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calmodulin-dependent protein kinase II in cerebellar granule cells by N-methyl-d -aspartate receptor activation
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Activation of Ca*~+/Calmodulin-Dependent Protein Kinase II in Cerebellar Granule Cells by A/-Methyl-D-aspartate Receptor Activation’
Cultured cerebellar granule cells were studied to determine if the excitatory neurotransmitter glutamate acting through the N-methyl-D-aspartate (NMDA) receptor could stimulate autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to generate its Ca2+-independent form. Glutamate did elevate Ca2’-independent CaM-kinase II through autophosphorylation when granule cells were incubated in Mg2+free buffer, and this response was potentiated by 1 WM glycine. Extracellular Ca”+ was required, and specific antagonists of the NMDA receptor blocked the response. These results support the hypothesis that postsynaptic Ca” influx through the NMDA receptor-gated ion channel, as occurs during induction of long-term potentiation, may convert CaM-kinase II to a constitutively active, Ca’ -independent form. I. 1990 Arademic Press. Inc. ~-___
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INTRODUCTION
The multifunctional Ca”/calmodulin-dependent protein kinase II (CaM-kinase II) has widespread tissue distribution as oligomeric isozyme forms and is particularly abundant in brain where it const,itutes up to l-2% of total protein in hippocampus (reviewed in (1,2)). Purified rat brain CaM-kinase II exhibit,s complete dependence on (‘&J / /CaM for initial phosphorylation of substrate proteins as well as its own intramolecular autophosphory.:itilJn. .~utophnr;pllc)r~lwtior? on Thr”“‘. whic,h occurs ,i ithin seconds, converts CaM-kinase II to a form which is 50.-80% Ca”-independent for activity (3-5). Thr’s” is located in a multifunctional regulatory domain that contains autoinhibitory motifs (residues B-302) and the C’aM-binding region (residues 296-309) (1). These studies, plus site-directed mutation experiments, have led to the formulation of a regulatory model for raM-kinase II (1,6).
Considerable attention has been given to putative involvement of CaM-kinase II in mechanisms of synaptic plasticity (7, 8) such as long-term potentiation (LTP), a popular cellular model for learning and memory. Induction of LTP in the CA1 region of rat hippocampus requires postsynaptic Ca2+-influx through the N-methyl-D-aspartate (NMDA) class of glutamate-receptor ion channels. Although the mechanisms involved in the induction, expression, and maintenance of LTP are not well understood, recent experiments strongly implicate the involvement of CaM-kinase II and/or protein kinase C (9, 10). For example, induction of LTP is blocked by CaM-antagonists (11-13) and a variety of inhibitors of protein kinase C and CaM-kinase II (14, 15). Most notably, postsynaptic injection of peptide inhibitors of these two Ca’+dependent protein kinases block the induction of LTP (13, 16). Other studies have suggested that during the induction of LTP a protein kinase is converted to a Ca’+independent form that is constitutively active (15). These studies make CaM-kinase II a very strong candidate for participation in LTP since (i) this kinase constitutes the major postsynaptic density protein (PSD) (17, 18), especially in hippocampus (19), at excitatory synapses using glutamate, and (ii) autophosphorylation in vitro of PSD CaM-kinase II converts it to a Ca” -independent enzyme (20). The PSD is a good candidate for modulating synaptic strength (21). An attractive hypothesis is that during induction of LTP the postsynaptic Ca” -influx through the YMDA-receptor ion channel promotes CaM-kinase II autophosphorylation to produce the Ca’ ’ -independent form (22). This constitutively active CaM-kinase II might then phosphorylate and potentiate a non-NMDA ion channel that is enhanced during expression of LTP (23, 24). Alteration of ion channel properties (e.g., mean open time) by protein phosphorylation is a common regulatory mechanism (25). This hypothesis would be greatly strengthened by demonstration that NMDA-receptor activation by glutamate can generate the Ca’+-independent form of CaM-kinase II. We chose cultured cerebellar granule cells for this study since they do have NMDA-
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receptors (26), and we previously demonstrated transient generation of the Ca ‘+-independent form of CaM-kinase II in response to K+-depolarization (27). MATERIALS
AND
METHODS
Materials. The following chemicals and reagents were obtained from the indicated sources: fetal calf serum, HyClone Labs; [32P]P0, and [y-32P]ATP, DuPont-New England Nuclear; okadaic acid, Moana Bioproducts, Inc. (Honolulu); glutamate, aspartate, kainate, quisqualate, NMDA, D-2-amino-5phosphonovalerate (APV), GAMS, and nitrindepine, Sigma; and CPP, Research Biochemicals, Inc. (Natick, MA). The CaM-kinase II substrate peptide syntide-2 was synthesized (28), and the peptide inhibitor of CAMP-kinase was from Pennisula Labs. Cell cultures. Cerebella were dissociated from g-dayold Sprague-Dawley rats, and cells were dissociated and plated (2.5-3.0 X lo6 cells/35mm dish) on Falcon dishes coated with calf skin collagen (0.1 mg/35-mm dish) (29). The cells were grown in basal modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, gentamicin (100 pg/ml), and 25 mM KC1 at 37’C in humified 94% air, 6% COZ (29). After 18-20 h, 10 PM cytosine arabinoside was added to the culture medium to prevent the replication of nonneuronal cells. The culture medium was replaced at 2 days of culture, and cells were maintained in culture for 8-9 days before use. The culture dishes were washed twice with room temperature Krebs-Ringer Hepes (KRH) solution which contained 128 mM NaCl, 5 mM KCl, 2.7 mM CaC&, 1 mM NaHP04, 10 mM glucose, and 20 mM Hepes (pH 7.4) with or without 1.2 mM MgS04. After incubation for 1 h in KRH, cells were incubated at 30°C for the specified time with KRH f MgSO, without (controls) or with the specified test agents. After the indicated time of incubation, the medium was quickly aspirated, and the plated cells were frozen on liquid NZ. CaM-kinase II assays. The frozen granule cells were scraped from the dishes and solubilized at 0°C in 0.3 ml of 50 mM Hepes (pH 7.4), 0.1% Triton X-100, 4 mA4 EGTA, 10 mM EDTA, 15 mM Na4P207, 100 mM /3-glycerophosphate, 25 mM NaF, 0.1 mM leupeptin, 75 PM pepstatin A, and 0.1 mg/ml aprotinin (27). After sonication with a Branson Sonifier 250, the insoluble materials were removed by centrifugation at 15,000g for 5 min. More than 95% of the CaM-kinase II activity was in the supernatant. The standard kinase assay contained 50 mM Hepes (pH 7.5), 10 m&f magnesium acetate, 0.1 mM [y32P]ATP (3000-5000 cpm/pmol), 1 mg/ml bovine serum albumin, 40 PM syntide-2, and 0.1 mM peptide inhibitor of CAMP-kinase in a volume of 25 ~1. To measure total CaM-kinase II activity the assay contained 1 mA4 CaClz and 3 PM CaM, whereas 1 mM EGTA was present to determine the Ca’+-independent activity. The reaction was initiated by addition of 1.5 ~1 of the supernatant frac-
SODERLING
tion; after 2 min a 15-~1 aliquot was spotted on P-cellulose paper and processed as described (30). Usually the Ca2+independent activity is expressed as a percentage of total CaM-kinase II activity. Results are means + standard errors. Analysis of 32P-labeling. The 32P-labeling of the cells was done as described previously (27). After homogenization and centrifugation, the 32P-labeled CaM-kinase II was immunoprecipitated and analyzed by SDS/PAGE as detailed elsewhere (27). 32P-incorporation into Thr2s’ was determined as described previously (3, 27). RESULTS
AND
DISCUSSION
Effects of extracellular Mgzi and glycine on responses to In initial experiments a number of glutamate agonists. excitatory amino acid agonists were tested for their abilities to generate the Ca2+ -independent form of CaM-kinase II. A specific assay (28) for CaM-kinase II activity in the absence (Ca*+-independent) and presence (total activity) of Ca2+ was utilized, and the ratio of these two assays was used to calculate the percentage Ca2+-independent activity. We have previously shown that this assay in cerebellar granule cells is specific for CaM-kinase II (27). Furthermore, the percentage Ca*+-independent activity in immunoprecipitates, using antibody to purified brain CaM-kinase II, was the same as in the extract, thereby establishing that the measured Ca2+-independent kinase activity is due to CaM-kinase II (27). Total CaMkinase II activity was not altered by any of the manipulations performed in this paper. In normal KRH buffer only kainic acid elevated the percentage Ca*+-independent activity of CaM-kinase II (Fig. 1A). This observation is consistent with a previous study of cultured cerebellar granule cells where kainic acid consistently evoked elevations in intracellular Ca*+, whereas responses to glutamate were inconsistent and varied from cell to cell (31). We have previously shown in these cells that depolarization-dependent formation of the Ca2+-independent activity of CaM-kinase II requires influx of extracellular Ca*+ (27). Since the NMDA-receptor ion channel is subject to a voltage-dependent Mg” blockage (32, 33), we switched to a Mg*+-free buffer system for the agonist treatments. We noted that in the Mg*+-free buffer the basal Ca*+-independence of CaMkinase II was variable between 4 and 7%, perhaps due to endogenously released glutamate, and we therefore pretreated the cells with 10 PM APV, an antagonist of the NMDA-receptor (34, 35). Under these conditions the basal Ca2+-independence of CaM-kinase II was consistently about 4%, and glutamate, aspartate, NMDA, and quisqualate were now able to promote elevations in Ca*+independent CaM-kinase II activity (Fig. 1B). This result is very similar to a study in cerebellar granule cells which investigated elevations in cGMP in response to excitatory amino acids. In the presence of Mg*+ only kainic acid
ACTIVATION
C
KA
L-GIL
OF
(‘a”/(‘AI,MOD~‘LIN~I)F,PF,NDENT
NMDA L-Asp
PROTEIN
KINASE
135
II
PA
1--_-
10-G
10-5
lo- 4
L-GLUTAMATE
(M)
FIG. 2. Effect of glycine on the response tamate. Granule cells were preincubated as in of the indicated concentrations of I,-Glu in the 10) of 1 gM glycine. The incubation was for calculated from forlr separate experiments.
0.0 C
KA
L-Glu
NMDA L-Asp
PA
FIG. 1. Effects of glutamate receptor agonists on generation of the Ca’+-independent form of CaM-kinase II. Cultured cerehellar granule cells (253.0 X 10” cells/:~5mm dish) were prepared as described under Materials and Methods. (A) The granule cells were washed twice in Krebs Ringer Hepes (KKH) + MgSO, and incubated for 1 h at 30°C. Cells were then incubated for an additional 30 s with the indicated agonist at 100 GM. (H) C;ranule cells were preincubated for 1 h as in A with the addition of 10 pM I)-amino-5-phosphonovalerate (AF’V). The solution was then changed to KRH-MgSO, without APV, and the indlcated agonists were added for the :(0-s incubation. In both A and R the Ca”-independent activity of the cell supernatant (Materials and Methods) is reported as a percentage of total kinase activity. Results ;ire means + standard errors of six separate experiments. Ahhreviations: (‘. vent rol: KA. kainate: I.-(~~II. I.-glutamate: I:Asp. I.-aspartate: NMDA. .~-rnrth~l~r)-asl)artnte: QA. quisqualate
gave large increases in cGMP content, whereas glutamate, aspartate, and NMDA produced equally large increases in cGMP only in the absence of extracellular Mg”’ (36). (ilutamate was chosen for further study since it is a natural excitatory neurotransmitter. A dose -response curve (Fig. 2) established that the maximally effective c IJncentration of glutamate (,:- 100 PM j ele\.ated the (‘a’ ’ independent activity of GM-kinase II to 7.5 f 0.3”; corn pared to the basal value of 4.0 k 0.3% (P < 0.01). When I PM glycine was included, the dose-response curve for glutamate was shifted to t,he left such that 10 pM glutamate was now maximally effective. Enhancement of the glutamate response by glycine is again consistent with the known effect of glycine to potentiate the NMDA-receptor ion channel response to agonists (37). Effects of glutamate antagonists. To ascertain if the glutamate response was mediated by the NMDA-receptor ion channel. antagonist treatment of the granule cells was performed. Incubation of the cells with either 100 pM
10-3
of CaM-kinase II to gluFig. 1B prior to addition absence (0) or presence 30 s, and the results are
APV or 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), both of which are selective competitive inhibitors of the NMDA-receptor (34, 35), completely blocked the stimulatory response to 10 pM glutamate plus 1 pM glycine (Fig. 3). However, y-D-glutamylaminomethylsulfonate (GAMS), a relatively selective inhibitor of non-NMDA-receptors (38), had little effect. Likewise, nitrendipine, an antagonist of the voltage-dependent Ca2+ channel, had little inhibitory effect. These pharmacological characteristics of the glutamate response to elevate the Ca’+-independent form of CaM-kinase II are consistent with mediation by the NMDA-receptor ion channel. Efieectsof extracellular Ca” and okadaic acid. Next we analyzed the time course of the glutamate response, the requirement for extracellular Ca” ’ , and the potentiat,ion
i
FIG.
I
c
+
APV
CPP
GAMS
&It
3. Inhibitor:; effect? of glutamate and calcium channel antagonists. (iranule cells were preincubated as in Fig. 1A for 50 min. and then the indicated glutamate antagonist (100 PM) or nitrendipine (10 PM) was added for the last 10 min of preincubation. The cells were then incubated at 30°C for ~10 P in Mg”+-free KRH without ( ) or with (+) 10 pM I.-C,lu plus 1 pM glq‘rine in the presence of the indicated antagonist (100 UM 1. Results are expressed as in Fig. 1 for four separate experiments.
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by okadaic acid, an inhibitor of protein phosphatases 1 and 2A (39). The cerebellar granule cells were preincubated with 5 PM okadaic acid in the absence of extracellular Ca*+. We have previously shown that upon removal of extracellular Ca”‘, the Ca*+-independence of CaM-kinase II decreases from a basal value of 4-5% to a new equilibrium value of l-2% (27), and this observation was confirmed in the experiment of Fig. 4. This decrease is interesting in that it indicates that basal intracellular Ca2+ in these cells (30-60 nM, Ref. (15)) is sufficient to partially stimulate CaM-kinase II. Exposure of the granule cells to 10 PM glutamate plus 1 PM glycine and 5 PM okadaic acid in the presence of extracellular Ca2+ produced a seven-fold increase in the Ca2+-independence of CaMkinase II to 18 -t 0.6% within 30 s, and this response was largely blocked by APV (Fig. 4). The elevation in Ca’+independence was relatively stable at 5 min, but it had declined to about 10% at 10 min. The increase in Ca’+independence of CaM-kinase II required extracellular Ca*+ since the control incubation without Ca*+ remained at the basal value of 2% independence (Fig. 4). The partial potentiation by okadaic acid is consistent with our previous results which indicate that these granule cells contain both okadaic acid-sensitive and -insensitive protein phosphatases which dephosphorylate CaM-kinase II and reverse the Ca2+-independence (13). In the absence of preincubation with okadaic acid, glutamate plus glycine increased the Ca2+-independence to about 7-870 at 30 s, and this value was also stable for 5-10 min. (data not shown). The relative stability of the increase in Ca2+independent activity in response to glutamate plus glycine is different than the response to K+-depolarization which is more transient (27), presumably because of inactivation of the voltage-gated Ca*+ channel. When the exposure to glutamate plus glycine was for only 30 s, the elevation in Ca’+-independent CaM-kinase II activity observed at 30 s (7-8%) returned to near basal values at 5 min (not shown). Activation of NMDA-receptors in rat striatal slices has been shown recently to promote dephosphorylation of DARPP-32, presumably by activation of calcineurin, a Ca*‘/CaM-dependent protein phosphatase (40). Thus, it is interesting that NMDA-receptor activation stimulates another Ca*+/CaM-dependent enzyme, but there is no evidence that calcineurin, which is insensitive to okadaic acid, can dephosphorylate CaM-kinase II. Autophosphorylation of CaM-kinase II. To document that the increase in Ca*+-independent activity seen in the top line of Fig. 4 was due to autophosphorylation of CaMkinase II, granule cells were prelabeled with 32P04 to label the ATP pool, and aliquots were removed just prior to addition (0 time value) and at 30 s and 5 min after addition of 10 PM glutamate, 1 PM glycine, 2.6 mM Ca*+, and 5 PM okadaic acid. The 32P-labeled CaM-kinase II in the homogenate was immunoprecipitated with antibody to the
SODERLING
A s o.o Ibtld
O TIME (min)
FIG. 4. Temporal relationship between formation of Ca’+-independent activity and autophosphorylation of CaM-kinase II. Dotted lines. Granule cells were washed once with phosphate-free and serum-free basal modified Eagle’s medium and cultured in this medium containing carrier-free 32P0, (0.5 mCi/ml) for 5 h (27). The cells were then washed with KRH without Mg” or Ca’+ plus 0.1 mM EGTA for 45 min at 30°C before addition of 5 PM okadaic acid for an additional 18 min. The incubation medium was changed to Mg”-free KRH containing 10 pM L-Glu plus 1 pM glycine and 5 WM okadaic acid for the indicated times at which the medium was aspirated and the cells were frozen. The cells were sonicated, centrifuged, and subjected to immunoprecipitation using antibody against rat forebrain CaM-kinase II as described (27). The immunoprecipitated %labeled CaM-kinase II was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS/PAGE), and the 58. to 60.kDa subunit was cut from the gel, quantitated by liquid scintillation counting (Cl), and cleaved with CNBr, and the CNBr peptides were separated by urea/SDS/PAGE as described (3, 27). The 32P-labeled CBl fragment containing Three7 was cut from the gel and counted (m). Each value was derived from 7.5 X 10s cells (n = 4). Results are presented as percentage increases over the 0 time values. Solid lines. Granule cells were incubated as above except the 32P04 was deleted from the 5-h preincubation, and the percentage Ca’+independence of CaM-kinase II was determined. At 0 time of Fig. 4, cells were exposed to Mg*+- and Cazt-free KRH containing 10 FM LGlu and 1 FM glycine (A), or Mg ‘+-free KRH (containing 2.7 mM Ca”) plus 10 FM L-Glu and 1 pM glycine without (e) or with 100 pM APV (0). (n = 6)
brain kinase and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (27). The radioactivity in the 58- to 60-kDa subunit of CaMkinase II was extracted from the gel and counted. Glutamate/glycine/okadaic acid/Ca*+ treatment increased the “*P-labeling of CaM-kinase II by only 1.44- and 1.39fold at 30 s and 5 min, respectively, over the 0 time value of 1.00 (Fig. 4). The small increase in phosphorylation of CaM-kinase II was probably due to the relatively high phosphorylation state of the multiple sites in the enzyme under basal conditions (27) and emphasizes that analyses of total ‘“P-incorporation into CaM-kinase II may not be useful. Since it is only the autophosphorylation of Thr287 in the 58- to 60-kDa subunit in the cerebellar CaM-kinase II that is responsible for formation of the Ca*+-independent form (3-5), we examined the 32P-labeling of the CNBr fragment (CBl, Ref. (3)) containing this regulatory phosphorylation site. The immunoprecipitated 32P-labeled CaM-kinase II was digested with CNBr and subjected to
ACTIVATION
OF
Ca’+/CAL,MOL~~~T,IN-DEI’ENI)ENT
electrophoresis to separate the CNBr ‘jZP-labeled peptides (see Fig. 4 of Ref. (27)). CBl, containing Thr”s7, was cut out of the gel and counted for radioactivity. As shown in Fig. 4, the seven-fold increase in ““P-labeling of CBl correlated quite closely with the increase in Ca*+-independent CaM-kinase II activity. This phosphorylation in CB1 is almost certainly on Thr”” since (i) it is associated with form of CaM-kinase generation of the Ca’ r -independent II, and (ii) autophosphorylation of the other Thr residues in CBl (residues 306/307) causes a loss of CaM-kinase II total activity. No loss of total CaM-kinase II activity was observed under any of the treatment conditions. In. situ autophosphorylation of CaM-kinase II has been examined in two other systems. Ohta et al. (41) observed increased phosphorylation of CaM-kinase II in a fibroblast cell line upon treatment with serum and growth factors. Formation of the Ca’ ’ -independent form of CaM-kinase II was not assessed, but it probably did not occur since only “‘P-serine was detected. Using isolated brain synaptosomes, Gorelick et al. (42) demonstrated that the transient phosphorylation of Thr in CaM-kinase II closely paralleled generation of Ca *+-independence under depolarizing conditions, Summary. The results of these experiments demonstrate that the excitatory amino acid glutamate can stimulate formation of the constitut,ively active, Ca”’ -independent form of CaM-kinase II in rat cerebellar granule cells. This effect of glutamate is mediated by the NMDAreceptor-gated ion channel since (i) it is blocked by extracellular Mg”, (ii) it is potentiated by 1 pM glycine, (iii) APV and CPP block the eff’ect, and (iv) it requires extracellular Ca’+-influx. The ohservation that NMDAreceptor activation results in formation of the Ca” -independent form of CaM-kinase II is generally consistent with and adds further evidence supporting the hypothesis that CaM-kinase II may be involved in usage-dependent forms of learning and memory such as LTP (IO, 22). Since brief exposure (30 s) of granule cells to glutamate resulted in a relatively transient (~5 min) increase in Ca”‘-independent CaM-kinase II, due to high protein phosphatase trctivity. one would not expect to observe induction of 1.‘I’P in these cells if st able arltophosphorylation of’ CaMkillabe 11 is part of the induction mechanism. Indeed, there a~ no reports of’ L’I‘P induction in cultured cerebellar granule cells, perhaps for t,his reason. Therefore, we are now investigating the CaM-kinase II autophosphorylation system in hippocampus where the high concentration of (:aM-kinase II and relatively low amount of protein phosphatases (43) in the PSD should favor stable formation of’ the (‘a’ -independent CaM-kinase II. ACKNOWLEDCMENT
PROTEIN
KINASE
137
II
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Activation of Ca*~+/Calmodulin-Dependent Protein Kinase II in Cerebellar Granule Cells by A/-Methyl-D-aspartate Receptor Activation’
Cultured cerebellar granule cells were studied to determine if the excitatory neurotransmitter glutamate acting through the N-methyl-D-aspartate (NMDA) receptor could stimulate autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to generate its Ca2+-independent form. Glutamate did elevate Ca2’-independent CaM-kinase II through autophosphorylation when granule cells were incubated in Mg2+free buffer, and this response was potentiated by 1 WM glycine. Extracellular Ca”+ was required, and specific antagonists of the NMDA receptor blocked the response. These results support the hypothesis that postsynaptic Ca” influx through the NMDA receptor-gated ion channel, as occurs during induction of long-term potentiation, may convert CaM-kinase II to a constitutively active, Ca’ -independent form. I. 1990 Arademic Press. Inc. ~-___
-__.
INTRODUCTION
The multifunctional Ca”/calmodulin-dependent protein kinase II (CaM-kinase II) has widespread tissue distribution as oligomeric isozyme forms and is particularly abundant in brain where it const,itutes up to l-2% of total protein in hippocampus (reviewed in (1,2)). Purified rat brain CaM-kinase II exhibit,s complete dependence on (‘&J / /CaM for initial phosphorylation of substrate proteins as well as its own intramolecular autophosphory.:itilJn. .~utophnr;pllc)r~lwtior? on Thr”“‘. whic,h occurs ,i ithin seconds, converts CaM-kinase II to a form which is 50.-80% Ca”-independent for activity (3-5). Thr’s” is located in a multifunctional regulatory domain that contains autoinhibitory motifs (residues B-302) and the C’aM-binding region (residues 296-309) (1). These studies, plus site-directed mutation experiments, have led to the formulation of a regulatory model for raM-kinase II (1,6).
Considerable attention has been given to putative involvement of CaM-kinase II in mechanisms of synaptic plasticity (7, 8) such as long-term potentiation (LTP), a popular cellular model for learning and memory. Induction of LTP in the CA1 region of rat hippocampus requires postsynaptic Ca2+-influx through the N-methyl-D-aspartate (NMDA) class of glutamate-receptor ion channels. Although the mechanisms involved in the induction, expression, and maintenance of LTP are not well understood, recent experiments strongly implicate the involvement of CaM-kinase II and/or protein kinase C (9, 10). For example, induction of LTP is blocked by CaM-antagonists (11-13) and a variety of inhibitors of protein kinase C and CaM-kinase II (14, 15). Most notably, postsynaptic injection of peptide inhibitors of these two Ca’+dependent protein kinases block the induction of LTP (13, 16). Other studies have suggested that during the induction of LTP a protein kinase is converted to a Ca’+independent form that is constitutively active (15). These studies make CaM-kinase II a very strong candidate for participation in LTP since (i) this kinase constitutes the major postsynaptic density protein (PSD) (17, 18), especially in hippocampus (19), at excitatory synapses using glutamate, and (ii) autophosphorylation in vitro of PSD CaM-kinase II converts it to a Ca” -independent enzyme (20). The PSD is a good candidate for modulating synaptic strength (21). An attractive hypothesis is that during induction of LTP the postsynaptic Ca” -influx through the YMDA-receptor ion channel promotes CaM-kinase II autophosphorylation to produce the Ca’ ’ -independent form (22). This constitutively active CaM-kinase II might then phosphorylate and potentiate a non-NMDA ion channel that is enhanced during expression of LTP (23, 24). Alteration of ion channel properties (e.g., mean open time) by protein phosphorylation is a common regulatory mechanism (25). This hypothesis would be greatly strengthened by demonstration that NMDA-receptor activation by glutamate can generate the Ca’+-independent form of CaM-kinase II. We chose cultured cerebellar granule cells for this study since they do have NMDA-
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AND
receptors (26), and we previously demonstrated transient generation of the Ca ‘+-independent form of CaM-kinase II in response to K+-depolarization (27). MATERIALS
AND
METHODS
Materials. The following chemicals and reagents were obtained from the indicated sources: fetal calf serum, HyClone Labs; [32P]P0, and [y-32P]ATP, DuPont-New England Nuclear; okadaic acid, Moana Bioproducts, Inc. (Honolulu); glutamate, aspartate, kainate, quisqualate, NMDA, D-2-amino-5phosphonovalerate (APV), GAMS, and nitrindepine, Sigma; and CPP, Research Biochemicals, Inc. (Natick, MA). The CaM-kinase II substrate peptide syntide-2 was synthesized (28), and the peptide inhibitor of CAMP-kinase was from Pennisula Labs. Cell cultures. Cerebella were dissociated from g-dayold Sprague-Dawley rats, and cells were dissociated and plated (2.5-3.0 X lo6 cells/35mm dish) on Falcon dishes coated with calf skin collagen (0.1 mg/35-mm dish) (29). The cells were grown in basal modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, gentamicin (100 pg/ml), and 25 mM KC1 at 37’C in humified 94% air, 6% COZ (29). After 18-20 h, 10 PM cytosine arabinoside was added to the culture medium to prevent the replication of nonneuronal cells. The culture medium was replaced at 2 days of culture, and cells were maintained in culture for 8-9 days before use. The culture dishes were washed twice with room temperature Krebs-Ringer Hepes (KRH) solution which contained 128 mM NaCl, 5 mM KCl, 2.7 mM CaC&, 1 mM NaHP04, 10 mM glucose, and 20 mM Hepes (pH 7.4) with or without 1.2 mM MgS04. After incubation for 1 h in KRH, cells were incubated at 30°C for the specified time with KRH f MgSO, without (controls) or with the specified test agents. After the indicated time of incubation, the medium was quickly aspirated, and the plated cells were frozen on liquid NZ. CaM-kinase II assays. The frozen granule cells were scraped from the dishes and solubilized at 0°C in 0.3 ml of 50 mM Hepes (pH 7.4), 0.1% Triton X-100, 4 mA4 EGTA, 10 mM EDTA, 15 mM Na4P207, 100 mM /3-glycerophosphate, 25 mM NaF, 0.1 mM leupeptin, 75 PM pepstatin A, and 0.1 mg/ml aprotinin (27). After sonication with a Branson Sonifier 250, the insoluble materials were removed by centrifugation at 15,000g for 5 min. More than 95% of the CaM-kinase II activity was in the supernatant. The standard kinase assay contained 50 mM Hepes (pH 7.5), 10 m&f magnesium acetate, 0.1 mM [y32P]ATP (3000-5000 cpm/pmol), 1 mg/ml bovine serum albumin, 40 PM syntide-2, and 0.1 mM peptide inhibitor of CAMP-kinase in a volume of 25 ~1. To measure total CaM-kinase II activity the assay contained 1 mA4 CaClz and 3 PM CaM, whereas 1 mM EGTA was present to determine the Ca’+-independent activity. The reaction was initiated by addition of 1.5 ~1 of the supernatant frac-
SODERLING
tion; after 2 min a 15-~1 aliquot was spotted on P-cellulose paper and processed as described (30). Usually the Ca2+independent activity is expressed as a percentage of total CaM-kinase II activity. Results are means + standard errors. Analysis of 32P-labeling. The 32P-labeling of the cells was done as described previously (27). After homogenization and centrifugation, the 32P-labeled CaM-kinase II was immunoprecipitated and analyzed by SDS/PAGE as detailed elsewhere (27). 32P-incorporation into Thr2s’ was determined as described previously (3, 27). RESULTS
AND
DISCUSSION
Effects of extracellular Mgzi and glycine on responses to In initial experiments a number of glutamate agonists. excitatory amino acid agonists were tested for their abilities to generate the Ca2+ -independent form of CaM-kinase II. A specific assay (28) for CaM-kinase II activity in the absence (Ca*+-independent) and presence (total activity) of Ca2+ was utilized, and the ratio of these two assays was used to calculate the percentage Ca2+-independent activity. We have previously shown that this assay in cerebellar granule cells is specific for CaM-kinase II (27). Furthermore, the percentage Ca*+-independent activity in immunoprecipitates, using antibody to purified brain CaM-kinase II, was the same as in the extract, thereby establishing that the measured Ca2+-independent kinase activity is due to CaM-kinase II (27). Total CaMkinase II activity was not altered by any of the manipulations performed in this paper. In normal KRH buffer only kainic acid elevated the percentage Ca*+-independent activity of CaM-kinase II (Fig. 1A). This observation is consistent with a previous study of cultured cerebellar granule cells where kainic acid consistently evoked elevations in intracellular Ca*+, whereas responses to glutamate were inconsistent and varied from cell to cell (31). We have previously shown in these cells that depolarization-dependent formation of the Ca2+-independent activity of CaM-kinase II requires influx of extracellular Ca*+ (27). Since the NMDA-receptor ion channel is subject to a voltage-dependent Mg” blockage (32, 33), we switched to a Mg*+-free buffer system for the agonist treatments. We noted that in the Mg*+-free buffer the basal Ca*+-independence of CaMkinase II was variable between 4 and 7%, perhaps due to endogenously released glutamate, and we therefore pretreated the cells with 10 PM APV, an antagonist of the NMDA-receptor (34, 35). Under these conditions the basal Ca2+-independence of CaM-kinase II was consistently about 4%, and glutamate, aspartate, NMDA, and quisqualate were now able to promote elevations in Ca*+independent CaM-kinase II activity (Fig. 1B). This result is very similar to a study in cerebellar granule cells which investigated elevations in cGMP in response to excitatory amino acids. In the presence of Mg*+ only kainic acid
ACTIVATION
C
KA
L-GIL
OF
(‘a”/(‘AI,MOD~‘LIN~I)F,PF,NDENT
NMDA L-Asp
PROTEIN
KINASE
135
II
PA
1--_-
10-G
10-5
lo- 4
L-GLUTAMATE
(M)
FIG. 2. Effect of glycine on the response tamate. Granule cells were preincubated as in of the indicated concentrations of I,-Glu in the 10) of 1 gM glycine. The incubation was for calculated from forlr separate experiments.
0.0 C
KA
L-Glu
NMDA L-Asp
PA
FIG. 1. Effects of glutamate receptor agonists on generation of the Ca’+-independent form of CaM-kinase II. Cultured cerehellar granule cells (253.0 X 10” cells/:~5mm dish) were prepared as described under Materials and Methods. (A) The granule cells were washed twice in Krebs Ringer Hepes (KKH) + MgSO, and incubated for 1 h at 30°C. Cells were then incubated for an additional 30 s with the indicated agonist at 100 GM. (H) C;ranule cells were preincubated for 1 h as in A with the addition of 10 pM I)-amino-5-phosphonovalerate (AF’V). The solution was then changed to KRH-MgSO, without APV, and the indlcated agonists were added for the :(0-s incubation. In both A and R the Ca”-independent activity of the cell supernatant (Materials and Methods) is reported as a percentage of total kinase activity. Results ;ire means + standard errors of six separate experiments. Ahhreviations: (‘. vent rol: KA. kainate: I.-(~~II. I.-glutamate: I:Asp. I.-aspartate: NMDA. .~-rnrth~l~r)-asl)artnte: QA. quisqualate
gave large increases in cGMP content, whereas glutamate, aspartate, and NMDA produced equally large increases in cGMP only in the absence of extracellular Mg”’ (36). (ilutamate was chosen for further study since it is a natural excitatory neurotransmitter. A dose -response curve (Fig. 2) established that the maximally effective c IJncentration of glutamate (,:- 100 PM j ele\.ated the (‘a’ ’ independent activity of GM-kinase II to 7.5 f 0.3”; corn pared to the basal value of 4.0 k 0.3% (P < 0.01). When I PM glycine was included, the dose-response curve for glutamate was shifted to t,he left such that 10 pM glutamate was now maximally effective. Enhancement of the glutamate response by glycine is again consistent with the known effect of glycine to potentiate the NMDA-receptor ion channel response to agonists (37). Effects of glutamate antagonists. To ascertain if the glutamate response was mediated by the NMDA-receptor ion channel. antagonist treatment of the granule cells was performed. Incubation of the cells with either 100 pM
10-3
of CaM-kinase II to gluFig. 1B prior to addition absence (0) or presence 30 s, and the results are
APV or 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), both of which are selective competitive inhibitors of the NMDA-receptor (34, 35), completely blocked the stimulatory response to 10 pM glutamate plus 1 pM glycine (Fig. 3). However, y-D-glutamylaminomethylsulfonate (GAMS), a relatively selective inhibitor of non-NMDA-receptors (38), had little effect. Likewise, nitrendipine, an antagonist of the voltage-dependent Ca2+ channel, had little inhibitory effect. These pharmacological characteristics of the glutamate response to elevate the Ca’+-independent form of CaM-kinase II are consistent with mediation by the NMDA-receptor ion channel. Efieectsof extracellular Ca” and okadaic acid. Next we analyzed the time course of the glutamate response, the requirement for extracellular Ca” ’ , and the potentiat,ion
i
FIG.
I
c
+
APV
CPP
GAMS
&It
3. Inhibitor:; effect? of glutamate and calcium channel antagonists. (iranule cells were preincubated as in Fig. 1A for 50 min. and then the indicated glutamate antagonist (100 PM) or nitrendipine (10 PM) was added for the last 10 min of preincubation. The cells were then incubated at 30°C for ~10 P in Mg”+-free KRH without ( ) or with (+) 10 pM I.-C,lu plus 1 pM glq‘rine in the presence of the indicated antagonist (100 UM 1. Results are expressed as in Fig. 1 for four separate experiments.
136
FUKUNAGA
AND
by okadaic acid, an inhibitor of protein phosphatases 1 and 2A (39). The cerebellar granule cells were preincubated with 5 PM okadaic acid in the absence of extracellular Ca*+. We have previously shown that upon removal of extracellular Ca”‘, the Ca*+-independence of CaM-kinase II decreases from a basal value of 4-5% to a new equilibrium value of l-2% (27), and this observation was confirmed in the experiment of Fig. 4. This decrease is interesting in that it indicates that basal intracellular Ca2+ in these cells (30-60 nM, Ref. (15)) is sufficient to partially stimulate CaM-kinase II. Exposure of the granule cells to 10 PM glutamate plus 1 PM glycine and 5 PM okadaic acid in the presence of extracellular Ca2+ produced a seven-fold increase in the Ca2+-independence of CaMkinase II to 18 -t 0.6% within 30 s, and this response was largely blocked by APV (Fig. 4). The elevation in Ca’+independence was relatively stable at 5 min, but it had declined to about 10% at 10 min. The increase in Ca’+independence of CaM-kinase II required extracellular Ca*+ since the control incubation without Ca*+ remained at the basal value of 2% independence (Fig. 4). The partial potentiation by okadaic acid is consistent with our previous results which indicate that these granule cells contain both okadaic acid-sensitive and -insensitive protein phosphatases which dephosphorylate CaM-kinase II and reverse the Ca2+-independence (13). In the absence of preincubation with okadaic acid, glutamate plus glycine increased the Ca2+-independence to about 7-870 at 30 s, and this value was also stable for 5-10 min. (data not shown). The relative stability of the increase in Ca2+independent activity in response to glutamate plus glycine is different than the response to K+-depolarization which is more transient (27), presumably because of inactivation of the voltage-gated Ca*+ channel. When the exposure to glutamate plus glycine was for only 30 s, the elevation in Ca’+-independent CaM-kinase II activity observed at 30 s (7-8%) returned to near basal values at 5 min (not shown). Activation of NMDA-receptors in rat striatal slices has been shown recently to promote dephosphorylation of DARPP-32, presumably by activation of calcineurin, a Ca*‘/CaM-dependent protein phosphatase (40). Thus, it is interesting that NMDA-receptor activation stimulates another Ca*+/CaM-dependent enzyme, but there is no evidence that calcineurin, which is insensitive to okadaic acid, can dephosphorylate CaM-kinase II. Autophosphorylation of CaM-kinase II. To document that the increase in Ca*+-independent activity seen in the top line of Fig. 4 was due to autophosphorylation of CaMkinase II, granule cells were prelabeled with 32P04 to label the ATP pool, and aliquots were removed just prior to addition (0 time value) and at 30 s and 5 min after addition of 10 PM glutamate, 1 PM glycine, 2.6 mM Ca*+, and 5 PM okadaic acid. The 32P-labeled CaM-kinase II in the homogenate was immunoprecipitated with antibody to the
SODERLING
A s o.o Ibtld
O TIME (min)
FIG. 4. Temporal relationship between formation of Ca’+-independent activity and autophosphorylation of CaM-kinase II. Dotted lines. Granule cells were washed once with phosphate-free and serum-free basal modified Eagle’s medium and cultured in this medium containing carrier-free 32P0, (0.5 mCi/ml) for 5 h (27). The cells were then washed with KRH without Mg” or Ca’+ plus 0.1 mM EGTA for 45 min at 30°C before addition of 5 PM okadaic acid for an additional 18 min. The incubation medium was changed to Mg”-free KRH containing 10 pM L-Glu plus 1 pM glycine and 5 WM okadaic acid for the indicated times at which the medium was aspirated and the cells were frozen. The cells were sonicated, centrifuged, and subjected to immunoprecipitation using antibody against rat forebrain CaM-kinase II as described (27). The immunoprecipitated %labeled CaM-kinase II was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS/PAGE), and the 58. to 60.kDa subunit was cut from the gel, quantitated by liquid scintillation counting (Cl), and cleaved with CNBr, and the CNBr peptides were separated by urea/SDS/PAGE as described (3, 27). The 32P-labeled CBl fragment containing Three7 was cut from the gel and counted (m). Each value was derived from 7.5 X 10s cells (n = 4). Results are presented as percentage increases over the 0 time values. Solid lines. Granule cells were incubated as above except the 32P04 was deleted from the 5-h preincubation, and the percentage Ca’+independence of CaM-kinase II was determined. At 0 time of Fig. 4, cells were exposed to Mg*+- and Cazt-free KRH containing 10 FM LGlu and 1 FM glycine (A), or Mg ‘+-free KRH (containing 2.7 mM Ca”) plus 10 FM L-Glu and 1 pM glycine without (e) or with 100 pM APV (0). (n = 6)
brain kinase and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (27). The radioactivity in the 58- to 60-kDa subunit of CaMkinase II was extracted from the gel and counted. Glutamate/glycine/okadaic acid/Ca*+ treatment increased the “*P-labeling of CaM-kinase II by only 1.44- and 1.39fold at 30 s and 5 min, respectively, over the 0 time value of 1.00 (Fig. 4). The small increase in phosphorylation of CaM-kinase II was probably due to the relatively high phosphorylation state of the multiple sites in the enzyme under basal conditions (27) and emphasizes that analyses of total ‘“P-incorporation into CaM-kinase II may not be useful. Since it is only the autophosphorylation of Thr287 in the 58- to 60-kDa subunit in the cerebellar CaM-kinase II that is responsible for formation of the Ca*+-independent form (3-5), we examined the 32P-labeling of the CNBr fragment (CBl, Ref. (3)) containing this regulatory phosphorylation site. The immunoprecipitated 32P-labeled CaM-kinase II was digested with CNBr and subjected to
ACTIVATION
OF
Ca’+/CAL,MOL~~~T,IN-DEI’ENI)ENT
electrophoresis to separate the CNBr ‘jZP-labeled peptides (see Fig. 4 of Ref. (27)). CBl, containing Thr”s7, was cut out of the gel and counted for radioactivity. As shown in Fig. 4, the seven-fold increase in ““P-labeling of CBl correlated quite closely with the increase in Ca*+-independent CaM-kinase II activity. This phosphorylation in CB1 is almost certainly on Thr”” since (i) it is associated with form of CaM-kinase generation of the Ca’ r -independent II, and (ii) autophosphorylation of the other Thr residues in CBl (residues 306/307) causes a loss of CaM-kinase II total activity. No loss of total CaM-kinase II activity was observed under any of the treatment conditions. In. situ autophosphorylation of CaM-kinase II has been examined in two other systems. Ohta et al. (41) observed increased phosphorylation of CaM-kinase II in a fibroblast cell line upon treatment with serum and growth factors. Formation of the Ca’ ’ -independent form of CaM-kinase II was not assessed, but it probably did not occur since only “‘P-serine was detected. Using isolated brain synaptosomes, Gorelick et al. (42) demonstrated that the transient phosphorylation of Thr in CaM-kinase II closely paralleled generation of Ca *+-independence under depolarizing conditions, Summary. The results of these experiments demonstrate that the excitatory amino acid glutamate can stimulate formation of the constitut,ively active, Ca”’ -independent form of CaM-kinase II in rat cerebellar granule cells. This effect of glutamate is mediated by the NMDAreceptor-gated ion channel since (i) it is blocked by extracellular Mg”, (ii) it is potentiated by 1 pM glycine, (iii) APV and CPP block the eff’ect, and (iv) it requires extracellular Ca’+-influx. The ohservation that NMDAreceptor activation results in formation of the Ca” -independent form of CaM-kinase II is generally consistent with and adds further evidence supporting the hypothesis that CaM-kinase II may be involved in usage-dependent forms of learning and memory such as LTP (IO, 22). Since brief exposure (30 s) of granule cells to glutamate resulted in a relatively transient (~5 min) increase in Ca”‘-independent CaM-kinase II, due to high protein phosphatase trctivity. one would not expect to observe induction of 1.‘I’P in these cells if st able arltophosphorylation of’ CaMkillabe 11 is part of the induction mechanism. Indeed, there a~ no reports of’ L’I‘P induction in cultured cerebellar granule cells, perhaps for t,his reason. Therefore, we are now investigating the CaM-kinase II autophosphorylation system in hippocampus where the high concentration of (:aM-kinase II and relatively low amount of protein phosphatases (43) in the PSD should favor stable formation of’ the (‘a’ -independent CaM-kinase II. ACKNOWLEDCMENT
PROTEIN
KINASE
137
II
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