Proc. Natl. Acad. Sci. USA Vol. 89, pp. 109-113, January 1992 Neurobiology
Antibody specific for the Thr-286-autophosphorylated a subunit of Ca2+/calmodulin-dependent protein kinase II (autophosphorylation/long-term potentiation/postsynaptic density)
TATSUO SUZUKI*t, KUNIKO OKUMURA-NOJI*, AKIHIKO OGURAt, YOSHIHISA KUDOt, AND Ryo TANAKA* *Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan; and tMitsubishi Kasei Institute of Life Sciences, Minamiooya 11, Machida, Tokyo 194, Japan
Communicated by Philip Siekevitz, September 12, 1991
ABSTRACT We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KU) autophosphorylated only at Thr-286 of the a subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys289 of a subunit of CaM-KIH)] was synthesized and phosphorylated by the CaM-KH endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase a subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KU both in purified and synaptic cytoskeletonassociated form. Appearance of CaM-KU subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca2+-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr286- or Thr-287-autophosphorylated form of CaM-KU in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KU.
(7, 8). Thus, the kinase activity and properties are controlled by its autophosphorylation state. In this report we describe the production of a specific antibody against CaM-KII autophosphorylated only at the Thr-286 site in a subunit (and at corresponding sites in other subunits), and we discuss usefulness of the antibody.
MATERIALS AND METHODS Materials. ATP, dithiothreitol, EGTA, high-molecularweight standards (SDS-6H), and hemocyanin from keyhole limpets (H-2133) were purchased from Sigma; rabbit normal IgG was from Jackson ImmunoResearch; CaM and lowmolecular-weight standards were from Pharmacia; Hepes was from Dojindo Laboratory, Kumamoto, Japan; [y- 32P]_ ATP and anti-microtubule-associated protein 2 monoclonal antibody were from Amersham; poly(vinylidene difluoride) membrane (Immobilon) was from Millipore. Tetramethylrhodamine-conjugated anti-rabbit antibody and fluoresceinconjugated anti-mouse goat IgG were from Chemicon. Peptide Y-66 (MHRQETVDC, 1119 Da) and syntide-2 (PLARTLSVAGLPGKK, 1508 Da) were synthesized by Fujiya BioLaboratory (Hatano, Japan). Other chemicals were all reagent grade. Purification of Proteins. Triton-insoluble cytoskeleton of synaptosome was prepared from rat cerebral cortex, forebrain, hippocampus, or cerebellum, according essentially to the method of Wu et al. (9) by using 0.5 mM Hepes-KOH buffer, pH 7.4 instead of 1 mM sodium bicarbonate and including or not including protease inhibitors. We call this preparation synaptic cytoskeleton in this paper. The protein composition and proteins phosphorylated by the endogenous CaM-KII were essentially the same as that of isolated postsynaptic density material. Preparations were suspended in a solution of 5 mM Hepes-KOH, pH 7.4/20% (vol/vol) glycerol kept at -80°C until use. CaM-KII was purified from rat forebrain by using a phosphocellulose column and as CaM-affinity column, according to the method of Goldenring et al. (10). Phosphorylation of Y-66 Peptide or Proteins of Synaptic Cytoskeleton. Y-66 peptide (180 ,uM) was phosphorylated by the CaM-KII endogenous to the synaptic cytoskeleton (50 ,ug of protein) prepared from cerebral cortex or forebrain of rat in 200 ,l of 50 mM Hepes-KOH buffer, pH 7.4/5 mM
It is important to know the autophosphorylation state of
Ca2+/calmodulin-dependent protein kinase II (CaM-KII) in vivo, especially that of Thr-286 autophosphorylation in the a subunit of the enzyme (the site corresponds to Thr-287 in the 13 subunit) because autophosphorylation of the residue is responsible for generating a Ca2+-independent activity (1); it has been suggested that the Ca2+-independent activity is related to the relatively long-term modulation of synaptic activity, such as long-term potentiation which is a memory model (2, 3). In vitro experiments demonstrated that a conformational change of the kinase caused by binding of Ca2+/calmodulin (CaM) to the kinase made ATP accessible to the kinase and that autophosphorylation first occurred at the Thr-286 site in the a subunit of the kinase (4, 5). This autophosphorylation potentiated the kinase and simultaneously endowed a Ca2+/CaM-independent activity to the kinase (4, 5). Subsequent Ca2+-independent autophosphorylation, either of Thr-305 or of Thr-306, inhibited binding of CaM to the kinase, whereas that of Ser-314 did not inhibit this binding (6). The Ca2+-independent autophosphorylation has been reported to inhibit both Ca2+/CaM-dependent and independent activities, but the independent activity could be kept stably high when the Ca2+/CaM-dependent autophosphorylation prior to the Ca2+-independent one was prolonged
MgCl2/1 mM dithiothreitol/l mM EGTA/1 mM CaCl2/1.5 ,uM CaM/1.5 mM ATP at 25°C. The reaction was stopped after 20 min by adding EDTA (final 1 mM) followed by immediate centrifugation at 10,000 x g for 5 min to remove synaptic cytoskeleton as a pellet. Abbreviations: CaM, calmodulin; CaM-KII, Ca2+/CaM-dependent protein kinase II; NMDA, N-methyl-D-aspartate; PY-66, phosphorylated Y-66 peptide. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Phosphorylation of Y-66 peptide (100 ,uM) was also done in A.l of solution by using 10 Aug of synaptic cytoskeleton protein and 1 mM [y-32P]ATP, analyzed either by HPLC or by counting 32p incorporated into the peptide that was spotted on phosphocellulose filter membrane. Purified CaM-KII (2.5 ,ug per 40 Al) was autophosphorylated or synaptic cytoskeleton (25 Ag ofprotein per 40 ,ul) was phosphorylated either with or without Ca2+/CaM. The reaction was stopped by adding 0.125 M Tris HCI, pH 6.8/3% SDS/5% (vol/vol) 2-mercaptoethanol, and the mixture was solubilized at 100'C for SDS/PAGE. For time course study, autophosphorylation of CaM-KII was done in low MgCI2 (0.1 mM) and low ATP (5 A.M) at 25TC. Autophosphorylation of the enzyme occurred, and Ca2+-independent activity was generated under the conditions used (11). Aliquots were taken, and kinase activities were assayed as incorporation of 32p into 20 ,uM (25 1.d) of syntide 2 for 30 sec at 250C with (total activity) or without (Ca2+-independent activity) Ca2+/CaM in the buffer containing 5 mM MgCl2 and 200 ,M [y-32P]ATP. Aliquots were also used for immunoblotting with antiphosphorylated Y-66 antibody. Preparation of Antiserum and Purification of Immunoglobulin. Phosphorylated Y-66 peptide (PY-66) was separated from unphosphorylated peptide by HPLC with a Vydac C18 column. The peptide was eluted by gradient of 5-15% acetonitrile over 25 min. Peak fractions of PY-66 were collected and pooled. The PY-66 was chemically coupled to hemocyanin by glutaraldehyde (12). Rabbit was primed immunologically with PY-66-hemocyanin complex, and antiserum was obtained after several times of booster either with PY-66hemocyanin complex or with PY-66. Titer of the antiserum was measured by ELISA (13) at each booster. Antihemocyanin antibody was removed by centrifugation after incubation at 37°C for 2 hr with hemocyanin (4.75 mg/ml of serum). This step was repeated by using 1.5 mg of hemocyanin per ml of serum. Antibody crossreacting with unphosphorylated Y-66 peptide was removed by incubation with nitrocellulose membrane that was blotted with Y-66 peptide and blocked with 2% bovine serum albumin. The adsorption was repeated. IgG (67.2 mg of protein per ml) was obtained after incubation of the serum in 33% ammonium sulfate, resuspension of the pellet in 10 mM phosphate-buffered saline (pH 7.5), and dialysis against the saline. For immunoblot analyses the antibody against unphosphorylated Y-66 peptide was adsorbed further from the IgG solution by incubation at 4°C overnight with nitrocellulose membrane that was blotted with purified CaM-KII (4.3 ,ug/10 pl of IgG solution). Immunocytochemical Methods. Hippocampal cells obtained from Wistar rat embryos of gestational day 18 were cultured on glass coverslips for 14 days, as described by Ogura et al. (14). The cells were exposed to 100 ,uM N-methyl-D-aspartate (NMDA) in the presence of 5.0 mM Ca2" for 15 min at 25°C, then fixed with acetone at -20°C, and transferred sequentially to a permeative (0.1% Triton X-100/5% preimmune goat serum), primary antibodies [antiPY66 (1:100 dilution), anti-PY66 preadsorbed with antigen (60 ,.g/ml), or anti-microtubule-associated protein 2 antibody (1:500 dilution)], and secondary antibodies (1:100 dilution). Neurons were identified by staining with the anti-microtubule-associated protein 2 antibody. Additional Techniques. Electrophoresis was done with the discontinuous SDS/buffer system of Laemmli (15). Immunoblotting was done as described (16) with Immobilon. Protein was assayed by the method of Lowry et al. (17) with bovine serum albumin as standard. Proteins of synaptic cytoskeleton were solubilized for protein assay by 1 M NaOH at 1000C for 10 min. The amount of Y-66 peptide was measured by absorption at 226 nm (0.004/pg of Y-66 peptide).
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Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 1. Amino acid sequence of Y-66 peptide. This sequence is boxed in the a subunit; amino acid numbers are those of a subunit. Thr-286 is indicated by an asterisk. Amino acid residues that differ from those in a subunit are indicated by underlining. Gr represents a Ca2W/CaM-dependent protein kinase enriched in cerebellar granule cells (CaM-kinase-Gr) (18). Sequences of y and 8 forms of the kinase and of CaM-kinase-Gr were from refs. 18 and 19.
RESULTS Synthesized Y-66 peptide (see Fig. 1) was phosphorylated by the CaM-KII endogenous to the synaptic cytoskeleton prepared from rat cerebral cortex or from rat forebrain. Time course of peptide (100 AM in 40 Al) phosphorylation is shown in Fig. 2b: =85% of the peptide was phosphorylated in 15 min
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FIG. 2. Time course of phosphorylation of Y-66 peptide by the CaM-K11 endogenous to synaptic cytoskeleton. (a) Changes of peaks 4 (PY-66) and 5 (unphosphorylated Y-66 peptide) in Fig. 3. Peptide (100 ,uM in 40 jul) was incubated at 250C with Ca2+/CaM, synaptic cytoskeleton prepared from rat forebrain, and 1 mM ATP. The phosphorylation reaction was started by ATP addition and stopped by adding 1 mM EDTA. Synaptic cytoskeleton was removed by centrifugation, and the supernatant was analyzed by HPLC. Peak heights of PY-66 (o) and of unphosphorylated peptide (o) were plotted. Peak height was expressed as percentage of the sum of both peak heights. (b) Phosphorylation of Y-66 peptide. Phosphorylation was done by using 1 mM of ['t-32P]ATP (A). After removal of synaptic cytoskeleton by centrifugation, the supernatant was spotted on phosphocellulose papers, and 32p incorporation into the peptides was counted by a scintillation counter.
Neurobiology: Suzuki et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 3. HPLC analysis of PY-66. (a) Y-66 peptide (2 ,ug) dissolved in H20. (b) Supernatant after phosphorylation of Y-66 peptide (36 Ag) by CaM-KIT endogenous to synaptic cytoskeleton. (c) Collected peak 4 material. This material was used for immunization after being coupled to hemocyanin. Gradient condition, which was the same in a, b, and c, is shown only in a.
at 1 mM ATP. The amount of 32p that could be incorporated into the peptide by incubation with 1 mM ATP was estimated at 1 mol/mol of peptide (Fig. 2b). Phosphorylation of the peptides was also analyzed by HPLC. Fig. 3 shows elution profiles of the peptides dissolved in H20 (a) and after phosphorylation at 1.5 mM ATP (b). Peaks 1-3 of Fig. 3b indicated (i) ATP and ADP, (ii) dithiothreitol, and (iii) Hepes, respectively. ADP was produced from ATP by the CaM-KIT. Peak 4 appeared, and peak 5 (unphosphorylated Y-66 peptide) decreased in height after 20 min of phosphorylation reaction (compare a and b of Fig. 3). Heights of peaks 4 and 5 were plotted as percentage of the sum of heights of both
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FIG. 4. Specificity of the antibody assessed by ELISA. ELISA done as described by using diaminobenzidine as substrate for peroxidase coupled to the second antibody. *, Reaction against PY-66; o, reaction against unphosphorylated Y-66 peptide, and v, reaction against hemocyanin. was
FIG. 5. Specificity of antibody assessed by immunoblot analysis. Phosphorylation reaction was done in either the presence (+) or the absence (-) of Ca2+/CaM with either purified CaM-KII (2.5 ,g) or synaptic cytoskeleton (25 /Ag for protein staining and 30 ,ug for immunoblotting) prepared from rat cerebral cortex (CTX), hippocampus (Hipp), or cerebellum (CBL). Proteins were separated by SDS/PAGE (10%o for two gels at left and 9% gel for others), transferred to poly(vinylidene fluoride) membrane, and stained with Coomassie brilliant blue or incubated with anti-PY-66 antibody. Pr, protein staining; WB, immunoblotting. Antibody used for ELISA of Fig. 4 was used for the immunostaining of WB1, and the antibody was further adsorbed with CaM-K11 as described for the staining of WB2. Dilutions of antibody were 1:100 for purified CaM-KII and 1:300 for synaptic cytoskeletons. Arrows indicate positions of either a (lowest arrow), (upper arrow), or (3' subunits (middle arrow) of CaM-KI.
peaks at each incubation time (Fig. 2a). The increment of peak 4 and the decrement of peak 5 paralleled the time course of 32p incorporation into the peptide (Fig. 2b). These results suggested that peak 4 was constituted of Y-66 peptide phosphorylated at threonine. The suggestion was confirmed by amino acid analysis and amino acid sequencing of peak 4 material (data not shown). Preparation of PY-66 was repeated, and peak 4 material was pooled. The collected material showed a single peak as in Fig. 3c. The peptide was lyophilized and resuspended in either H20 or 10 mM phosphate-buffered saline, pH 7.5. Antiserum obtained after each booster injection was examined by ELISA. The serum showed a strong reactivity against hemocyanin, a weak reactivity against unphosphorylated Y-66 peptide, and an intermediate reactivity against PY-66 (data not shown). ELISA analysis of the IgG obtained after adsorption of the antibodies against hemocyanin and unphosphorylated Y-66 peptide showed specific and strong reactivities against PY-66 (Fig. 4). Cross-reactivity against both unphosphorylated Y-66 peptide and hemocyanin was considered almost completely eliminated because the dosedependent curves for both antigens in ELISA analysis were nearly identical with those using normal IgG (starting protein concentration was set at 67.2 mg/ml) instead of the IgG against PY-66 (undiluted protein concentration was 67.2 mg/ml) in the simultaneous assay. Immunoblot analysis of the antibody used for ELISA of Fig. 4 to purified CaM-KII showed reactivity to CaM-K11 autophosphorylated not only in a subunit but also in /3 subunit (Fig. 5, CaM-KII WB1+) and very weak reactivity to the kinase not autophosphorylated in vitro (Fig. 5, CaM-KII WB1-). Reactivity to the unphosphorylated CaM-KII disappeared nearly completely when the antibody was further adsorbed by purified unphosphorylated CaM-KII (Fig. 5, CaM-KII WB2-). Antibody further adsorbed by unphosphorylated Y-66-hemocyanin complex, instead of purified CaM-KII, also was not reactive to the unphosphorylated CaM-KII at all (data not shown).
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Ca2+/CaM (Fig. 5, WB2- of cortex and hippocampus). Weak reactivity was also detected in the a subunit of the kinase endogenous to the synaptic cytoskeleton with which in vitro phosphorylation had not been done (data not shown). This weak reactivity probably reflects the autophosphorylation of the enzyme in vivo. No reactivity was detected to the substrates endogenous to the synaptic cytoskeleton. The relationship between generation of the Ca2+independent kinase activity and development of the band immunoreactive to anti-PY-66 antibody is shown in Fig. 6. For time course study, autophosphorylation was done with low MgCl2 and low ATP (5 ,tM) to slow the reaction (11). The Ca2+-independent activity appeared and reached nearly maximum level at 1 min of autophosphorylation. Level of Ca2+independent activity remained maximal until 10 min of prephosphorylation (autophosphorylation) but decreased at 20 min of prephosphorylation. The a subunit of CaM-KII became visible by anti-PY-66 staining after 30 sec of prephosphorylation (data not shown) and was clearly visible thereafter (Fig. 6). Density of the immunoreactive 8 subunit was weak but appeared to parallel that of a subunit. Thus, generation of the Ca2+-independent activity correlated with appearance of the bands immunoreactive to anti-PY-66 antibody. Decrease of Ca2'-independent activity in spite of maintained level of immunoreactivity at 20 min of prephosphorylation may be due, at least partly, to the instability of the autophosphorylated kinase at 25°C (20). Applicability of anti-PY66 antibody to immunohistochemical studies was tested by using cultured hippocampal cells (Fig. 7). Both neurons and nonneurons were weakly stained without NMDA treatment; this is not a nonspecific staining because preadsorption of the anti-PY-66 antibody with PY-66 eliminated the staining (compare Fig. 7b with a). The weak staining suggests a partial phosphorylation of CaM-KII in the basal state. Treatment with NMDA enhanced the PY-66 immunoactivity in both perikarya and dendrites of some (but not all) neurons (Fig. 7d), which agrees well with the fact that NMDA treatment can generate Ca2+-independent activity of
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FIG. 6. Time course of kinase activities and development of bands immunoreactive to anti-PY-66 antibody. (Upper) Total kinase activity (o) or Ca2l-independent kinase activity (e) was assayed with or without Ca2+/CaM, respectively, after prephosphorylation with Ca2+/CaM/0.1 mM MgCl2/5 ,uM ATP. Values are averages of duplicate assays from which background values (946 cpm) were subtracted (assayed without CaM-KII). Ordinate value is expressed in cpm x 10-3. (Lower) Development of bands immunoreactive to anti-PY-66 antibody after prephosphorylation. Preincubation time is indicated in min. a and ,f, a and , subunits of CaM-KII, respectively.
Reactivity to the autophosphorylated CaM-KII was eliminated by adding PY-66 (0.54 ,ug/,l of anti-PY-66) to the first immunoreaction mixture (data not shown). Antibody further adsorbed by purified CaM-KII also reacted strongly with the proteins corresponding to a and P31,' subunits of CaM-KII endogenous to the synaptic cytoskeletons prepared from cerebral cortex, hippocampus, or cerebellum after in vitro phosphorylation with Ca2+/CaM (Fig. 5, WB2+ of cerebral cortex, hippocampus, and cerebellum). The antibody reacted weakly with the a subunit endogenous to the synaptic cytoskeletons from cerebral cortex and hippocampus with which phosphorylation reaction had been done without
II
FIG. 7. Indirect immunocytochemical observation of hippocampal cells maintained 14 days under culture. (a) Unstimulated cells stained with anti-PY-66 antibody preadsorbed with PY-66 (nonspecific staining). (b) Unstimulated cells stained with anti-PY-66. (c) Cells stimulated with NMDA and stained with preadsorbed anti-PY-66 (nonspecific staining). (d) Cells stimulated with NMDA and stained with anti-PY-66. Phase-contrast views of a-d are essentially the same. (Bar = 100 ,uM.)
Neurobiology: Suzuki et al. CaM-KII in cultured cerebellar granule cells (21). Immunostaining of neurons in Fig. 6b (untreated one) was not enhanced by anti-PY-66 not because of the CaM-KII absence in the cells but probably because of the lack of autophosphorylation without any stimuli of Ca2l influx. Interestingly, the nuclear region was not stained under the conditions used. The limited staining in some neuronal-cell populations may be from the selective expression of NMDA receptors in neurons (22). Similar results were obtained in the cells treated with ionomycin, a Ca2+ ionophore (unpublished work). To clarify the nature of the anti-PY-66 antibody, we conducted immunoblotting and immunocytochemical experiments with different anti-CaM-KII antibodies (monoclonal antibody 2D5 and an antibody raised against a synthetic peptide with the sequence Met-281 to Ala-309 of CaM-KII a subunit). The anti-CaM-KII antibodies stained both unphosphorylated and autophosphorylated CaM-KII in immunoblots and enhanced immunostaining of neurons untreated with NMDA as well as those that had been treated (data not shown). Results are essentially the same as reported by Scholz et al. (23). These results indicate that anti-CaM-KII antibodies other than anti-PY-66 react with CaM-KII, irrespective of the phosphorylation state of the kinase.
DISCUSSION The antibody we obtained (anti-PY-66) reacted specifically with autophosphorylated subunits (at least, a, f3, and f3' subunits, as predicted from the amino acid sequences shown in Fig. 1) of CaM-KII, both in purified and synaptic cytoskeleton-associated form. The antibody can actually discriminate the autophosphorylation state of CaM-KI. Our antibody is applicable both to immunoblotting and to immunohistochemical studies. Reports on the relationship between kinase activity and autophosphorylation suggest that the antibody detects stably active CaM-KII with Ca2+-independent activity. Autophosphorylation of Thr-286 residue (the residue corresponds to Thr-287 in /3 subunit) is the initial step of regulatory events of the kinase activity by autophosphorylation (4, 5). Autophosphorylation of the residue hinders interaction of the autoinhibitory domain with the ATPbinding catalytic domain, regardless of the presence or absence of Ca2+/CaM complex and keeps the catalytic domain accessible to substrates (4, 5, 24). Thus, the kinase autophosphorylated at the site is stably active and has a Ca2+_ independent activity. Therefore, the antibody reacting specifically with CaM-KII, the Thr-286 residue of which has been autophosphorylated, is a useful reagent to detect a Ca2+-independent form of the kinase in vivo. Detection of the active CaM-KII with Ca2+-independent activity in vivo may provide valuable information about physiological functions of the kinase. Occurrence of CaMKII in the postsynaptic density material (25, 26) suggests its synaptic function, especially in modulating neuronal signals at the postsynaptic site. It has been proposed that the independent and persistent activity of CaM-KII plays a crucial role in relatively long-term modulation of synaptic function (3, 27-29), such as seen in long-term potentiation, as indicated first by Miller and Kennedy (2). It has been speculated that CaM-KII activated by either a strong stimulus or high-frequency stimuli can be autophosphorylated to the threshold to become a Ca2+-independent form and that this form can prolong and enhance the effectiveness of intermittent or transient elevation of Ca2' by continuing phosphorylation of substrate(s), such as glutamate-activated channel(s) (3, 4). In fact, activation of postsynaptic CaM and CaM-KII has been shown by Malenka et al. (30) to be a critical requirement for generation of long-term potentiation, although contribution of the Ca2"/CaM-independent form of the kinase to long-term potentiation remained unknown in
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their experiment. In experimentally kindled animals, in which certain neuronal circuits remain potentiated for life once kindling is established, the activity of postsynaptic CaM-KII was kept low as assessed by in vitro phosphorylation of the postsynaptic density proteins (31). This result might be due to extensive phosphorylation of substrates and autophosphorylation in vivo. The antibody specificity reported here may have a wide range of use to ascertain involvement of the kinase in these phenomena. We thank Dr. P. T. Kelly, University of Texas Health Science Center at Houston, for the generous gift of anti-CaM-KII antibodies. We thank Ms. M. Nakazawa for her technical assistance. This research was supported, in part, by The Ishida Foundation and by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture. 1. Thiel, G., Czernik, A. J., Gorelick, F., Nairn, A. & Greengard, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6337-6341. 2. Miller, S. G. & Kennedy, M. B. (1986) Cell 44, 861-870. 3. Lisman, J. (1989) Proc. Natl. Acad. Sci. USA 86, 9574-9578. 4. Colbran, R. J., Schworer, C. M., Hashimoto, Y., Fong, Y.-L., Rich, D. P., Smith, M. K. & Soderling, T. R. (1989) Biochem. J. 258, 313-325. 5. Schulman, H. & Lou, L. L. (1989) Trends Biochem. Sci. 14, 62-66. 6. Colbran, R. J. & Soderling, T. R. (1990) J. Biol. Chem. 265, 11213-11219. 7. Lou, L. L. & Schulman, H. (1989) J. Neurosci. 9, 2020-2032. 8. Hashimoto, Y., Schworer, C. M., Colbran, R. J. & Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055. 9. Wu, K., Carlin, R. & Siekevitz, P. (1986) J. Neurochem. 46, 831-841. 10. Goldenring, J. R., Gonzalez, B., McGuire, J. S., Jr., & DeLorenzo, R. J. (1983) J. Biol. Chem. 258, 12632-12640. 11. Lickteig, R., Shenolikar, S., Denner, L. & Kelly, P. T. (1988) J. Biol. Chem. 263, 19232-19239. 12. Collawn, J. F. & Paterson, Y. (1989) in Current Protocols in Molecular Biology, eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene and Wiley-Interscience), pp. 11.15.1-11.15.4. 13. Takahashi, M. & Hurrell, J. G. R. (1989) in Current Protocols in Molecular Biology, eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene and Wiley-Interscience), pp. 11.4.1-11.4.4. 14. Ogura, A., Miyamoto, M. & Kudo, Y. (1988) Exp. Brain Res. 73, 447-458. 15. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 16. Suzuki, T., Sato, C. & Tanaka, R. (1988) Neurochem. lnt. 13, 53-61. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. Ohmstede, C.-A., Jensen, K. F. & Sahyoun, N. E. (1989) J. Biol. Chem. 264, 5866-5875. 19. Tobimatsu, T. & Fujisawa, H. (1989) J. Biol. Chem. 264, 1790717912. 20. Lai, Y., Nairn, A. C. & Greengard, P. (1986) Proc. Natl. Acad. Sci. USA 83, 4253-4257. 21. Fukunaga, K., Rich, D. P. & Soderling, T. R. (1989) J. Biol. Chem. 264, 21830-21836. 22. Cull-Candy, S. G., Howe, J. R. & Ogden, D. C. (1988) J. Physiol. (London) 400, 189-222. 23. Scholz, W. K., Baitinger, C., Schulman, H. & Kelly, P. T. (1988) J. Neurosci. 8, 1039-1051. 24. Waldmann, R., Hanson, P. I. & Schulmann, H. (1990) Biochemistry 29, 1679-1684. 25. Ouimet, C. C., McGuiness, T. L. & Greengard, P. (1984) Proc. Natl. Acad. Sci. USA 81, 5604-5608. 26. Fukunaga, K., Goto, S. & Miyamoto, E. (1988) J. Neurochem. 51, 1070-1076. 27. Malinow, R., Madison, D. V. & Tsien, R. W. (1988) Nature (London) 335, 820-824. 28. Lovinger, D. M., Wong, K. L., Murakami, K. & Routtenberg, A. (1987) Brain Res. 436, 177-183. 29. Suzuki, T., Fujii, T. & Tanaka, R. (1987) J. Neurochem. 48, 1716-1724. 30. Malenka, R. C., Kauer, J. A., Perkel, D. J., Mauk, M. D., Kelly, P. T., Nicoll, R. A. & Waxham, M. N. (1989) Nature (London) 340, 554-557. 31. Wu, K., Wasterlain, C., Sachs, L. & Siekevitz, P. (1990) Proc. Natl. Acad. Sci. USA 87, 5298-5302.
Antibody specific for the Thr-286-autophosphorylated a subunit of Ca2+/calmodulin-dependent protein kinase II (autophosphorylation/long-term potentiation/postsynaptic density)
TATSUO SUZUKI*t, KUNIKO OKUMURA-NOJI*, AKIHIKO OGURAt, YOSHIHISA KUDOt, AND Ryo TANAKA* *Department of Biochemistry, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan; and tMitsubishi Kasei Institute of Life Sciences, Minamiooya 11, Machida, Tokyo 194, Japan
Communicated by Philip Siekevitz, September 12, 1991
ABSTRACT We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KU) autophosphorylated only at Thr-286 of the a subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys289 of a subunit of CaM-KIH)] was synthesized and phosphorylated by the CaM-KH endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase a subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KU both in purified and synaptic cytoskeletonassociated form. Appearance of CaM-KU subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca2+-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr286- or Thr-287-autophosphorylated form of CaM-KU in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KU.
(7, 8). Thus, the kinase activity and properties are controlled by its autophosphorylation state. In this report we describe the production of a specific antibody against CaM-KII autophosphorylated only at the Thr-286 site in a subunit (and at corresponding sites in other subunits), and we discuss usefulness of the antibody.
MATERIALS AND METHODS Materials. ATP, dithiothreitol, EGTA, high-molecularweight standards (SDS-6H), and hemocyanin from keyhole limpets (H-2133) were purchased from Sigma; rabbit normal IgG was from Jackson ImmunoResearch; CaM and lowmolecular-weight standards were from Pharmacia; Hepes was from Dojindo Laboratory, Kumamoto, Japan; [y- 32P]_ ATP and anti-microtubule-associated protein 2 monoclonal antibody were from Amersham; poly(vinylidene difluoride) membrane (Immobilon) was from Millipore. Tetramethylrhodamine-conjugated anti-rabbit antibody and fluoresceinconjugated anti-mouse goat IgG were from Chemicon. Peptide Y-66 (MHRQETVDC, 1119 Da) and syntide-2 (PLARTLSVAGLPGKK, 1508 Da) were synthesized by Fujiya BioLaboratory (Hatano, Japan). Other chemicals were all reagent grade. Purification of Proteins. Triton-insoluble cytoskeleton of synaptosome was prepared from rat cerebral cortex, forebrain, hippocampus, or cerebellum, according essentially to the method of Wu et al. (9) by using 0.5 mM Hepes-KOH buffer, pH 7.4 instead of 1 mM sodium bicarbonate and including or not including protease inhibitors. We call this preparation synaptic cytoskeleton in this paper. The protein composition and proteins phosphorylated by the endogenous CaM-KII were essentially the same as that of isolated postsynaptic density material. Preparations were suspended in a solution of 5 mM Hepes-KOH, pH 7.4/20% (vol/vol) glycerol kept at -80°C until use. CaM-KII was purified from rat forebrain by using a phosphocellulose column and as CaM-affinity column, according to the method of Goldenring et al. (10). Phosphorylation of Y-66 Peptide or Proteins of Synaptic Cytoskeleton. Y-66 peptide (180 ,uM) was phosphorylated by the CaM-KII endogenous to the synaptic cytoskeleton (50 ,ug of protein) prepared from cerebral cortex or forebrain of rat in 200 ,l of 50 mM Hepes-KOH buffer, pH 7.4/5 mM
It is important to know the autophosphorylation state of
Ca2+/calmodulin-dependent protein kinase II (CaM-KII) in vivo, especially that of Thr-286 autophosphorylation in the a subunit of the enzyme (the site corresponds to Thr-287 in the 13 subunit) because autophosphorylation of the residue is responsible for generating a Ca2+-independent activity (1); it has been suggested that the Ca2+-independent activity is related to the relatively long-term modulation of synaptic activity, such as long-term potentiation which is a memory model (2, 3). In vitro experiments demonstrated that a conformational change of the kinase caused by binding of Ca2+/calmodulin (CaM) to the kinase made ATP accessible to the kinase and that autophosphorylation first occurred at the Thr-286 site in the a subunit of the kinase (4, 5). This autophosphorylation potentiated the kinase and simultaneously endowed a Ca2+/CaM-independent activity to the kinase (4, 5). Subsequent Ca2+-independent autophosphorylation, either of Thr-305 or of Thr-306, inhibited binding of CaM to the kinase, whereas that of Ser-314 did not inhibit this binding (6). The Ca2+-independent autophosphorylation has been reported to inhibit both Ca2+/CaM-dependent and independent activities, but the independent activity could be kept stably high when the Ca2+/CaM-dependent autophosphorylation prior to the Ca2+-independent one was prolonged
MgCl2/1 mM dithiothreitol/l mM EGTA/1 mM CaCl2/1.5 ,uM CaM/1.5 mM ATP at 25°C. The reaction was stopped after 20 min by adding EDTA (final 1 mM) followed by immediate centrifugation at 10,000 x g for 5 min to remove synaptic cytoskeleton as a pellet. Abbreviations: CaM, calmodulin; CaM-KII, Ca2+/CaM-dependent protein kinase II; NMDA, N-methyl-D-aspartate; PY-66, phosphorylated Y-66 peptide. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Phosphorylation of Y-66 peptide (100 ,uM) was also done in A.l of solution by using 10 Aug of synaptic cytoskeleton protein and 1 mM [y-32P]ATP, analyzed either by HPLC or by counting 32p incorporated into the peptide that was spotted on phosphocellulose filter membrane. Purified CaM-KII (2.5 ,ug per 40 Al) was autophosphorylated or synaptic cytoskeleton (25 Ag ofprotein per 40 ,ul) was phosphorylated either with or without Ca2+/CaM. The reaction was stopped by adding 0.125 M Tris HCI, pH 6.8/3% SDS/5% (vol/vol) 2-mercaptoethanol, and the mixture was solubilized at 100'C for SDS/PAGE. For time course study, autophosphorylation of CaM-KII was done in low MgCI2 (0.1 mM) and low ATP (5 A.M) at 25TC. Autophosphorylation of the enzyme occurred, and Ca2+-independent activity was generated under the conditions used (11). Aliquots were taken, and kinase activities were assayed as incorporation of 32p into 20 ,uM (25 1.d) of syntide 2 for 30 sec at 250C with (total activity) or without (Ca2+-independent activity) Ca2+/CaM in the buffer containing 5 mM MgCl2 and 200 ,M [y-32P]ATP. Aliquots were also used for immunoblotting with antiphosphorylated Y-66 antibody. Preparation of Antiserum and Purification of Immunoglobulin. Phosphorylated Y-66 peptide (PY-66) was separated from unphosphorylated peptide by HPLC with a Vydac C18 column. The peptide was eluted by gradient of 5-15% acetonitrile over 25 min. Peak fractions of PY-66 were collected and pooled. The PY-66 was chemically coupled to hemocyanin by glutaraldehyde (12). Rabbit was primed immunologically with PY-66-hemocyanin complex, and antiserum was obtained after several times of booster either with PY-66hemocyanin complex or with PY-66. Titer of the antiserum was measured by ELISA (13) at each booster. Antihemocyanin antibody was removed by centrifugation after incubation at 37°C for 2 hr with hemocyanin (4.75 mg/ml of serum). This step was repeated by using 1.5 mg of hemocyanin per ml of serum. Antibody crossreacting with unphosphorylated Y-66 peptide was removed by incubation with nitrocellulose membrane that was blotted with Y-66 peptide and blocked with 2% bovine serum albumin. The adsorption was repeated. IgG (67.2 mg of protein per ml) was obtained after incubation of the serum in 33% ammonium sulfate, resuspension of the pellet in 10 mM phosphate-buffered saline (pH 7.5), and dialysis against the saline. For immunoblot analyses the antibody against unphosphorylated Y-66 peptide was adsorbed further from the IgG solution by incubation at 4°C overnight with nitrocellulose membrane that was blotted with purified CaM-KII (4.3 ,ug/10 pl of IgG solution). Immunocytochemical Methods. Hippocampal cells obtained from Wistar rat embryos of gestational day 18 were cultured on glass coverslips for 14 days, as described by Ogura et al. (14). The cells were exposed to 100 ,uM N-methyl-D-aspartate (NMDA) in the presence of 5.0 mM Ca2" for 15 min at 25°C, then fixed with acetone at -20°C, and transferred sequentially to a permeative (0.1% Triton X-100/5% preimmune goat serum), primary antibodies [antiPY66 (1:100 dilution), anti-PY66 preadsorbed with antigen (60 ,.g/ml), or anti-microtubule-associated protein 2 antibody (1:500 dilution)], and secondary antibodies (1:100 dilution). Neurons were identified by staining with the anti-microtubule-associated protein 2 antibody. Additional Techniques. Electrophoresis was done with the discontinuous SDS/buffer system of Laemmli (15). Immunoblotting was done as described (16) with Immobilon. Protein was assayed by the method of Lowry et al. (17) with bovine serum albumin as standard. Proteins of synaptic cytoskeleton were solubilized for protein assay by 1 M NaOH at 1000C for 10 min. The amount of Y-66 peptide was measured by absorption at 226 nm (0.004/pg of Y-66 peptide).
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Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 1. Amino acid sequence of Y-66 peptide. This sequence is boxed in the a subunit; amino acid numbers are those of a subunit. Thr-286 is indicated by an asterisk. Amino acid residues that differ from those in a subunit are indicated by underlining. Gr represents a Ca2W/CaM-dependent protein kinase enriched in cerebellar granule cells (CaM-kinase-Gr) (18). Sequences of y and 8 forms of the kinase and of CaM-kinase-Gr were from refs. 18 and 19.
RESULTS Synthesized Y-66 peptide (see Fig. 1) was phosphorylated by the CaM-KII endogenous to the synaptic cytoskeleton prepared from rat cerebral cortex or from rat forebrain. Time course of peptide (100 AM in 40 Al) phosphorylation is shown in Fig. 2b: =85% of the peptide was phosphorylated in 15 min
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FIG. 2. Time course of phosphorylation of Y-66 peptide by the CaM-K11 endogenous to synaptic cytoskeleton. (a) Changes of peaks 4 (PY-66) and 5 (unphosphorylated Y-66 peptide) in Fig. 3. Peptide (100 ,uM in 40 jul) was incubated at 250C with Ca2+/CaM, synaptic cytoskeleton prepared from rat forebrain, and 1 mM ATP. The phosphorylation reaction was started by ATP addition and stopped by adding 1 mM EDTA. Synaptic cytoskeleton was removed by centrifugation, and the supernatant was analyzed by HPLC. Peak heights of PY-66 (o) and of unphosphorylated peptide (o) were plotted. Peak height was expressed as percentage of the sum of both peak heights. (b) Phosphorylation of Y-66 peptide. Phosphorylation was done by using 1 mM of ['t-32P]ATP (A). After removal of synaptic cytoskeleton by centrifugation, the supernatant was spotted on phosphocellulose papers, and 32p incorporation into the peptides was counted by a scintillation counter.
Neurobiology: Suzuki et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 3. HPLC analysis of PY-66. (a) Y-66 peptide (2 ,ug) dissolved in H20. (b) Supernatant after phosphorylation of Y-66 peptide (36 Ag) by CaM-KIT endogenous to synaptic cytoskeleton. (c) Collected peak 4 material. This material was used for immunization after being coupled to hemocyanin. Gradient condition, which was the same in a, b, and c, is shown only in a.
at 1 mM ATP. The amount of 32p that could be incorporated into the peptide by incubation with 1 mM ATP was estimated at 1 mol/mol of peptide (Fig. 2b). Phosphorylation of the peptides was also analyzed by HPLC. Fig. 3 shows elution profiles of the peptides dissolved in H20 (a) and after phosphorylation at 1.5 mM ATP (b). Peaks 1-3 of Fig. 3b indicated (i) ATP and ADP, (ii) dithiothreitol, and (iii) Hepes, respectively. ADP was produced from ATP by the CaM-KIT. Peak 4 appeared, and peak 5 (unphosphorylated Y-66 peptide) decreased in height after 20 min of phosphorylation reaction (compare a and b of Fig. 3). Heights of peaks 4 and 5 were plotted as percentage of the sum of heights of both
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FIG. 4. Specificity of the antibody assessed by ELISA. ELISA done as described by using diaminobenzidine as substrate for peroxidase coupled to the second antibody. *, Reaction against PY-66; o, reaction against unphosphorylated Y-66 peptide, and v, reaction against hemocyanin. was
FIG. 5. Specificity of antibody assessed by immunoblot analysis. Phosphorylation reaction was done in either the presence (+) or the absence (-) of Ca2+/CaM with either purified CaM-KII (2.5 ,g) or synaptic cytoskeleton (25 /Ag for protein staining and 30 ,ug for immunoblotting) prepared from rat cerebral cortex (CTX), hippocampus (Hipp), or cerebellum (CBL). Proteins were separated by SDS/PAGE (10%o for two gels at left and 9% gel for others), transferred to poly(vinylidene fluoride) membrane, and stained with Coomassie brilliant blue or incubated with anti-PY-66 antibody. Pr, protein staining; WB, immunoblotting. Antibody used for ELISA of Fig. 4 was used for the immunostaining of WB1, and the antibody was further adsorbed with CaM-K11 as described for the staining of WB2. Dilutions of antibody were 1:100 for purified CaM-KII and 1:300 for synaptic cytoskeletons. Arrows indicate positions of either a (lowest arrow), (upper arrow), or (3' subunits (middle arrow) of CaM-KI.
peaks at each incubation time (Fig. 2a). The increment of peak 4 and the decrement of peak 5 paralleled the time course of 32p incorporation into the peptide (Fig. 2b). These results suggested that peak 4 was constituted of Y-66 peptide phosphorylated at threonine. The suggestion was confirmed by amino acid analysis and amino acid sequencing of peak 4 material (data not shown). Preparation of PY-66 was repeated, and peak 4 material was pooled. The collected material showed a single peak as in Fig. 3c. The peptide was lyophilized and resuspended in either H20 or 10 mM phosphate-buffered saline, pH 7.5. Antiserum obtained after each booster injection was examined by ELISA. The serum showed a strong reactivity against hemocyanin, a weak reactivity against unphosphorylated Y-66 peptide, and an intermediate reactivity against PY-66 (data not shown). ELISA analysis of the IgG obtained after adsorption of the antibodies against hemocyanin and unphosphorylated Y-66 peptide showed specific and strong reactivities against PY-66 (Fig. 4). Cross-reactivity against both unphosphorylated Y-66 peptide and hemocyanin was considered almost completely eliminated because the dosedependent curves for both antigens in ELISA analysis were nearly identical with those using normal IgG (starting protein concentration was set at 67.2 mg/ml) instead of the IgG against PY-66 (undiluted protein concentration was 67.2 mg/ml) in the simultaneous assay. Immunoblot analysis of the antibody used for ELISA of Fig. 4 to purified CaM-KII showed reactivity to CaM-K11 autophosphorylated not only in a subunit but also in /3 subunit (Fig. 5, CaM-KII WB1+) and very weak reactivity to the kinase not autophosphorylated in vitro (Fig. 5, CaM-KII WB1-). Reactivity to the unphosphorylated CaM-KII disappeared nearly completely when the antibody was further adsorbed by purified unphosphorylated CaM-KII (Fig. 5, CaM-KII WB2-). Antibody further adsorbed by unphosphorylated Y-66-hemocyanin complex, instead of purified CaM-KII, also was not reactive to the unphosphorylated CaM-KII at all (data not shown).
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Ca2+/CaM (Fig. 5, WB2- of cortex and hippocampus). Weak reactivity was also detected in the a subunit of the kinase endogenous to the synaptic cytoskeleton with which in vitro phosphorylation had not been done (data not shown). This weak reactivity probably reflects the autophosphorylation of the enzyme in vivo. No reactivity was detected to the substrates endogenous to the synaptic cytoskeleton. The relationship between generation of the Ca2+independent kinase activity and development of the band immunoreactive to anti-PY-66 antibody is shown in Fig. 6. For time course study, autophosphorylation was done with low MgCl2 and low ATP (5 ,tM) to slow the reaction (11). The Ca2+-independent activity appeared and reached nearly maximum level at 1 min of autophosphorylation. Level of Ca2+independent activity remained maximal until 10 min of prephosphorylation (autophosphorylation) but decreased at 20 min of prephosphorylation. The a subunit of CaM-KII became visible by anti-PY-66 staining after 30 sec of prephosphorylation (data not shown) and was clearly visible thereafter (Fig. 6). Density of the immunoreactive 8 subunit was weak but appeared to parallel that of a subunit. Thus, generation of the Ca2+-independent activity correlated with appearance of the bands immunoreactive to anti-PY-66 antibody. Decrease of Ca2'-independent activity in spite of maintained level of immunoreactivity at 20 min of prephosphorylation may be due, at least partly, to the instability of the autophosphorylated kinase at 25°C (20). Applicability of anti-PY66 antibody to immunohistochemical studies was tested by using cultured hippocampal cells (Fig. 7). Both neurons and nonneurons were weakly stained without NMDA treatment; this is not a nonspecific staining because preadsorption of the anti-PY-66 antibody with PY-66 eliminated the staining (compare Fig. 7b with a). The weak staining suggests a partial phosphorylation of CaM-KII in the basal state. Treatment with NMDA enhanced the PY-66 immunoactivity in both perikarya and dendrites of some (but not all) neurons (Fig. 7d), which agrees well with the fact that NMDA treatment can generate Ca2+-independent activity of
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FIG. 6. Time course of kinase activities and development of bands immunoreactive to anti-PY-66 antibody. (Upper) Total kinase activity (o) or Ca2l-independent kinase activity (e) was assayed with or without Ca2+/CaM, respectively, after prephosphorylation with Ca2+/CaM/0.1 mM MgCl2/5 ,uM ATP. Values are averages of duplicate assays from which background values (946 cpm) were subtracted (assayed without CaM-KII). Ordinate value is expressed in cpm x 10-3. (Lower) Development of bands immunoreactive to anti-PY-66 antibody after prephosphorylation. Preincubation time is indicated in min. a and ,f, a and , subunits of CaM-KII, respectively.
Reactivity to the autophosphorylated CaM-KII was eliminated by adding PY-66 (0.54 ,ug/,l of anti-PY-66) to the first immunoreaction mixture (data not shown). Antibody further adsorbed by purified CaM-KII also reacted strongly with the proteins corresponding to a and P31,' subunits of CaM-KII endogenous to the synaptic cytoskeletons prepared from cerebral cortex, hippocampus, or cerebellum after in vitro phosphorylation with Ca2+/CaM (Fig. 5, WB2+ of cerebral cortex, hippocampus, and cerebellum). The antibody reacted weakly with the a subunit endogenous to the synaptic cytoskeletons from cerebral cortex and hippocampus with which phosphorylation reaction had been done without
II
FIG. 7. Indirect immunocytochemical observation of hippocampal cells maintained 14 days under culture. (a) Unstimulated cells stained with anti-PY-66 antibody preadsorbed with PY-66 (nonspecific staining). (b) Unstimulated cells stained with anti-PY-66. (c) Cells stimulated with NMDA and stained with preadsorbed anti-PY-66 (nonspecific staining). (d) Cells stimulated with NMDA and stained with anti-PY-66. Phase-contrast views of a-d are essentially the same. (Bar = 100 ,uM.)
Neurobiology: Suzuki et al. CaM-KII in cultured cerebellar granule cells (21). Immunostaining of neurons in Fig. 6b (untreated one) was not enhanced by anti-PY-66 not because of the CaM-KII absence in the cells but probably because of the lack of autophosphorylation without any stimuli of Ca2l influx. Interestingly, the nuclear region was not stained under the conditions used. The limited staining in some neuronal-cell populations may be from the selective expression of NMDA receptors in neurons (22). Similar results were obtained in the cells treated with ionomycin, a Ca2+ ionophore (unpublished work). To clarify the nature of the anti-PY-66 antibody, we conducted immunoblotting and immunocytochemical experiments with different anti-CaM-KII antibodies (monoclonal antibody 2D5 and an antibody raised against a synthetic peptide with the sequence Met-281 to Ala-309 of CaM-KII a subunit). The anti-CaM-KII antibodies stained both unphosphorylated and autophosphorylated CaM-KII in immunoblots and enhanced immunostaining of neurons untreated with NMDA as well as those that had been treated (data not shown). Results are essentially the same as reported by Scholz et al. (23). These results indicate that anti-CaM-KII antibodies other than anti-PY-66 react with CaM-KII, irrespective of the phosphorylation state of the kinase.
DISCUSSION The antibody we obtained (anti-PY-66) reacted specifically with autophosphorylated subunits (at least, a, f3, and f3' subunits, as predicted from the amino acid sequences shown in Fig. 1) of CaM-KII, both in purified and synaptic cytoskeleton-associated form. The antibody can actually discriminate the autophosphorylation state of CaM-KI. Our antibody is applicable both to immunoblotting and to immunohistochemical studies. Reports on the relationship between kinase activity and autophosphorylation suggest that the antibody detects stably active CaM-KII with Ca2+-independent activity. Autophosphorylation of Thr-286 residue (the residue corresponds to Thr-287 in /3 subunit) is the initial step of regulatory events of the kinase activity by autophosphorylation (4, 5). Autophosphorylation of the residue hinders interaction of the autoinhibitory domain with the ATPbinding catalytic domain, regardless of the presence or absence of Ca2+/CaM complex and keeps the catalytic domain accessible to substrates (4, 5, 24). Thus, the kinase autophosphorylated at the site is stably active and has a Ca2+_ independent activity. Therefore, the antibody reacting specifically with CaM-KII, the Thr-286 residue of which has been autophosphorylated, is a useful reagent to detect a Ca2+-independent form of the kinase in vivo. Detection of the active CaM-KII with Ca2+-independent activity in vivo may provide valuable information about physiological functions of the kinase. Occurrence of CaMKII in the postsynaptic density material (25, 26) suggests its synaptic function, especially in modulating neuronal signals at the postsynaptic site. It has been proposed that the independent and persistent activity of CaM-KII plays a crucial role in relatively long-term modulation of synaptic function (3, 27-29), such as seen in long-term potentiation, as indicated first by Miller and Kennedy (2). It has been speculated that CaM-KII activated by either a strong stimulus or high-frequency stimuli can be autophosphorylated to the threshold to become a Ca2+-independent form and that this form can prolong and enhance the effectiveness of intermittent or transient elevation of Ca2' by continuing phosphorylation of substrate(s), such as glutamate-activated channel(s) (3, 4). In fact, activation of postsynaptic CaM and CaM-KII has been shown by Malenka et al. (30) to be a critical requirement for generation of long-term potentiation, although contribution of the Ca2"/CaM-independent form of the kinase to long-term potentiation remained unknown in
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their experiment. In experimentally kindled animals, in which certain neuronal circuits remain potentiated for life once kindling is established, the activity of postsynaptic CaM-KII was kept low as assessed by in vitro phosphorylation of the postsynaptic density proteins (31). This result might be due to extensive phosphorylation of substrates and autophosphorylation in vivo. The antibody specificity reported here may have a wide range of use to ascertain involvement of the kinase in these phenomena. We thank Dr. P. T. Kelly, University of Texas Health Science Center at Houston, for the generous gift of anti-CaM-KII antibodies. We thank Ms. M. Nakazawa for her technical assistance. This research was supported, in part, by The Ishida Foundation and by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture. 1. Thiel, G., Czernik, A. J., Gorelick, F., Nairn, A. & Greengard, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6337-6341. 2. Miller, S. G. & Kennedy, M. B. (1986) Cell 44, 861-870. 3. Lisman, J. (1989) Proc. Natl. Acad. Sci. USA 86, 9574-9578. 4. Colbran, R. J., Schworer, C. M., Hashimoto, Y., Fong, Y.-L., Rich, D. P., Smith, M. K. & Soderling, T. R. (1989) Biochem. J. 258, 313-325. 5. Schulman, H. & Lou, L. L. (1989) Trends Biochem. Sci. 14, 62-66. 6. Colbran, R. J. & Soderling, T. R. (1990) J. Biol. Chem. 265, 11213-11219. 7. Lou, L. L. & Schulman, H. (1989) J. Neurosci. 9, 2020-2032. 8. Hashimoto, Y., Schworer, C. M., Colbran, R. J. & Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055. 9. Wu, K., Carlin, R. & Siekevitz, P. (1986) J. Neurochem. 46, 831-841. 10. Goldenring, J. R., Gonzalez, B., McGuire, J. S., Jr., & DeLorenzo, R. J. (1983) J. Biol. Chem. 258, 12632-12640. 11. Lickteig, R., Shenolikar, S., Denner, L. & Kelly, P. T. (1988) J. Biol. Chem. 263, 19232-19239. 12. Collawn, J. F. & Paterson, Y. (1989) in Current Protocols in Molecular Biology, eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene and Wiley-Interscience), pp. 11.15.1-11.15.4. 13. Takahashi, M. & Hurrell, J. G. R. (1989) in Current Protocols in Molecular Biology, eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene and Wiley-Interscience), pp. 11.4.1-11.4.4. 14. Ogura, A., Miyamoto, M. & Kudo, Y. (1988) Exp. Brain Res. 73, 447-458. 15. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 16. Suzuki, T., Sato, C. & Tanaka, R. (1988) Neurochem. lnt. 13, 53-61. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. Ohmstede, C.-A., Jensen, K. F. & Sahyoun, N. E. (1989) J. Biol. Chem. 264, 5866-5875. 19. Tobimatsu, T. & Fujisawa, H. (1989) J. Biol. Chem. 264, 1790717912. 20. Lai, Y., Nairn, A. C. & Greengard, P. (1986) Proc. Natl. Acad. Sci. USA 83, 4253-4257. 21. Fukunaga, K., Rich, D. P. & Soderling, T. R. (1989) J. Biol. Chem. 264, 21830-21836. 22. Cull-Candy, S. G., Howe, J. R. & Ogden, D. C. (1988) J. Physiol. (London) 400, 189-222. 23. Scholz, W. K., Baitinger, C., Schulman, H. & Kelly, P. T. (1988) J. Neurosci. 8, 1039-1051. 24. Waldmann, R., Hanson, P. I. & Schulmann, H. (1990) Biochemistry 29, 1679-1684. 25. Ouimet, C. C., McGuiness, T. L. & Greengard, P. (1984) Proc. Natl. Acad. Sci. USA 81, 5604-5608. 26. Fukunaga, K., Goto, S. & Miyamoto, E. (1988) J. Neurochem. 51, 1070-1076. 27. Malinow, R., Madison, D. V. & Tsien, R. W. (1988) Nature (London) 335, 820-824. 28. Lovinger, D. M., Wong, K. L., Murakami, K. & Routtenberg, A. (1987) Brain Res. 436, 177-183. 29. Suzuki, T., Fujii, T. & Tanaka, R. (1987) J. Neurochem. 48, 1716-1724. 30. Malenka, R. C., Kauer, J. A., Perkel, D. J., Mauk, M. D., Kelly, P. T., Nicoll, R. A. & Waxham, M. N. (1989) Nature (London) 340, 554-557. 31. Wu, K., Wasterlain, C., Sachs, L. & Siekevitz, P. (1990) Proc. Natl. Acad. Sci. USA 87, 5298-5302.
