281-309) was synthesized and characterized as described previously (50) . Calmodulin was purified from bovine brain (51) . RII and C-subunit were gifts from Dr. J. D. Corbin (Vanderbilt University); purified RII contains about 0.5 mol of phosphate/mol of RII subunit (52) . Tubulin (53) and mixed MAPs (54) were gifts from Dr. R. C. Williams, Jr. (Vanderbilt University). Postsynaptic densities (55) were a gift from Drs. S. E. Tan and T. Soderling (Vollum Institute).


RESULTS

As a first test of the hypothesis that the subcellular localization of CaMKII may be determined by association with specific anchoring protein(s), a gel overlay assay was developed to detect proteins capable of binding to CaMKII. Discrete patterns of [P]mCaMKII-binding proteins were detected in various central nervous system and peripheral tissues (Fig. 1, top). The most abundant binding of [P]mCaMKII was detected in forebrain extracts, where [P]mCaMKII-binding proteins of 140 and 190 kDa were particularly prominent, but several other [P]mCaMKII-binding proteins were also detected. Cerebellum and pons/medulla extracts contained generally lower [P]mCaMKII binding activities, and the pattern of binding was significantly different. The pattern of [P]mCaMKII binding to proteins in peripheral tissue extracts was different. For example, kidney extracts contain very few prominent [P]mCaMKII-binding proteins, whereas ovary, spleen, and lung contain a major [P]mCaMKII-binding protein of about 65 kDa. Heart extracts contained several [P]mCaMKII-binding proteins between 40 and 220 kDa. Generally, similar patterns of CaMKII-binding proteins were detected when [P]xCaMKII was used as a probe (Fig. 1, bottom). However, the 65-kDa CaMKII-binding protein detected in ovary, lung, and spleen was apparently specific for the -isoform. Binding of P-labeled and isoforms to all proteins could be largely competed by excess nonradioactive autophosphorylated ligand (see below). The pattern of [P]CaMKII-binding proteins did not correspond to the Coomassie Blue staining pattern of the proteins in any of the tissue extracts (data not shown).


Figure 1: Survey of rat tissues for CaMKII-binding proteins. The indicated whole tissue extracts (100 µg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and then probed with either [P]mCaMKII ( top) or [P]xCaMKII ( bottom) as described under ``Experimental Procedures.'' Approximate molecular masses (kDa) are shown on the left.



In order to determine whether the [P]mCaMKII-binding proteins might play a role in localizing rat forebrain CaMKII, extracts were separated into soluble (S1), Triton X-100 soluble (S2), and Triton X-100 insoluble (P2) fractions (see ``Experimental Procedures''). The three fractions were subjected to overlay analysis using [P]mCaMKII or probed with a polyclonal antibody to CaMKII. The most abundant binding of [P]mCaMKII was detected in the P2 fraction, where proteins of 140 and 190 kDa (p140 and p190) were most prominent (Fig. 2, left). The pattern of [P]mCaMKII-binding proteins in P2 was very similar to the [P]mCaMKII-binding protein pattern detected in whole forebrain extracts (Fig. 1, top). Consistent with this observation, [P]mCaMKII-binding proteins were barely detectable in S1 and S2. In addition, the relative abundance of [P]mCaMKII-binding proteins in the particulate fraction (P2) parallels the localization of endogenous CaMKII immunoreactivity to this fraction, as determined by Western blot (Fig. 2, middle).


Figure 2: Subcellular distribution of rat forebrain CaMKII-binding proteins. Rat forebrain extracts were fractionated as described under ``Experimental Procedures.'' Fractions were separated by SDS-PAGE (100 µg of protein/lane), transferred to PVDF membranes, and then probed with either [P]mCaMKII ( left) or antibodies to CaMKII ( middle). Purified PSDs (20 µg), purified tubulin (5 µg), and a mixed MAPs sample (20 µg) were separated by SDS-PAGE, transferred to PVDF membrane, and then probed with [P]mCaMKII ( right).



The P2 fraction is a crude cytoskeletal preparation which likely contains postsynaptic densities (PSDs), tubulin, neurofilaments, various microtubule-associated proteins (MAPs), and other cytoskeletal proteins. In order to further characterize the location of the [P]mCaMKII-binding proteins, overlay assays were performed using purified tubulin, a mixed MAP fraction (which contains tubulin, neurofilament proteins, , and microtubule-associated proteins; see Ref. 54), and purified PSDs. The pattern of [P]mCaMKII-binding proteins detected in PSDs was very similar to the pattern in the P2 fraction and in whole forebrain extracts (compare Fig. 2, left, with Fig. 2 , right, and Fig. 1). Much weaker binding of [P]mCaMKII to tubulin and to several MAPs was also detected (Fig. 2, right).

As a first step toward understanding the possible physiological relevance of these observations, the affinity for the interaction of [P]mCaMKII with its binding proteins was determined. Strips of PVDF membrane containing rat forebrain P2 proteins were incubated with increasing concentrations of [P]mCaMKII. Binding of [P]mCaMKII to p190 was detectable at 25 nM [P]mCaMKII subunit and increased with the concentration of [P]mCaMKII (Fig. 3, top). Other bands were detectable in the affinity experiments; however, they were only apparent at higher concentrations of [P]mCaMKII. The portion of each membrane corresponding to p190 was cut out, and the amount of [P]mCaMKII bound to p190 was determined in a scintillation counter (Fig. 3, bottom). The data appear to follow a generally hyperbolic form approaching saturation at 3 µM [P]mCaMKII. However, almost double the [P]mCaMKII was bound at the 4 µM data point, and there was considerably more variability in the amount of binding detected, as indicated by the larger standard error. This might indicate that a second binding component was being detected at 4 µM [P]mCaMKII, or that nonspecific binding was significantly increased. However, these alternatives could not be investigated further because 4 µM was the maximum concentration of [P]mCaMKII possible in these experiments. Scatchard transformation of the data obtained up to 3 µM [P]mCaMKII yielded a straight line (Fig. 3, bottom, inset), indicating that [P]mCaMKII bound to p190 with a Kof 609 nM mCaMKII subunit (61 nM holoenzyme) and a Bof 7.0 pmol of [P]mCaMKII subunit bound per mg of P2 protein. Very similar binding parameters were obtained when the raw data (up to 3 µM) were fitted using nonlinear regression techniques (data not shown).


Figure 3: Determination of the affinity for [P]mCaMKII binding to p190. Rat forebrain P2 fraction was fractionated by preparative SDS-PAGE and then transferred to PVDF membranes. Strips (5 mm) of the blocked membrane were incubated with the indicated concentration of [P]mCaMKII in a total volume of 0.5 ml. Top, representative autoradiograms from a single experiment. The panel on the left was exposed to film for 4 h, and the panel on the right was exposed for 14 h. Bottom, quantitation of [P]mCaMKII binding to p190 (see ``Experimental Procedures''). Data were obtained in 10 experiments, and each point is the mean ± S.E. of at least 3 determinations. The hyperbola is plotted according to parameters derived from the Scatchard plot ( inset) of the binding data (), excluding the 4 µM data point () (see text). Since the amount of [P]mCaMKII bound to the membranes was less than 0.5% of the total present at all concentrations, the amount of free ligand was assumed to be equal to the total concentration.



In order to establish the specificity of the interaction between [P]mCaMKII and p190, competition experiments were performed. Strips of membrane containing P2 proteins were incubated with 100 nM [P]mCaMKII, autophosphorylated in the presence of Ca/CaM, in the additional presence of potential nonradioactive competitors. Fig. 4shows competition by different autophosphorylated forms of mCaMKII. Representative experiments are shown in the top and middle panels, and quantitative data from three independent experiments are shown below. Competition by mCaMKII autophosphorylated with nonradioactive ATP in the presence of Ca/CaM (same conditions used to prepare [P]mCaMKII) was concentration-dependent, exhibiting an ECof about 200 nM and reducing binding by over 90% at 2 µM. However, if ATP was omitted from the competitor autophosphorylation reaction ( i.e. nonphosphorylated competitor), only a 20% reduction in binding was detected at 2 µM competitor. Therefore, autophosphorylation of mCaMKII in the presence of Ca/CaM increased the potency of competition by more than 10-fold.


Figure 4: Competition for [P]mCaMKII binding to p190 by mCaMKII autophosphorylated at different sites. Membrane strips containing fractionated P2 proteins were incubated with 100 nM [P]mCaMKII in the additional presence of nonradioactive mCaMKII that had previously been incubated in the presence (, ) or absence (, ) of ATP with either Ca/CaM (, ) or EGTA (, ). Top, autoradiograms from a representative experiment in which the competitor was incubated with Ca/CaM. Middle, autoradiograms from a representative experiment in which the competitor was incubated with EGTA. Bottom, quantitation of [P]mCaMKII binding to p190 (see ``Experimental Procedures'') in the presence of competitors. Each point represents the mean ± S.E. ( n > 3).



CaMKII can be autophosphorylated at multiple sites; with Ca/CaM present, Thris the primary site of autophosphorylation when incubations are performed on ice (22, 23, 24) as above. Additional competition experiments were performed using mutant mCaMKII in which Thrwas changed to Ala (T286A). T286A-mCaMKII was a poor competitor (ECof approximately 2 µM) for wild type [P]mCaMKII (data not shown), even following incubation on ice with ATP in the presence of Ca/CaM (stoichiometry < 0.05 mol of [P]phosphate/mol of T286A-mCaMKII subunit).

CaMKII undergoes slower basal autophosphorylation at Thrin the absence of Ca/CaM, and possibly other sites, but not at Thr(26, 27) . Therefore, the effect of basal autophosphorylation (with nonradioactive ATP) on competition was investigated. Binding of [P]mCaMKII was reduced by only about 45% at 2 µM basal autophosphorylated mCaMKII (Fig. 4). Furthermore, the omission of ATP from the basal autophosphorylation reaction had little effect on competition. Therefore, autophosphorylation of mCaMKII at the basal site(s) did not affect the potency of competition for [P]mCaMKII binding.

The data comparing competition by different autophosphorylated forms of mCaMKII suggest that potent binding of mCaMKII to p190 requires autophosphorylation at Thr. Autophosphorylation at Thris already known to convert the kinase to an open/active (Ca-independent) conformation (21, 22, 23, 24) , whereas nonphosphorylated CaMKII or basal autophosphorylated CaMKII adopts a closed/inactive conformation. Therefore, it seemed possible that [P]mCaMKII binding to p190 may require the open/active conformation, perhaps because p190 is a substrate for CaMKII. However, several observations argue against this possibility. 1) Competition for [P]mCaMKII binding by nonphosphorylated CaMKII is unaffected by the presence of CaCl(2.5 mM) plus CaM (1 µM), which maintains the competitor in an open/active conformation (not shown). 2) Binding of [P]mCaMKII is essentially unaffected by a variety of reagents that bind to, or affect binding of substrates to, the active site, including ATP (0.5 mM), ADP (0.5 mM), syntide-2 (0.25 mM), magnesium acetate (10 mM), EDTA (1 mM), EGTA (1 mM), CaCl(2.5 mM), and CaCl(2.5 mM) plus CaM (1 µM) (data not shown). These data suggest that the interaction of CaMKII with p190 is not mediated via the catalytic domain. Furthermore, no competition for binding of 100 nM [P]mCaMKII is observed using a 100-fold excess of a CaMKII regulatory domain peptide, CaMK-(281-309), that had been phosphorylated at Thr(not shown). Therefore, the regulatory domain itself is insufficient to compete for the interaction. However, since heat inactivation of Thr-autophosphorylated mCaMKII (15 min at 65 °C) prevents competition for [P]mCaMKII binding (not shown), the native conformation of the kinase appears to be necessary for binding.

Since binding of CaMKII to p190 appears to require autophosphorylation at Thr, another possibility is that p190 is a Thr-specific protein phosphatase. However, binding of 100 nM [P]mCaMKII is unaffected by the inclusion of protein phosphatase inhibitors ( e.g. 1 µM microcystin LR) (not shown). In addition, there are no reported protein phosphatase catalytic subunits in this molecular weight range.

As a final test of the binding specificity of p190, [P]mCaMKII overlay experiments were performed in the presence of other phosphorylated competitors. Essentially no competition was observed using 10-fold molar excesses of glycogen phosphorylase a, phosphocasein, or type II regulatory subunit of protein kinase A, or using a 10,000-fold molar excess of free phosphoserine or phosphothreonine (not shown).


DISCUSSION

It is becoming increasingly clear that subcellular localization/compartmentation of specific proteins plays a significant role in the functioning of signal transduction pathways. The intracellular localization of CaMKII varies in different tissues; in cerebral cortex, about 88% of CaMKII activity is associated with a particulate (0.1% Triton X-100 insoluble) fraction (56) , probably containing cytoskeletal elements. Association of CaMKII with specific subcellular structures is likely to play an important role in determining specific functions of CaMKII. For example, to play a role in long term potentiation (20, 57, 58) , it is probably important for CaMKII to be localized to dendritic spines and postsynaptic densities, where Caconcentrations can be selectively regulated (59, 60) and the appropriate substrates ( e.g. receptors and/or ion channels) may also be localized ( e.g. Ref. 55). However, the mechanism(s) explaining the diverse localization of CaMKII are unclear.

One possible mechanism that may contribute to differential localization is the specific interaction of CaMKII with a diverse array of anchoring proteins. Preliminary reports have suggested that CaMKII can interact with several cellular proteins besides calmodulin. Using an overlay assay with I-labeled rat brain CaMKII as a probe, CaMKII was shown to interact with tubulin and unidentified postsynaptic density proteins of 115 and 155 kDa (61) . Binding of CaMKII to actin has also been reported (62) . Although these interactions may localize CaMKII to cytoskeletal components, they were not characterized in detail. More recently, synaptic vesicle CaMKII was shown to bind to synapsin I (63) , although this may be a mechanism for localizing synapsin I to the vesicles, rather than for localizing CaMKII to vesicles.

The present report describes experiments using recombinant mouse CaMKII isoform (P-autophosphorylated in the presence of Ca/calmodulin) ([P]mCaMKII) as a probe in a gel overlay assay. Multiple [P]mCaMKII-binding proteins were present in whole tissue extracts, and the pattern and amount of [P]mCaMKII binding varied between tissues (Fig. 1, top). Although one could question the physiological relevance of screening peripheral tissues extracts with a neuronal isoform of CaMKII, the fact that similar CaMKII binding patterns were observed with the isoform (Fig. 1, bottom) might suggest that a conserved domain is involved in binding. All four known isoform classes (, , , ) share highly conserved amino-terminal catalytic and regulatory (residues 1-315; 90% identical) domains. The major differences arise from the presence of multiple variable insertions immediately following the regulatory domain (6) . The remainder of the carboxyl-terminal domain is 75% homologous in all isoforms. Little data are available concerning the function of the insertion and carboxyl-terminal domains, although the deletion of both domains results in a monomer, rather than the normal holoenzyme (64) .

The forebrain CaMKII-binding proteins were chosen for further investigation because more is known about the localization of neuronal CaMKII, and the isoform is primarily and highly expressed in forebrain. Analysis of crude subcellular fractions revealed that the [P]mCaMKII-binding proteins were primarily present in the P2 (cytoskeletal) fraction (Fig. 2). In addition, purified PSD preparations exhibited a [P]mCaMKII binding protein pattern similar to the P2 fraction. Weak binding to tubulin was detected, as previously observed (61) , and to several unidentified proteins in the mixed MAP sample. These observations are consistent with the high concentration of CaMKII at PSDs (13, 14, 28, 29, 30) and its association with microtubules and neurofilaments (65, 66) .

The [P]mCaMKII-binding protein that had the strongest signal in the forebrain P2 fraction (p190) had a Kof 609 nM for the CaMKII subunit (61 nM holoenzyme) (Fig. 3), 20-fold lower than the concentration of CaMKII in rat forebrain.() Therefore, the affinity data are consistent with the possibility that CaMKII and p190 can interact in intact neurons. However, one should use care extrapolating affinities from immobilized ligand assays to physiological conditions. Binding to p190 exhibited a Bof 7.0 pmol of [P]mCaMKII per mg of P2 protein, suggesting that p190 may be capable of binding 30-40% of the CaMKII in the P2 fraction, when saturated.()

Competition experiments suggested that prior autophosphorylation of CaMKII at Thrwas required for binding of CaMKII to p190 (Fig. 4; see ``Results''). Therefore, if p190 is responsible for localizing CaMKII to PSDs, one would expect CaMKII in isolated PSDs to be Thr-autophosphorylated and thus partially Ca-independent. Although CaMKII is abundant in isolated PSDs (28, 29, 30) , substantial Ca-independent CaMKII activity has not been detected without Ca/CaM-dependent phosphorylation in vitro (67) . This may be because autophosphorylated CaMKII is less stable than nonphosphorylated kinase (68) , and, therefore, active autophosphorylated CaMKII may not survive the isolation procedure. In agreement with this possibility, the specific CaMKII activity in isolated PSDs is about 10-fold lower than expected based on its abundance (67) . Another possibility is that nonphosphorylated CaMKII may be localized to PSDs by other mechanisms, possibly another class of anchoring proteins. Therefore, activation and autophosphorylation of PSD CaMKII may result in its ``translocation'' to p190. Indeed, previous studies have suggested that the subcellular distribution of CaMKII in invertebrate neurons is regulated by autophosphorylation (69, 70) . Recent studies also suggest that the association of the RII subunit of protein kinase A with AKAPs is regulated by phosphorylation (71) . Alternatively, it has recently been suggested that CaMKII translocates to PSDs from a soluble fraction following euthanasia (72) . Therefore, it is possible that the high concentration of nonphosphorylated CaMKII in isolated PSDs is artifactual, as suggested previously (2) .

The data reported in this manuscript suggest that p190 may be a PSD protein that specifically anchors activated CaMKII. However, its precise identity remains unknown. Association of CaMKII with p190 following activation/autophosphorylation in response to dendritic Camobilization may be an important point of regulation in the Ca/CaM-dependent phosphorylation of PSD proteins, such as glutamate receptors (55) . These phosphorylations are thought to play a critical role in the regulation of synaptic plasticity. In addition, the diversity of CaMKII-binding proteins in other tissues suggests that there may be a variety of anchoring proteins, analogous to AKAPs and RACKs, which specifically localize CaMKII to discrete subcellular compartments. These interactions may be important in determining other physiological processes regulated by Camobilization.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM 47973 and by the Vanderbilt University Diabetes Research and Training Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Funded by the Molecular Endocrinology Training Program through Grant 5T32DK07563-07.

To whom correspondence and reprint requests should be addressed: Dept. of Molecular Physiology and Biophysics, Rm. 762, Medical Research Bldg., Vanderbilt University School of Medicine, Nashville, TN 37232-0615. Tel./Fax: 615-936-1630; E-mail: roger.colbran@mcmail.vanderbilt.edu.

The abbreviations used are: CaM, calmodulin; CaMKII, Ca/calmodulin-dependent protein kinase II; protein kinase A, cyclic AMP-dependent protein kinase; C-subunit, catalytic subunit of protein kinase A; DTT, dithiothreitol; MAPs, microtubule-associated proteins; mCaMKII, recombinant baculovirus-expressed mouse isoform of CaMKII; PAGE, polyacrylamide gel electrophoresis; PSD, postsynaptic densities; PVDF membranes, polyvinylidene difluoride; RII, type II regulatory subunit of protein kinase A; TBS, Tris-buffered saline; xCaMKII, recombinant baculovirus-expressed Xenopus isoform of CaMKII.

From our yield of purified CaMKII from rat forebrain (48) (0.1 mg purified per g of tissue with a yield of about 14% by activity), rat forebrain contains about 0.7 mg of CaMKII per g of tissue and constitutes about 1% of total forebrain protein. This agrees with estimates of the subunit concentration in whole rat brain extracts by radioimmunoassay (0.7% of total protein) (15). Assuming 1 g of tissue has a volume of 1 ml and an average subunit molecular mass of 54 kDa for CaMKII, the average concentration of CaMKII subunit in forebrain is about 13 µM.

Assuming stoichiometric binding of [P]mCaMKII at B, there is about 7.0 pmol of p190 (1.31 µg) per mg of P2. This estimate for the abundance of p190 is consistent with the fact that no 190-kDa protein can be detected in the P2 fraction by Coomassie Blue staining of SDS-PAGE gels, but should be considered a minimal estimate, since it assumes 100% efficiency for both the electrophoretic transfer and renaturation steps of the protocol. By semiquantitative Western blotting, there is about 1 µg (approximately 19 pmol) of CaMKII per mg of P2 protein (R. J. Colbran, unpublished). Thus, p190 may be capable of binding about 37% of the CaMKII in the P2 fraction when saturated.


ACKNOWLEDGEMENTS

We express our appreciation to James Bann and Susanne Kloeker for performing a few of the experiments reported; Drs. Jackie Corbin, Sharron Francis, Lee Limbird, and Brian Wadzinski for critically reading an initial version of the manuscript and for invaluable discussions; and Mary Ann Barban for excellent technical assistance.


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