(Received for publication, August 15, 1994; and in revised form, November 14, 1994)
From the
The active 30-kDa chymotryptic fragment of calmodulin-dependent
protein kinase II (CaM kinase II), devoid of the autoinhibitory domain,
and the enzyme, autothiophosphorylated at
Thr/Thr
, were much more labile than was the
original native enzyme. They were markedly stabilized by synthetic
peptides, designed after the sequence around the autophosphorylation
site in the autoinhibitory domain, such as autocamtide-2 and
CaMK-(281-309), but such marked stabilizations were not observed
with the ordinary exogenous substrates, such as syntide-2. These
results suggest that the autoinhibitory domain of CaM kinase II plays a
crucial role in stabilizing the enzyme. A nonphosphorylatable analog of
autocamtide-2, AIP, strongly inhibited the activity of the 30-kDa
fragment. Kinetic analysis revealed that the inhibition by AIP was
competitive with respect to autocamtide-2 and CaMK-(281-289) and
noncompetitive with respect to syntide-2 and ATP/Mg
,
suggesting that CaM kinase II possesses at least two distinct
substrate-binding sites; one for ordinary exogenous substrates such as
syntide-2 and the other for an endogenous substrate, the
autophosphorylation site (Thr
/Thr
) in the
autoinhibitory domain. Fluorescence analysis of the binding of
7-nitrobenz-2-oxa-1,3-diazole-4-yl labeled AIP to the 30-kDa fragment
also supported this contention. Thus, the autoinhibitory domain appears
to play a crucial role in keeping the enzyme stable by binding to the
substrate-binding site for the autophosphorylation site.
Calmodulin-dependent protein kinase II (CaM kinase II) ()is known to be a second-messenger-responsive
multifunctional protein kinase which plays important roles in
controlling a variety of cellular functions in response to an increase
in intracellular Ca
(reviewed in (1, 2, 3) ). CaM kinase II is widely
distributed in many tissues and organisms, but most abundantly in
brain. CaM kinase II is thought to play important roles in the central
nervous system, and possible involvements of CaM kinase II in the
regulation of neuronal functions such as neurotransmitter
synthesis(4, 5, 6) , neurotransmitter
release(7, 8) , long-term
potentiation(9, 10, 11) , and formation of
spatial learning (12) have so far been suggested.
Rat brain
CaM kinase II is an oligomeric enzyme with a molecular weight of about
540,000 and is composed of subunits ranging in molecular weight from
about 55,000 to 60,000(1, 2, 3) . The primary
structure of four rat brain CaM kinase II isoforms, (13) ,
(14, 15) ,
(16) , and
(17) , have been determined from the cDNA clones. Each
subunit has the consensus ATP-binding domain near the amino terminus,
the catalytic domain in the amino-terminal region, and the regulatory
domain consisting of the autoinhibitory and calmodulin-binding domains
in the central region of the
protein(1, 2, 3) . Upon binding
Ca
/calmodulin in the presence of
ATP/Mg
, CaM kinase II undergoes a rapid
autophosphorylation on Thr
/Thr
(in
/
,
, and
isoforms) located within the
autoinhibitory domain, leading to the full activation of the enzyme (18, 19) as well as the generation of the
Ca
/calmodulin-independent
activity(20, 21, 22, 23, 24) .
This autophosphorylation has recently been reported to cause a
dramatic increase in the affinity of the enzyme for
Ca
/ calmodulin(25) .
Further
autophosphorylation of the enzyme following the initial rapid
autophosphorylation on Thr results in a gradual loss of
the enzymatic activity(26, 27, 28) . Lai et al.(29) have suggested that the inactivation may
be a consequence of a decrease in the thermal stability of the enzyme.
Colbran (30) has reported that basal autophosphorylation at
Thr
in the calmodulin-binding site blocks calmodulin
binding, resulting in inactivation of the enzyme.
We have recently
reported that incubation of the enzyme with
Ca/calmodulin causes a marked inactivation of the
enzyme and that the inactivation was prevented by autocamtide-2, a
synthetic substrate peptide modeled after the sequence around the
autophosphorylation site (Thr
) within the autoinhibitory
domain, but not prevented by syntide-2, a synthetic substrate peptide
modeled after a phosphorylation site of glycogen synthase(31) .
These observations led us to a postulation that there are two distinct
substrate-binding sites in CaM kinase II, one for ordinary exogenous
substrates and the other for the endogenous substrate, the
autophosphorylation site (Thr
) in the autoinhibitory
domain, and that the latter may be involved in stabilizing the enzyme.
The present paper describes several experimental data supporting this
contention. Thus, the autoinhibitory domain of CaM kinase II appears to
play a crucial role in stabilizing the enzyme as well as in inhibiting
the catalytic activity of the enzyme, keeping the enzyme inactive and
stable. Similar stabilizing and inhibitory effects have been observed
with the pseudosubstrate domain of smooth muscle myosin light chain
kinase(32) .
where F and F
are the relative fluorescence intensities at 521.6 nm before and
after the addition of the 30-kDa fragment, respectively. The values
were corrected for dilution.
Figure 1:
Effect of temperature on thermal
stability of the 30-kDa fragment of CaM kinase II and
autothiophosphorylated CaM kinase II. A, the 30-kDa fragment
(163 nM) of CaM kinase II () or native CaM kinase II
(16.7 nM as holoenzyme) (
) was incubated at the indicated
temperatures for 3 min, and then aliquots were assayed for the kinase
activity. The CaM kinase II fragment was assayed in the absence of
Ca
/calmodulin, and the native CaM kinase II was
assayed in the presence of Ca
/calmodulin. B,
CaM kinase II autothiophosphorylated as described under
``Experimental Procedures'' (
) or the
nonthiophosphorylated enzyme, which was prepared by adding EDTA/EGTA
before starting the autothiophosphorylation reaction as described under
``Experimental Procedures'' (
), was incubated at the
indicated temperatures for 3 min, and aliquots were assayed for the
kinase activity in the presence of
Ca
/calmodulin.
Figure 2: Analyses of the inactivated 30-kDa fragment of CaM kinase II and its susceptibility to tryptic digestion on SDS-polyacrylamide gel electrophoresis. A, the 30-kDa fragment (407 nM) of CaM kinase II was incubated at 30 °C for inactivation, and after incubation for 0, 2, 5, and 10 min (lanes 1-4, respectively), aliquots were subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel, followed by silver staining. B, the 30-kDa fragment (167 nM) was incubated at 0, 10, 20, 30, and 40 °C (lanes 1-5, respectively) for 3 min, and aliquots were subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel, followed by silver staining. C, approximately 980 ng of the active 30-kDa fragment (lanes 1-5) or the inactivated 30-kDa fragment (lanes 6-10), obtained by incubation for 10 min at 30 °C as described under ``Experimental Procedures,'' was incubated with 21 ng of N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin at 0 °C. After incubation for 0 (lanes 1 and 6), 5 (lanes 2 and 7), 30 (lanes 3 and 8), 60 (lanes 4 and 9), and 120 min (lanes 5 and 10), aliquots were mixed with phenylmethanesulfonyl fluoride (10 mM) and subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel, followed by silver staining.
Fig. 1B shows that autothiophosphorylation of CaM kinase II resulted in a
decrease in the thermal stability of the enzyme. Under the experimental
conditions used, autothiophosphorylation should occur only at
Thr(24) , which is located in the autoinhibitory
domain of the enzyme. Thus, not only the removal of the autoinhibitory
domain but also the thiophosphorylation of Thr
in the
autoinhibitory domain led to a large decrease in the thermal stability
of CaM kinase II.
Figure 3:
Effect of syntide-2 or autocamtide-2 on
thermal stability of the 30-kDa fragment of CaM kinase II and
autothiophosphorylated CaM kinase II. A, the 30-kDa fragment
(163 nM) of CaM kinase II (,
,
) or native
CaM kinase II (17 nM as holoenzyme) (
) was incubated in
the inactivation mixture with additions of none (
,
), 1
µM autocamtide-2 (
), or 0.3 mM syntide-2
(
) at 30 °C for the indicated times, and then aliquots were
assayed for the kinase activity. B, CaM kinase II
autothiophosphorylated as described under ``Experimental
Procedures'' (
,
,
) or the
nonthiophosphorylated enzyme, which was prepared as described in the
legend for Fig. 1(
), was incubated at 30 °C for the
indicated times with the addition of none (
,
), 5 µM autocamtide-2 (
), or 0.3 mM syntide-2 (
),
and aliquots were assayed for the kinase
activity.
In order to estimate affinities of CaMK-(281-309) and autocamtide-2 for the 30-kDa fragment, the effect of varying their concentrations on the thermal stability of the fragment was examined as shown in Fig. 4. Under our experimental conditions, the concentration of the 30-kDa fragment required for accurate determination of the remaining activity was at least 85 nM. Both CaMK-(281-309) and autocamtide-2 almost completely protected 85 nM enzyme against inactivation at concentrations as low as about 100 nM, indicating that the two peptides have very high affinities for the enzyme.
Figure 4: Effect of varying the concentration of autocamtide-2 and CaMK-(281-309) on thermal stability of the 30-kDa fragment of CaM kinase II. A, the 30-kDa fragment (85 nM) of CaM kinase II was incubated at 30 °C for 10 min with the indicated concentrations of autocamtide-2, and then aliquots were assayed for the kinase activity. B, the 30-kDa fragment (85 nM) of CaM kinase II was incubated at 30 °C for 10 min with the indicated concentrations of CaMK-(281-309), and aliquots were assayed for the kinase activity.
Figure 5:
Kinetic analyses of the inhibition of the
30-kDa fragment of CaM kinase II by AIP. The activity of the 30-kDa
fragment of CaM kinase II was measured at varying concentrations of the
indicated substrates at four or five different fixed concentrations of
AIP, and the data were plotted as a double-reciprocal plot. A,
assayed at varying concentrations of syntide-2 in the presence of 0
(), 0.01 (
), 0.02 (
), 0,03 (
), and 0.05
µM (
) AIP. B, assayed at varying
concentrations of autocamtide-2 in the presence of 0 (
), 0.01
(
), 0.02 (
), 0.05 (
), and 0.08 µM (
) of AIP. C, assayed at varying concentrations
of CaMK-(281-289) in the presence of 0 (
), 0.003 (
),
0.007 (
), and 0.01 µM (
) of AIP. D,
assayed at varying concentrations of ATP in the presence of 0 (
),
0.01 (
), 0.015 (
), 0.025 (
), and 0.03 µM (
) of AIP, using syntide-2 (200 µM) as a
peptide substrate.
Figure 6:
Kinetic analyses of the inhibition of the
30-kDa fragment of CaM kinase II by CaMK-(281-309). The activity
of the 30-kDa fragment of CaM kinase II was measured at varying
concentrations of the indicated substrates at four or five different
fixed concentrations of CaMK-(281-309), and the data were plotted
as a double-reciprocal plot. A, assayed at varying
concentrations of syntide-2 in the presence of 0 (), 0.05
(
), 0.1 (
), and 0.2 µM (
) of
CaMK-(281-309). B, assayed at varying concentrations of
autocamtide-2 in the presence of 0 (
), 0.1 (
), 0.2 (
),
0.3 (
), and 0.5 µM (
) of
CaMK-(281-309). C, assayed at varying concentrations of
ATP in the presence of 0 (
), 0.05 (
), 0.1 (
), 0.2
(
), and 0.3 µM (
) of CaMK-(281-309),
using syntide-2 (200 µM) as a peptide
substrate.
Further
experimental support for our contention was provided by fluorescence
analysis of the binding of NBD-AIP to the 30-kDa fragment. Fig. 7shows the emission spectra of NBD-AIP at an excitation
wavelength of 472 nm before and after the addition of the 30-kDa
fragment. Both spectra showed maxima at 521.6 nm, but the addition of
the 30-kDa fragment caused a significant decrease in the fluorescence,
while bovine serum albumin caused no fluorescence change (data not
shown), indicating the specific interaction of the fragment with
NBD-AIP. Effects of various substrates on the interaction between
NBD-AIP and the fragment were examined by monitoring the changes in the
fluorescence intensity at 521.6 nm at an excitation wavelength of 472
nm, as summarized in Table 2. The addition of micromolar
concentrations of autocamtide-2 and CaMK-(281-309) completely
blocked the quenching of the NBD-AIP fluorescence induced by the
binding of the 30-kDa fragment, whereas syntide-2, synapsin I site 3
peptide, and ATP/Mg did not significantly affect the
quenching even at millimolar concentrations, suggesting that
autocamtide-2 and CaMK-(281-309) competed with NBD-AIP for
binding to the 30-kDa fragment but the other substrates did not, in
good agreement with the results of the kinetic analyses as shown in Fig. 5and 6.
Figure 7: A change in the fluorescence emission spectrum of NBD-AIP upon binding to the 30-kDa fragment of CaM kinase II. Fluorescence emission spectra of NBD-AIP were recorded at an excitation wavelength of 472 nm before (curve a) and after (curve b) the addition of the 30-kDa fragment of CaM kinase II, as described under ``Experimental Procedures.'' The spectra were corrected for dilution.
Figure 8:
Effect of syntide-2 and autocamtide-2 on
autothiophosphorylation of CaM kinase II. CaM kinase II (16.9 nM as holoenzyme) was autothiophosphorylated at 5 °C for 30 s (lane 1) or 1 min (lane 2) in the reaction mixture
containing 40 mM Hepes-NaOH (pH 8.0), 0.4 mM CaCl, 0.1 mM EGTA, 0.5 µM calmodulin, 5 mM magnesium acetate, 0.01% Tween 20, and
50 µM [
-
S]ATP
S (0.12
mCi/ml), with the following additions: A, none; B,
0.3 mM syntide-2; C, 9 µM autocamtide-2.
The autothiophosphorylation reaction was terminated by adding 16.3
mM EDTA, and the mixture was mixed with an equal volume of a
solution consisting of 125 mM Tris-HCl (pH 6.8), 4% SDS, 20%
glycerol, 0.002% bromphenol blue, and 130 mM
-mercaptoethanol, followed by boiling for 2 min, and then
aliquots were subjected to SDS-polyacrylamide gel electrophoresis on a
10% polyacrylamide gel. The gel was visualized by
fluorography.
Previous reports from our own and other
laboratories(26, 27, 28) demonstrated that
autophosphorylation of CaM kinase II, presumably occurring at multiple
sites of the enzyme, causes a progressive decrease in the enzyme
activity, and thereafter Lai et al.(29) suggested
that the inactivation is due to a decrease in the thermal stability of
the autophosphorylated enzyme. More recently, Colbran (30) reported that autophosphorylation at Thr,
gradually occurring in the absence of Ca
/calmodulin,
results in inactivation of the enzyme. In the present paper, we show
that autothiophosphorylation at Thr
, which is involved in
the activation of the
enzyme(18, 19, 20, 21, 22, 23, 24) ,
caused a marked decrease in the thermal stability. The active 30-kDa
chymotryptic fragment of CaM kinase II(31, 41) ,
devoid of the autoinhibitory domain containing the calmodulin-binding
and inhibitory domains and also the autophosphorylation site of
Thr
, was also very labile (Fig. 1). The thermal
inactivation of the two labile CaM kinase II preparations, the
autothiophosphorylated enzyme and the 30-kDa fragment, was prevented by
autocamtide-2 and CaMK-(281-289), synthetic peptides modeled
after the sequence around the autophosphorylation site of
Thr
, and CaMK-(281-309), a synthetic peptide
corresponding to the sequence of the autoinhibitory domain, but it was
not prevented by ordinary protein and peptide substrates such as MAP2,
myosin light chain, syntide-2, and synapsin I site 3 peptide, while it
was also prevented by ATP/Mg
( Fig. 3and 4,
and Table 1). These results, taken together with the general
contention that the autoinhibitory domain of CaM kinase II suppresses
the enzyme activity by interaction with the catalytic
site(50) , suggested that the autophosphorylation site located
in the autoinhibitory domain not only inhibits the enzyme activity but
also stabilizes the enzyme by interacting with the second
substrate-binding site specific for the autophosphorylation site, which
is distinct from the substrate-binding site for ordinary exogenous
substrates such as MAP2, myosin light chain, syntide-2, and so on. The
possibility of the existence of the two distinct substrate-binding
sites in the catalytic site of CaM kinase II, one for ordinary
exogenous protein and peptide substrates and the other for the
endogenous substrate, the autophosphorylation site (Thr
),
has been suggested from our previous observation (31) that the
inactivation of CaM kinase II by Ca
/calmodulin is
prevented by autocamtide-2 but not by syntide-2 or MAP2. In the present
study, this was confirmed by the kinetic data that the inhibitions of
the enzyme by AIP and CaMK-(281-309), respectively, were
competitive with respect to autocamtide-2 but noncompetitive with
respect to syntide-2 ( Fig. 5and 6), and was further confirmed
by the fluorescence analysis that the quenching of NBD-AIP induced by
the 30-kDa fragment of CaM kinase II was completely blocked by
autocamtide-2 or CaMK-(281-309) but was not affected by syntide-2
or synapsin I site 3 peptide at all (Table 2). This was also
suggested by the fact that autocamtide-2 inhibited the
autothiophosphorylation of Thr
much more strongly than
did syntide-2 (Fig. 8). The K
value for
autocamtide-2 was reduced from 2.3 µM to 0.4 µM by truncation of the autoinhibitory domain, whereas the K
for syntide-2 was not changed significantly.
This observation might be explained by two different substrate-binding
sites; the binding of autocamtide-2 to its binding site is inhibited by
the autoinhibitory domain in the native enzyme, but the binding of
syntide-2 to its binding site is not inhibited.
Of particular
interest is a novel synthetic peptide inhibitor, AIP (KKALRRQEAVDAL),
which was made by the substitution of Ala for the phosphorylation site
of autocamtide-2 (KKALRRQETVDAL), Thr. This peptide was a
potent inhibitor with a K
value as low as
2-8 nM for the 30-kDa fragment of the enzyme. The
kinetic data of the inhibition by AIP (Fig. 5) and the results
of fluorescence analysis with NBD-AIP (Table 2), together with
the results that autocamtide-2 inhibited the autothiophosphorylation of
Thr
more strongly than did syntide-2 (Fig. 8),
indicated that AIP may bind to the substrate-binding site for the
autophosphorylation site (Thr
) but not bind to the
substrate-binding site for the ordinary exogenous substrates, thereby
inhibiting the enzyme activity. Thus, AIP will be a useful tool for
studying the regulatory mechanism of CaM kinase II through the
autophosphorylation of Thr
in the autoinhibitory domain
of the enzyme. It will be also interesting to examine the question of
whether AIP is a specific inhibitor for CaM kinase II, because no real
specific inhibitor for CaM kinase II has so far been
reported(51) . The specificity and the detailed mechanism for
the inhibition by AIP are under investigation in this laboratory.
In
contrast to AIP, CaMK-(281-309), a synthetic peptide
corresponding to the sequence of the autoinhibitory domain containing
the autophosphorylation site and calmodulin-binding site, inhibited the
enzyme activity in a competitive manner with respect not only to
autocamtide-2 but also to ATP, while it inhibited the enzyme in a
noncompetitive manner with respect to syntide-2 (Fig. 6),
suggesting that the autoinhibitory domain of the enzyme may interact
with both the substrate-binding site for the autophosphorylation site
(Thr) and the ATP-binding site.
Pears et al.(52) found that deletion of the pseudosubstrate sequence
of protein kinase C made it so unstable that it was not possible to
purify the recombinant enzyme. More recently, Faux et al.(32) reported that the pseudosubstrate sequence of smooth
muscle myosin light chain kinase plays an important role in stabilizing
the enzyme, and they predicted that other enzymes regulated by
pseudosubstrate mechanism may also utilize the pseudosubstrate sequence
to stabilize the structure. Here we show that CaM kinase II was also
stabilized by a similar mechanism, utilizing the autophosphorylation
site, the endogenous substrate, instead of the endogenous
pseudosubstrate sequence. Knighton et al.(53) have
proposed a model for the regulatory mechanism of myosin light chain
kinase by the pseudosubstrate sequence that the pseudosubstrate
sequence occupies the substrate binding site. However, the results that
substrate peptides, such as MLC-(1-23) and
MLC-(11-23)A, can protect the enzyme from thermal
inactivation but their potency is not as great as that of the
pseudosubstrate sequence (32) may reflect the existence of the
endogenous pseudosubstrate-binding site different from the ordinary
substrate-binding site.