From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan
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ABSTRACT |
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A synthetic peptide corresponding to the
autophosphorylation site of
Ca2+/calmodulin-dependent protein kinase
II (CaMKII) (residues 281-289) was conjugated to paramagnetic
particles, and phosphorylated by a constitutively active CaMKII
fragment. Using this phosphopeptide conjugate as a substrate, a
calyculin A-insensitive, Mn2+-dependent, and
poly-L-lysine-stimulated protein phosphatase activity was
detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent
molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel
electrophoresis and gel-filtration analysis, respectively. It was not
inhibited by 100 nM calyculin A or 10 µM
okadaic acid. Mn2+, but not Mg2+, was
absolutely required for activity. CaMKIIPase was potently activated by
polycations. Autophosphorylated CaMKII was dephosphorylated by
CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and -casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase
a (phosphorylated by phosphorylase kinase) were not
significantly dephosphorylated. No other proteins than CaMKII in rat
brain extract which had been phosphorylated by CaMKII were
dephosphorylated. The stimulated Ca2+-independent activity
of autophosphorylated CaMKII was reversed by the action of CaMKIIPase.
Thus, CaMKIIPase appears to be a specialized protein phosphatase for
the regulation of CaMKII.
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INTRODUCTION |
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Calmodulin-dependent protein kinase II (CaMKII)1 occurs abundantly in the brain, and has a broad substrate specificity (1). It plays a number of roles in the functioning of the central nervous system in response to intracellular Ca2+ (reviewed in Refs. 2-4). The possible involvement of CaMKII in the regulation of neuronal functions such as neurotransmitter synthesis (5, 6), neurotransmitter release (7, 8), long term potentiation (9-11), and the formation of spatial learning (12) has so far been suggested. CaMKII is known to be regulated by autophosphorylation at multiple sites. Among them, autophosphorylation at Thr286 results in generation of Ca2+/calmodulin-independent activity (13-17), full activation of the total activity (18-20), and trapping of Ca2+/calmodulin (21, 22). Therefore, the protein phosphatases that dephosphorylate the residue may be important for regulation of CaMKII activity. It has so far been reported that protein phosphatase 1 (PP1) (23), protein phosphatase 2A (PP2A) (14, 16, 24, 25), and protein phosphatase 2C (PP2C) (26) dephosphorylate Thr286 in vitro and that the dephosphorylation is catalyzed by distinct phosphatases in distinct subcellular compartments (27). Recently, we developed a novel in-gel protein phosphatase assay, using polyacrylamide gels containing phosphorylated peptide conjugates, and found that at least three distinct phosphatases existed in the rat brain extract which catalyzed dephosphorylation of the residue corresponding to Thr286 of CaMKII in the gels (28).
In the present study, we achieved purification and characterization of the protein phosphatase with an apparent molecular weight of 54,000, which had been previously detected by the in-gel assay (28). The purified phosphatase, which was designated as CaMKII phosphatase (CaMKIIPase), was highly specific for autophosphorylated CaMKII, and actually reversed the activated Ca2+/calmodulin-independent activity of autophosphorylated CaMKII.
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EXPERIMENTAL PROCEDURES |
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Materials
ATP, poly-L-lysine (poly(Lys), average molecular
weight 87,000 and 9,600), poly-L-arginine (poly(Arg),
average molecular weight 40,000), poly-L-(glutamic
acid,lysine,tyrosine) 6:3:1 (poly(Glu,Lys,Tyr) 6:3:1, average molecular
weight 23,000), and heparin were purchased from Sigma. W-7 was from
Toronto Research Chemicals. Okadaic acid and calyculin A were from Wako
Pure Chemical Industries. -Glycerophosphate (disodium salt) was from
nacalai tesque. A hetero-bifunctional reagent,
N-(6-maleimidocaproyloxy)succinimide (EMCS), was obtained from Dojindo Laboratories. Amino paramagnetic particles (1-2 µm) was
from Polysciences, Inc. [
-32P]ATP (5,000 Ci/mmol) was from Amersham International. CaMKII(281-289) (MHRQETVDC) (29) was synthesized by a Shimadzu PSSM8 automated peptide
synthesizer and purified by reversed-phase HPLC.
Protein Preparations
CaMKII and its constitutively active 30-kDa fragment were
prepared as described previously (30, 31). Catalytic subunit of
cAMP-dependent protein kinase (PKA) was purified as
described (32). Protein phosphatase inhibitor 2 was obtained from
BIOMOL Research Labs., Inc. Protamine sulfate was from nacalai tesque. Calmodulin was prepared as described previously (33). Mixed histones
(type II-A), myelin basic protein (MBP), phosphorylase kinase,
-casein, myoglobin, transferrin,
-globulin, bovine serum albumin
(BSA), ovalbumin, and catalase were purchased from Sigma. Phosphorylase
b, cytochrome c, and ferritin were from
Boehringer Mannheim.
Purification of CaMKIIPase from Rat Brain Stem
All procedures were carried out at 4 °C. Approximately
20 g of rat brain stems, which had been frozen in liquid nitrogen
immediately after excision and stored at 80 °C, were homogenized
in 70 ml of 50 mM Tris-HCl (pH 7.5) containing 10 mM EDTA, 10 mM EGTA, 1 mM
dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), and 5 µg/ml each of pepstatin, leupeptin, antipain, and chymostatin with a Teflon/glass homogenizer. The homogenate was centrifuged at 100,000 × g for 60 min, and the
supernatant was applied to a phosphocellulose column (Whatman P11,
5.2 × 3.0 cm), which had been equilibrated with the
homogenization buffer. The flow through fraction was pooled and applied
to a DEAE-cellulose column (Whatman DE52, 5.2 × 3.0 cm), which
had been equilibrated with the homogenization buffer without PMSF,
pepstatin, leupeptin, antipain, and chymostatin. The column was washed
with buffer A (comprising 25 mM Tris-HCl (pH 7.5), 1 mM EGTA, 0.5 mM DTT, 0.05% Tween 40)
containing 1 mM PMSF and 0.1 M NaCl, and then
eluted with buffer A containing 0.25 M NaCl. To the active
fraction eluted, solid ammonium sulfate was added to give 60%
saturation, and it was allowed to stand for overnight while being
stirred. The precipitate, collected by centrifugation at 100,000 × g for 30 min, was dissolved in 25 mM Tris-HCl
(pH 7.5) containing 1 mM EGTA and 0.5 mM DTT (buffer B). To this solution, an equal volume of buffer B containing 2 M ammonium sulfate was added. The supernatant, obtained by
centrifugation at 100,000 × g for 30 min, was
filtrated by a cellulose acetate membrane filter (0.45 µm, Advantec)
and applied to a TSKgel phenyl-5PW column (0.5 × 5.0 cm, Tosoh)
attached to a non-metal HPLC system (Tosoh), previously equilibrated
with buffer B containing 1 M ammonium sulfate. The column
was eluted with a linear gradient from 1 to 0 M ammonium
sulfate. Fractions eluted as a major peak of CaMKIIPase activity were
pooled and made 60% saturation in ammonium sulfate. After overnight
stirring, the precipitate was collected by centrifugation at
100,000 × g for 40 min, and it was dissolved in buffer
B containing 0.05% Tween 40 and 0.1 M NaCl, followed by
centrifugation at 30,000 × g for 20 min. Subsequently, the supernatant was applied to a Hiload 26/60 Superdex 200 prep grade
column (Pharmacia) attached to a non-metal HPLC system (Tosoh), which
had been equilibrated with the same buffer, and active fractions eluted
with the same buffer were pooled. Ethylene glycol was added to the
pooled active solution to give 5% (v/v), and it was centrifuged at
30,000 × g for 10 min. The supernatant was applied to
a DEAE-NPR column (0.46 × 10 cm, Tosoh) attached to a HPLC system
(Tosoh), previously equilibrated with buffer C (comprising 40 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Tween 40, 5% ethylene glycol, and 0.5 mM DTT) containing 50 mM NaCl. The column was eluted, at a flow rate of 0.3 ml/min, with linear gradients of NaCl in
buffer C as follows: 0-15 min, 50 mM NaCl; 15-20 min, 50-120 mM NaCl; 20-85 min, 120-175 mM NaCl;
85-105 min, 175-300 mM NaCl. Fractions of 0.3 ml were
then collected. The purities of the active fractions were checked by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and essentially pure
fractions were combined. The purified enzyme was frozen in aliquots at
80 °C. The enzyme can be stored for at least several months
without detectable loss of activity under the storage conditions.
Preparations of Protein Phosphatase Substrates
Detailed procedures for preparation of [32P]PMC-KII(281-289) (peptide-magnetic particle conjugate of CaMKII(281-289)), a phosphopeptide corresponding to amino acid residues 281-289 of autophosphorylated CaMKII conjugated to paramagnetic particles, are described elsewhere (34). Briefly, poly(Lys) (Mr 87,000) was conjugated to amino paramagnetic particles by glutaraldehyde according to the manufacturer's instruction. To the particles, CaMKII(281-289) was conjugated using EMCS, a hetero-bifunctional reagent. The resulting peptide conjugate PMC-KII(281-289) was phosphorylated by a constitutively active 30-kDa proteolytic fragment of CaMKII to yield [32P]PMC-KII(281-289). After phosphorylation, the conjugate was exhaustively washed with 10 mM sodium phosphate buffer (pH 7.2) containing 0.85% NaCl (phosphate-buffered saline), 5 mM ATP, and 0.05% Tween 20, to remove unreacted radioactive ATP. The phosphopeptide conjugate was stored in H2O at 4 °C until used.
CaMKII (112.4 µg/ml) was autophosphorylated at 5 °C for 1 min in
the reaction mixture containing 40 mM Hepes-NaOH (pH 8.0), 5 mM Mg(CH3CO2)2, 0.1 mM EGTA, 5 µM calmodulin, 0.4 mM
CaCl2, 0.02% Tween 20, and 50 µM
[-32P]ATP (28,000 cpm/pmol). Excess EDTA (12.3 mM) and BSA (1 mg/ml) were added to stop the reaction, and
the reaction mixture was applied to a small column of Sephadex G-50
(Pharmacia Biotech Inc.) equilibrated with 50 mM Tris-HCl
(pH 7.5) containing 0.2 M NaCl, 0.05% Tween 40, and 1 mM DTT. The column was eluted with the same buffer by the
spin column method as described (35). Fractions of 50-100 µl were
collected, and the radioactive fractions eluted near the void volume of
the column were pooled and stored in aliquots at
80 °C.
Phosphorylase b (5.3 mg/ml) was phosphorylated by
phosphorylase kinase at 30 °C for 4 h in the reaction mixture
containing 66.4 mM -glycerophosphate (pH 8.5), 2 mM Mg(CH3CO2)2, 0.125 mM CaCl2, 0.218 mg/ml phosphorylase kinase, and
50 µM [
-32P]ATP (28,000 cpm/pmol). After
the reaction was terminated by adding 14.2 mM EDTA, the
resulting [32P]phosphorylase a was desalted by
a Sephadex G-50 spin column as described above and stored at
4 °C.
Mixed histones (1 mg/ml), MBP (1 mg/ml), -casein (4 mg/ml), and
phosphorylase kinase (1.6 mg/ml) were phosphorylated by PKA in the
reaction mixture containing 50 mM Hepes-NaOH (pH 7.0), 5 mM Mg(CH3CO2)2, 0.01%
Tween 20, 2 µg/ml PKA, and 50 µM
[
-32P]ATP (28,000 cpm/pmol). For phosphorylase kinase,
EGTA (1 mM) and EDTA (0.4 mM) were included in
the reaction mixture. The phosphorylation reactions were carried out at
30 °C for 4 h for histones, MBP, and
-casein, and for 4 min
for phosphorylase kinase. The reactions were terminated and the
resulting phosphoproteins were desalted, as described above. The
phosphoproteins were stored in aliquots at
80 °C. The substrate
concentrations presented in the text represent the concentrations of
32P bound to the substrate proteins.
A crude extract of rat cerebral cortex (407 µg/ml) was phosphorylated
by CaMKII at 30 °C for 1 min in the reaction mixture containing 40 mM Hepes-NaOH (pH 8.0), 5 mM
Mg(CH3CO2)2, 0.5 mM EGTA, 0.4 mM EDTA, 1 µM calmodulin, 1.2 mM CaCl2, 0.01% Tween 20, 0.96 µg/ml CaMKII,
and 50 µM [-32P]ATP (26,300 cpm/pmol).
Termination of the reaction and desalting of the phosphoproteins were
carried out as described for autophosphorylated CaMKII except that 100 nM calyculin A was included in the equilibration/elution buffer for the spin column. The phosphorylated sample was stored in
aliquots at
80 °C.
Protein Phosphatase Assay
CaMKIIPase activity was determined by the following four assay methods. Unless otherwise stated, poly(Lys) means Mr 87,000 species of poly(Lys). Basic proteins and polycations including poly(Lys) were initially dissolved in phosphate-buffered saline to yield 10 mg/ml solutions and they were diluted with H2O to appropriate concentrations prior to use.
Immobilized Phosphopeptide Assay-- For analysis with the purified enzyme, dephosphorylation of [32P]PMC-KII(281-289) was carried out at 30 °C for 5 min in a reaction mixture (30 µl) containing an appropriate amount of CaMKIIPase, 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 2 mM MgCl2, 2 mM MnCl2, 0.1 mM EGTA, 0.01% Tween 20, 10 µg/ml poly(Lys), and 89 nM [32P]PMC-KII(281-289) in a polypropylene microtube (Eppendorf). For purification of CaMKIIPase, the dephosphorylation of [32P]PMC-KII(281-289) was carried out at 30 °C for 10 min in the presence of 100 nM calyculin A. It should be noted that the peptide conjugate in the tube must be suspended uniformly by vortexing just prior to initiation of the reaction. The reaction was started by adding the enzyme, and terminated by adding 500 µl of 5% trichloroacetic acid, followed by vortexing and then brief centrifugation at room temperature. The 32P radioactivity of the supernatant (450 µl) was determined by liquid scintillation counting. The sedimented conjugate was attached to the bottom of the tube by a neodymium iron magnet (Polysciences, Inc.) when the supernatant was withdrawn. One unit of enzyme was defined as the amount that catalyzed the release of 1 µmol of phosphate/min at 30 °C.
Trichloroacetic Acid Precipitation Assay-- This method was used when phosphoproteins were used as substrates. Prior to addition of the substrate, the reaction mixture was preincubated for 30 s at 30 °C, and the reaction was started by adding the phosphoprotein substrate. After incubation for 30 s or 1 min, a 10-µl aliquot was withdrawn and transferred to a tube containing 100 µl of 20% trichloroacetic acid, and then 100 µl of 6 mg/ml BSA was added to the tube, followed by vortexing. The mixtures were allowed to stand for 10 min on ice, and then 300 µl of ice-cold 5% trichloroacetic acid was added. After centrifugation for 5 min at 4 °C in a microcentrifuge at maximum speed, 450 µl of the supernatant was withdrawn and counted for 32P radioactivity.
SDS-PAGE Assay--
After preincubation for 30 s at
30 °C, the reaction was started by adding the phosphoprotein. After
incubation for 1 min, the reaction was terminated by adding 22.7 mM EDTA, and the mixture was mixed with an equal volume of
a sample buffer consisting of 125 mM Tris-HCl (pH 6.8), 4%
SDS, 20% glycerol, 0.002% bromphenol blue, and 285 mM
-mercaptoethanol. The mixture was boiled for 2 min and centrifuged
for 2 min at room temperature in a microcentrifuge at maximum speed,
and an aliquot of the supernatant was subjected to SDS-PAGE on a 10%
acrylamide gel. The gel was dried and visualized by
autoradiography.
In-gel Assay-- In-gel phosphatase assay of CaMKIIPase was carried out as described previously (28).
Other Analytical Procedures
SDS-PAGE was carried out according to the method of Laemmli (36). Protein concentrations were determined by the method of Lowry et al. (37), as modified by Peterson (38).
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RESULTS |
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Purification of CaMKIIPase-- Since our previous in-gel assay for protein phosphatase with a synthetic peptide corresponding to the autophosphorylation site of CaMKII (residues 281-289) as a substrate suggested the existence of two protein phosphatases specific for the autophosphorylation site in rat brain (28), we attempted to purify and characterize them. Our preliminary experiments using immobilized phosphopeptide assay showed that a calyculin A-insensitive, Mn2+-dependent, and poly(Lys)-stimulated phosphatase activity toward [32P]PMC-KII(281-289) was present in a rat brain extract (data not shown). The possibility that the activity detected under such conditions was due to a protease could be eliminated, since almost all the radioactivities released into the supernatant of the assay mixture during the reaction were extracted with isobutyl alcohol/heptane (1/1, v/v) in the presence of ammonium molybdate/sulfuric acid (data not shown). Thus, we purified a protein phosphatase, which can dephosphorylate [32P]PMC-KII(281-289) in the presence of 100 nM calyculin A, 2 mM Mn2+, and 10 µg/ml poly(Lys), from rat brain stem, as described under "Experimental Procedures." Table I summarizes the results of a typical purification. The purified protein gave a single protein band corresponding to a molecular weight of about 54,000 on SDS-PAGE (Fig. 1A), and in-gel protein phosphatase assay also gave a single transparent band at the same position as the protein staining band (Fig. 1B), indicating that the protein purified to apparent homogeneity was CaMKIIPase. The molecular weight of the enzyme, as determined by gel filtration, was approximately 65,000 (Fig. 1C), indicating that CaMKIIPase consisted of a single polypeptide chain.
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Factors Affecting CaMKIIPase Activity-- We initially detected protein phosphatases capable of dephosphorylating a phosphopeptide corresponding to the autophosphorylation site of CaMKII (residues 281-289) in a reaction mixture containing 50 mM Tris-HCl (pH 7.0), 100 mM KCl, 2 mM MgCl2, 2 mM MnCl2, 0.5 mM EGTA, and 2 mM DTT (28). When purified CaMKIIPase was assayed by immobilized phosphopeptide method as described under "Experimental Procedures," it required Mn2+ (2 mM) for its activity and showed no activity in the absence of Mn2+ (data not shown). No other divalent cations tested, such as Mg2+, Zn2+, Co2+, Ca2+, Ni2+, and Ba2+, could replace Mn2+ but, in the presence of Mn2+, Mg2+ slightly stimulated the activity (109.7 ± 2.6%). When the pH profile of CaMKIIPase activity was examined using immobilized phosphopeptide assay, the maximal activity was observed over the pH range of 7 to 8. CaMKIIPase activity was markedly stimulated in the presence of poly(Lys). Fig. 2 shows the effect of varying the concentration of poly(Lys) on the activation of CaMKIIPase. Maximal activation occurred with 5-50 µg/ml poly(Lys), and higher concentrations of poly(Lys) resulted in decrease in the enzyme activity. The activation by poly(Lys) was enhanced 2-3-fold by the presence of 100 µM phosphate (data not shown).
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Dephosphorylation of Autophosphorylated CaMKII by CaMKIIPase-- In our experiments described so far, we had used [32P]PMC-KII(281-289), a synthetic phosphopeptide conjugate corresponding to residues 281-289 of autophosphorylated CaMKII, as a substrate for assay of CaMKIIPase. The important question of whether the purified enzyme can actually catalyze dephosphorylation of autophosphorylated CaMKII was examined as shown in Fig. 4. CaMKIIPase dephosphorylated autophosphorylated CaMKII in the presence of poly(Lys), and the dephosphorylation was almost completely blocked by the omission of poly(Lys). The dephosphorylation was not inhibited by calyculin A, but inhibited by NaF or EDTA, in good agreement with the results obtained with [32P]PMC-KII(281-289) as a substrate (Table II). The dephosphorylation of autophosphorylated CaMKII by CaMKIIPase was also confirmed by trichloroacetic acid precipitation assay. When [32P]autophosphorylated CaMKII (50 nM) was incubated with CaMKIIPase (64 ng/ml) in the presence of poly(Lys) (10 µg/ml) and then precipitated with trichloroacetic acid, a rapid release of [32P]radioactivity into the supernatant was observed, while no significant radioactivity was recovered in the supernatant when poly(Lys) was omitted from the incubation mixture (data not shown). When much higher concentrations of CaMKIIPase was used in this assay, however, a slow but significant dephosphorylation was observed in the absence of poly(Lys) (data not shown), indicating that CaMKIIPase possessed a low dephosphorylating activity toward autophosphorylated CaMKII even in the absence of poly(Lys). The activation by 10 µg/ml poly(Lys) under the assay conditions was estimated to be approximately 90-fold.
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Substrate Specificity of CaMKIIPase--
The next important
question of whether the action of CaMKIIPase is specific for CaMKII was
examined with several phosphorylated proteins known to be
dephosphorylated by various protein phosphatases as substrates
(39-41), as shown in Fig. 5A.
None of the proteins tested, such as phosphorylase kinase, histones,
MBP, and -casein, which had been phosphorylated by PKA, and
phosphorylase a, which had been phosphorylated by
phosphorylase kinase, was significantly dephosphorylated by CaMKIIPase,
while simultaneously added CaMKII which had been autophosphorylated was
strongly dephosphorylated, suggesting that CaMKIIPase might be a
specific protein phosphatase for autophosphorylated CaMKII. No
significant dephosphorylation of the phosphorylase kinase, histones,
MBP,
-casein, and phosphorylase a by CaMKIIPase was also
observed by trichloroacetic acid precipitation assay (data not
shown).
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Decrease of the Increased Ca2+/Calmodulin-independent Activity of Autophosphorylated CaMKII by CaMKIIPase-- Since autophosphorylation of CaMKII at Thr286 results in generation of Ca2+/calmodulin-independent activity (13-17) and full activation of the total activity (18-20), dephosphorylation of the autophosphorylated CaMKII by CaMKIIPase should restore the activated activities to the original levels. The facts that autophosphorylated CaMKII is much more labile than nonphosphorylated CaMKII (24, 31) and 10 µg/ml poly(Lys) markedly labilized autophosphorylated CaMKII2 made it difficult to measure changes in the activity of CaMKII produced by dephosphorylation by CaMKIIPase at 30 °C. Therefore, changes in the activity of autophosphorylated CaMKII by CaMKIIPase were studied at 5 °C and at a low concentration of poly(Lys) (1 µg/ml), as shown in Fig. 6. When autophosphorylated CaMKII was incubated with or without 1 µg/ml poly(Lys) in the absence of CaMKIIPase at 5 °C for 60 min, no significant changes in the autonomy were observed. On the other hand, incubation of autophosphorylated CaMKII with CaMKIIPase in the absence of poly(Lys) caused a relatively small but significant decrease in the autonomy. This decrease in the autonomy is probably due to the low basal activity of CaMKIIPase in the absence of poly(Lys), since a relatively high concentration of CaMKIIPase was used in this experiment. In contrast, the incubation with CaMKIIPase in the presence of poly(Lys) caused a marked decrease in the autonomy. Thus, the Ca2+/calmodulin-independent activity of CaMKII generated by autophosphorylation at Thr286 was reversed by incubation with CaMKIIPase in the presence of poly(Lys). When the dephosphorylated kinase was assayed after brief incubation (30 °C, 10 s) under the autophosphorylating conditions, generation of Ca2+/calmodulin-independent activity was observed again (44% of the total activity), suggesting that the dephosphorylation process is reversible.
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DISCUSSION |
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Since CaMKII, which is involved in controlling a variety of neuronal functions, is markedly activated through autophosphorylation at Thr286 (2-4, 13-22), the dephosphorylation of the Thr286 is thought to be very important for the regulation of the activity of CaMKII. Recently, the existence of protein phosphatases catalyzing the dephosphorylation of the Thr286 in the rat brain was suggested by in-gel protein phosphatase assay using a synthetic peptide CaMKII(281-289) (MHRQETVDC) corresponding to the autophosphorylation site of CaMKII (residues 281-289) as a substrate (28). In the present study, we purified and characterized a novel protein phosphatase, designated CaMKIIPase, catalyzing the dephosphorylation of [32P]PMC-KII(281-289), a phosphopeptide conjugate composed of phosphorylated CaMKII(281-289), poly(Lys) and paramagnetic particles. Table III compares some catalytic properties of CaMKIIPase with those of other well known protein Ser/Thr phosphatases, such as PP1, PP2A, PP2B, and PP2C. The most striking characteristics of CaMKIIPase are the activation by polycations and the requirement for Mn2+. The polycation activation has so far been considered as a characteristic of PP2A (39, 40). As judged by requirements for activity and sensitivities to various inhibitors summarized in Table III, CaMKIIPase is thought to be a protein phosphatase distinct from any of the four well known protein phosphatases. Several protein Ser/Thr phosphatases, which differ in their biochemical properties from PP1, PP2A, PP2B, and PP2C, have recently been isolated from mammalian tissues (39, 41-43), but no reports describing the properties similar to those of CaMKIIPase have appeared.
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It has been reported that some protein phosphatases having broad
substrate specificities, such as PP1 (23), PP2A (14, 16, 24, 25), and
PP2C (26), can dephosphorylate Thr286 of autophosphorylated
CaMKII in vitro. In contrast to these multifunctional protein phosphatases, CaMKIIPase dephosphorylated autophosphorylated CaMKII but none of the other phosphorylated proteins examined, such as
phosphorylase kinase, histones, MBP, -casein, and phosphorylase a (Fig. 5A), and dephosphorylated no other
proteins than CaMKII in the rat brain extract (Fig. 5B),
suggesting that CaMKIIPase might be a protein phosphatase specific for
autophosphorylated CaMKII, in good agreement with our previous finding
(28) that the band corresponding to CaMKIIPase detected by the in-gel
protein phosphatase assay was observed when CaMKII(281-289) was used
as a substrate but not observed when other phosphopeptides such as C-syntide-2 and CAMKAKS peptide were used as substrates. An apparent Km value of CaMKIIPase for autophosphorylated CaMKII estimated from double-reciprocal plots of the rates of the
dephosphorylation determined by trichloroacetic acid precipitation
assay as functions of the concentrations of CaMKII was in the range of
30-80 nM (data not shown). This value is much lower than
the Km values of PP2A ranging from 1 to 100 µM obtained for various substrates (44) or the
Km value of PP2C from turkey gizzard smooth muscle
of 7.9 µM estimated for myosin light chain (45),
suggesting that autophosphorylated CaMKII is a good specific substrate
for CaMKIIPase.
Since the autophosphorylation of CaMKII at Thr286 is known to generate the Ca2+/calmodulin-independent activity, the dephosphorylation of the phosphorylated Thr286 should result in a decrease in the activity. However, autophosphorylated CaMKII was very labile to poly(Lys) added for the reaction of CaMKIIPase, and therefore the reverse of the activity due to the dephosphorylation of Thr286 of CaMKII was monitored by a decrease in the autonomy of the activity of CaMKII (Fig. 6). A maximum decrease in the autonomy was observed after incubation with CaMKIIPase in the presence of poly(Lys), suggesting that CaMKIIPase dephosphorylated the autophosphorylated Thr286 of CaMKII, thereby resulting in a decrease in the autonomy. The results, taken together, suggest the possibility that CaMKIIPase purified in the present study is a specialized protein phosphatase for the regulation of CaMKII.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by grants from the Byotai Taisha Research Foundation, the Mitsubishi Foundation, and the Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The abbreviations used are: CaMKII, calmodulin-dependent protein kinase II; BSA, bovine serum albumin; CaMKIIPase, CaMKII phosphatase; DTT, dithiothreitol; EMCS, N-(6-maleimidocaproyloxy)succinimide; HPLC, high performance liquid chromatography; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; PKA, catalytic subunit of cAMP-dependent protein kinase; PMC, peptide-magnetic particle conjugate; PMSF, phenylmethanesulfonyl fluoride; poly(Lys), poly-L-lysine; poly(Arg), poly-L-arginine; poly(Glu,Lys,Tyr); poly-L-(glutamic acid,lysine,tyrosine); PP, protein phosphatase.
2 A. Ishida, I. Kameshita, and H. Fujisawa, unpublished observations.
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REFERENCES |
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