(Received for publication, February 10, 1995; and in revised form, January 18, 1996)
From the
The time-dependent loss in enzyme activity associated with the
autophosphorylation of Ca/calmodulin-dependent
protein kinase II (CaM-kinase) was altered by both pH and ATP
concentration. These parameters also influenced the extent to which
soluble CaM-kinase undergoes self-association to form large aggregates
of sedimentable enzyme. Specifically, autophosphorylation of CaM-kinase
in 0.01 mM ATP at pH 6.5 resulted in the formation of
sedimentable enzyme and a 70% loss of enzyme activity. Under similar
conditions at pH 7.5, the enzyme lost only 30% of its activity, and no
sedimentable enzyme was detected. In contrast to 0.01 mM ATP,
autophosphorylation of CaM-kinase at pH 6.5 in 1 mM ATP did
not result in a loss of activity or the production of sedimentable
enzyme, even though the stoichiometry of autophosphorylation was
comparable. Electron microscopy studies of CaM-kinase
autophosphorylated at pH 6.5 in 0.01 mM ATP revealed particles
100-300 nm in diameter that clustered into branched complexes.
Inactivation and self-association of CaM-kinase were influenced by the
conditions of autophosphorylation in vitro, suggesting that
both the catalytic and physical properties of the enzyme may be
sensitive to fluctuations in ATP concentration and pH in vivo.
Ca/calmodulin-dependent protein kinase II
(CaM-kinase) (
)is highly enriched in brain where it is
thought to perform a multifunctional role in the transduction of
Ca
signals. Substrates phosphorylated by CaM-kinase
are involved in neurotransmitter synthesis and release, cytoskeletal
function, intracellular Ca
homeostasis, carbohydrate
metabolism, ion channel function, and synaptic plasticity (for reviews,
see (1) and (2) ). Purified forebrain CaM-kinase is
isolated as a holoenzyme of approximately 460-650 kDa (3, 4) and estimated to contain 10-12 subunits
consisting primarily of 50 kDa (
) and 60 kDa (
) isozymes in a
3:1 ratio, respectively(3) . Although substrate phosphorylation
is stringently coupled to Ca
/CaM binding, the
activity and regulatory properties of CaM-kinase are altered by
autophosphorylation (for reviews, see Refs. 1, 2, and 5). Previously
reported autophosphorylation-associated changes in the functional
properties of CaM-kinase include generation of the
Ca
/CaM-independent (autonomous)
form(6, 7, 8, 9, 10) ,
Ca
/CaM
insensitivity(11, 12, 13, 14, 15) ,
CaM trapping(16, 17) , and
inactivation(18, 19, 20, 21, 22, 23, 24) .
We have demonstrated a loss of CaM-kinase activity in both in vivo and in situ models of ischemia(25, 26) . Coincident with inactivation in these models was a redistribution of soluble CaM-kinase to particulate fractions. One possible explanation for the loss of activity and redistribution of soluble CaM-kinase is that ischemic-induced alterations in the cellular environment alter the properties of the enzyme, potentially through an autophosphorylation-mediated process. Biochemical alterations observed during ischemia include ionic imbalances with marked reductions in intracellular pH and ATP concentrations (see ``Discussion''). Although several investigators have observed a time-dependent decrease in the activity of autophosphorylated CaM-kinase (i.e. inactivation) in vitro(18, 19, 20, 21, 22, 23, 24) , the physiological significance and role of autophosphorylation in this loss of activity is unknown. Lou et al. (22) reported that ATP concentration influences autophosphorylation-associated losses in the activity of CaM-kinase, with low ATP maximizing and high ATP preventing enzyme inactivation. The generation of autonomous activity and the sites of autophosphorylation also were influenced by ATP concentration(22) . Other parameters reported to influence the loss of enzyme activity and sites of autophosphorylation include reaction temperature (27) and the type and concentration of divalent ions(24, 27) . These findings indicate that the conditions of autophosphorylation are important determinants for alterations in the activity of CaM-kinase. The redistribution and loss of soluble CaM-kinase activity associated with ischemic conditions led us to further examine the influence of conditions of autophosphorylation, specifically pH and ATP concentration on the activity and physical characteristics of CaM-kinase in vitro. In this study, we observed that pH and ATP concentration influenced the inactivation of CaM-kinase associated with autophosphorylation. These parameters also influenced the self-association of soluble CaM-kinase into sedimentable enzyme complexes, suggesting that cells undergoing fluctuations in pH and ATP concentration could produce autophosphorylation-associated changes in both the physical and catalytic properties of the enzyme in vivo.
Electrophoresis was
performed as described elsewhere(30) , using a 10% resolving
gel. Following SDS-PAGE, the enzyme was visualized using either
Coomassie Blue staining or Western blotting. Coomassie staining of
proteins was accomplished by incubation of gels in fixative (20%
methanol, 7% acetic acid) containing 0.002% of Coomassie Brilliant Blue
R-250 for 30 min and destained overnight in fixative. For Western
blotting, the enzyme was transferred to nitrocellulose using a Genie
transfer apparatus (Idea Scientific). Transfer was performed at 4
°C in 20% methanol, 190 mM glycine, and 26 mM Tris, pH 8.0, for 1-2 h at a constant 15-20 V. After
transfer, the nitrocellulose was blocked overnight in 5% dry milk in
phosphate-buffered saline. Monoclonal antibodies to the 50- (2D5) (31) and 60- (Cb-1) (32) kDa subunits of
CaM-kinase were diluted 1:2000 in TBST (10 mM Tris, pH 8.0,
150 mM NaCl, and 0.05% Nonidet P-40), and the membrane was
incubated at room temperature for 1 h on a platform shaker. The
membrane was washed in TBST, followed by incubation in anti-mouse-IgG
AP-conjugated secondary antibody (Promega) diluted 1:5000 in TBST for 1
h. The membrane again was washed in TBST and the immunoreactive
proteins were visualized using the alkaline phosphatase substrates,
nitro blue tetrazolium and 5-bromo-1-chloro-3-indolyl phosphate as
described by Promega. Autoradiographs were produced by exposing Kodak
XAR-5 film at -80 °C for 1-2 h in the presence of an
intensifying screen. The antibody to the 60-kDa subunit (Cb
-1) was
kindly provided by Dr. Howard Schulman (Stanford University).
Autophosphorylation of CaM-kinase was associated with a
time-dependent decrease in Ca/CaM-dependent enzyme
activity at pH 7.5 (Fig. 1A). Activity measurements
from enzyme autophosphorylated at pH 7.0 and 6.5 indicated that a
greater percentage of enzyme activity was lost over time with a
decrease in reaction pH (Fig. 1A). Autophosphorylation
of CaM-kinase at pH 7.5 resulted in a 15% decrease in enzyme activity
at 2 min, whereas pH 7.0 and pH 6.5 were both associated with a 40%
loss of enzyme activity at 2 min. After 5 min, the extent of enzyme
inactivation at pH 7.5 (30%) was less than both pH 7.0 (50%) and pH 6.5
(70%). Activity measurements also were performed in the presence of
EGTA to monitor changes in Ca
/CaM-independent
activity (Fig. 1B). The time course of
Ca
/CaM-independent inactivation at pH 6.5 (65 and 40%
at 2 and 5 min, respectively) and pH 7.0 (80 and 58% at 2 and 5 min,
respectively) were similar to decreases in
Ca
/CaM-dependent activity over time. However, at pH
7.5 an increase in independent activity was observed at 2 min (120%)
and a modest decrease in activity observed at 5 min (90%). These data
suggested that maximal Ca
/CaM-independent activity
had not been obtained during the 0.5-min preautophosphorylation at pH
7.5. This conclusion was supported by measuring the percent
Ca
/CaM-independent activity generated at time 0 for
pH 6.5, 7.0, and 7.5, which were 55 ± 2.1, 54.2 ± 5.4,
and 42 ± 0.2, respectively (n = 3, ±S.D.)
following the standard 0.5-min preautophosphorylation protocol.
Extending the preautophosphorylation to 1, 2, or 3 min on ice increased
the percent Ca
/CaM-independent activity generated at
pH 7.5 at time 0 to 52.1 ± 0.09, 55.3 ± 0.06, and 54.6
± 0.04, respectively (n = 3, ±S.D.).
After extending the preautophosphorylation on ice to 2 min, we observed
no differences in the time course of autonomous inactivation for pH 6.5
and 7.0 conditions (data not shown). However, at pH 7.5, the percent
inactivation observed for Ca
/CaM-independent activity
(90 and 60% at 2 and 5 min, respectively) was similar to the
Ca
/CaM-dependent loss of activity over time (see solid bar in Fig. 1B). These data indicated
that the loss of CaM-kinase activity observed during
autophosphorylation was influenced by pH and that the extent of
inactivation observed is similar for both
Ca
/CaM-dependent and independent activities.
Figure 1:
Activity
measurements demonstrating that pH influences the inactivation of
CaM-kinase during autophosphorylation. Autophosphorylation was
initiated by the addition of 1.0 µg purified CaM-kinase to the
reaction mixture: 10 mM Pipes pH 6.5, 7.0 or 7.5, 0.4 mM DTT, 0.5 mM CaCl, 1 µM CaM, 10
mM MgCl
, 10 µM ATP, 10% glycerol and
0.1% Tween-20. Aliquots of the autophosphorylation mix were collected
at the indicated time points and A,
Ca
/CaM-dependent and B,
Ca
/CaM-independent activity were determined in
second-stage reactions as described under ``Experimental
Procedures.'' Percent initial activity represents syntide
phosphorylation over time, normalized to the time 0 point for enzyme
autophosphorylated at pH 6.5 (cross-hatched bar), pH 7.0 (striped bar) and pH 7.5 (open bar), (n = 3, ±S.D.). Specific activity for
Ca
/CaM-dependent activity at time 0 was 12.3 ±
0.7, 12.7 ± 1.2, and 12.0 ± 0.5 µmol/min/mg for pH
6.5, 7.0, and 7.5, respectively. The solid bar in B represents changes in Ca
/CaM-independent
activity at pH 7.5 following a 2-min preautophosphorylation on
ice.
At 0,
2, and 5 min of autophosphorylation, aliquots of the reaction mixture
were subjected to centrifugation for 30 min at 15,000 g to determine the influence of pH on the sedimentability of
autophosphorylated CaM-kinase. The Western blot in Fig. 2depicts the distribution of CaM-kinase in the supernatant
and pellet following autophosphorylation at pH 6.5, 7.0, and 7.5. As
expected, the 50- and 60-kDa subunits were detected in the supernatant
at time 0 in all pH conditions. Interestingly, after 2 min of
autophosphorylation at pH 6.5, the enzyme was completely in the pellet.
After 2 min of autophosphorylation at pH 7.0, the enzyme was detected
in both the supernatant and pellet fractions, while the enzyme
autophosphorylated at pH 7.5 remained in the supernatant. Although
enzyme activity continued to decrease (see Fig. 1), no further
changes in the distribution of CaM-kinase between supernatant and
pellet were observed at 5 min. The electrophoretic mobility of the
50-kDa subunit was altered during autophosphorylation and decreased to
a 54-kDa form in a time-dependent manner in all three pH conditions
(see Fig. 2). Anomalies in the electrophoretic mobility of
CaM-kinase subunits have been correlated with a high stoichiometry of
autophosphorylation (3, 18) and may result from
differential SDS binding to the autophosphorylated
subunits(22) . Although the immunostaining of the 60-kDa
subunit at the 2- and 5-min time points often was below the sensitivity
of the immunoassay, autophosphorylation-associated changes in
sedimentability and electrophoretic mobility were also observed for the
subunit. The loss of immunostaining is not due to a loss of the
60-kDa subunit (see autoradiogram in Fig. 3) or specific to any
reaction pH and may represent changes in the affinity of the monoclonal
antibody to the autophosphorylated subunit and/or a decrease in the
threshold for immunostaining. It should be noted that an increase in
the preautophosphorylation period from the standard 0.5 min to 2 min
did not alter the formation of sedimentable enzyme in the different pH
conditions, nor did omitting the preautophosphorylation step completely
(data not shown).
Figure 2:
Western
blot analysis demonstrating that pH influences the formation of
sedimentable CaM-kinase during autophosphorylation. Enzyme
autophosphorylated at pH 6.5, 7.0, or 7.5 as described in Fig. 1was subjected to sedimentation analysis. Aliquots of the
reaction mix were collected at the indicated times and diluted 6-fold
in 10 mM EDTA buffered with 10 mM Pipes, pH 7.2.
Samples were centrifuged at 15,000 g for 30 min and
supernatant (S) and pellet (P) fractions isolated for
each time point. A complete description of enzyme processing for
centrifugation and Western blotting is provided under
``Experimental Procedures.'' Western blotting was performed
using monoclonal antibodies to the 50- and 60-kDa subunits of
CaM-kinase. The relative position of 50- and 60-kDa subunits of
CaM-kinase are indicated by arrows.
Figure 3:
Western blot and autoradiogram showing the
time course in the formation of sedimentable enzyme during
autophosphorylation at pH 6.5. CaM-kinase was autophosphorylated in
standard conditions: 50 mM MES/Hepes, pH 6.5, 0.4 mM DTT, 0.5 mM CaCl, 1 µM CaM, 10
mM MgCl
, 10 µM ATP, 2 µCi of
[
-
P]ATP, 10% glycerol and 0.1% Tween-20. Western, at the indicated times, autophosphorylated enzyme was
subjected to centrifugation and processing as described in Fig. 2. Supernatant (S) and pellet (P)
fractions are indicated at each time point. Arrows mark the
positions of the 50- and 60-kDa subunits. Alternative molecular mass
forms of the 50- and 60-kDa subunits are indicated with arrows at 54 and 62 kDa, respectively, on the right side of the figure. Autoradiograph, the autoradiograph was produced from the
Western blot above by exposure to x-ray film for 2 h at -80
°C with an intensifying screen.
A more comprehensive time course was constructed
to observe changes in sedimentability and activity of CaM-kinase during
autophosphorylation at pH 6.5. The Western blot in Fig. 3demonstrates that, under standard reaction conditions,
CaM-kinase was detected in the pellet as early as 1 min. Similar to Fig. 2, changes in the electrophoretic mobilities of the
subunits of CaM-kinase occurred with autophosphorylation. A 54-kDa form
of the 50-kDa subunit was observed as early as 1 min, appearing
temporally with the detection of sedimentable enzyme. By 5 min, the
50-kDa form was predominantly shifted to the 54-kDa form. The mobility
of the 60-kDa subunit was also altered during autophosphorylation and
although not visible by immunostaining in Fig. 3, the formation
of a 62-kDa form was evident after 5 min of autophosphorylation on the
autoradiogram (Fig. 3). The autoradiogram of the Western blot in Fig. 3also demonstrates alterations in the distribution of
CaM-kinase between supernatant and pellet and altered mobilities of the
50- and 60-kDa subunits of the enzyme. The signal from the P-labeled phosphoproteins clearly illustrates both the
presence of the 60-kDa subunit throughout the period of
autophosphorylation and the generation of the 62-kDa form after 5 min (Fig. 3).
The time course of CaM-kinase inactivation during
autophosphorylation as described in Fig. 3, is shown in Fig. 4(left ordinate axis). At 1.5 min, the enzyme was
completely sedimentable (see Fig. 3), yet retained over 60% of
its initial activity. After 7 min, the enzyme retained less than 25% of
the initial activity. Quantitative analysis of PO
incorporation into CaM-kinase during autophosphorylation
indicated an increase in the incorporation of
PO
over time, with approximately 2.3 mol of phosphate incorporated
per mol of enzyme after 7 min of autophosphorylation (Fig. 4, right ordinate axis). The time course of enzyme inactivation
parallels an increase in the stoichiometry of autophosphorylation,
suggesting that autophosphorylation contributes to the time-dependent
loss in enzyme activity.
Figure 4:
Time course of inactivation and
stoichiometry of autophosphorylation at pH 6.5. Stoichiometry ()
and Ca
/CaM-dependent activity (
) of CaM-kinase
autophosphorylated as described in Fig. 3. Left ordinate
axis (percent initial activity) indicates CaM-kinase
phosphorylation of the substrate syntide normalized to the time 0
point. Aliquots of the reaction mix were taken at 0, 0.5, 1, 1.5, 2, 5,
and 7 min of autophosphorylation and second-stage activity measurements
were performed as described under ``Experimental Procedures'' (n = 18, ±S.D.). Specific activity for the time
0 point was 15.6 ± 3.9 µmol/min/mg. Right ordinate axis indicates moles of phosphate incorporated per mol of CaM-kinase
during autophosphorylation. Stoichiometry of autophosphorylation was
determined as described under ``Experimental Procedures'' (n = 4, ±S.D.).
The autophosphorylation dependence of the
formation of sedimentable CaM-kinase at pH 6.5 was investigated by
substituting a nonhydrolyzable analogue for ATP (AMP-PCP) in the
reaction mixture (Fig. 5A). In the absence of
autophosphorylation, sedimentable enzyme was not formed. Similar
results were obtained when the reaction mix contained 0.01 mM ADP substituted for ATP (data not shown). A decrease in the
activity of enzyme incubated in AMP-PCP was observed over time in
parallel second-stage reactions (Fig. 5B). The enzyme
retained approximately 60% of its initial activity after 5 min in the
presence of AMP-PCP. We observed that enzyme incubated in similar
reaction conditions, yet in the absence of Ca/CaM
retained 90% of its activity after 5 min (data not shown). This loss of
substrate phosphorylation by CaM-kinase in the presence of
Ca
/CaM is similar to previous reports and may reflect
a ``thermal instability'' of activated enzyme in the absence
of ATP(21, 23) .
Figure 5:
Western blot and activity measurements
depicting the effects of no autophosphorylation and limited
autophosphorylation on the sedimentability and inactivation of
CaM-kinase at pH 6.5. A, CaM-kinase was incubated in standard
reaction conditions with the changes indicated. Aliquots of enzyme were
processed for sedimentation analysis and Western blotting as described
in Fig. 2. Supernatant (S) and pellet (P)
fractions are indicated at each time point. Arrows indicate
the 50- and 60-kDa subunits of CaM-kinase. AMP-PCP, standard reaction
conditions with 10 µM AMP-PCP substituted for ATP and
[-
P]ATP omitted. 4 °C, standard
autophosphorylation conditions performed at 4 °C.
Mn
, standard autophosphorylation conditions with the
substitution of 10 mM MnCl
for 10 mM MgCl
. B, Ca
/CaM-dependent
activity was determined in second-stage reaction mixtures as described
under ``Experimental Procedures.'' Percent initial activity
(±S.D.) represents syntide phosphorylation, normalized to the
time 0 point for enzyme incubated in [
AMP-PCP (n = 10),
ice (n = 9), and
Mn
(n = 5). In addition, the loss of
activity during autophosphorylation in
standard reaction
conditions (10 mM MgCl
, 10 µM ATP at
30 °C, n = 11) was performed in parallel for
comparison. Specific activity at time 0 was 10.3 ± 1.9, 9.8
± 1.8, 2.3 ± 0.5, and 11.3 ± 4.3 µmol/min/mg
(±S.D.) for AMP-PCP, ice, Mn
, and standard
reaction conditions, respectively.
The role of autophosphorylation in the inactivation and formation of sedimentable enzyme was further investigated by altering the conditions of autophosphorylation. Autophosphorylation on ice previously was demonstrated to produce the autonomous form of CaM-kinase without enzyme inactivation(21, 22) . Lou et al. (22) suggested that this condition not only slows the kinase reaction, but also modifies the sites autophosphorylated compared to similar conditions at 30 °C. Fig. 5, A and B, demonstrates that autophosphorylation at 4 °C does not result in the formation of sedimentable enzyme or in a significant loss of activity. Limited autophosphorylation in these experiments was supported by a calculated stoichiometry for autophosphorylation of 0.4 ± 0.04, 0.4 ± 0.06, and 0.4 ± 0.05 mol of phosphate per mol of CaM-kinase after 0, 2, and 5 min, respectively (n = 5, ±S.D.). The absence of mobility shifts for the subunits of CaM-kinase during SDS-PAGE further indicates a restricted state of autophosphorylation(22) .
To explore the
effects of temperature during autophosphorylation, limited
autophosphorylation was accomplished at 30 °C by substituting the
divalent Mn for Mg
(27) .
The Western blot in Fig. 5A demonstrates that
autophosphorylation in the presence of 10 mM Mn
does not result in the formation of sedimentable enzyme. In
addition, no change was observed in the relative mobility of the 50-kDa
subunit. The 60-kDa subunit clearly shifted to a 62-kDa form after 2
min and a 64-kDa form after 5 min of autophosphorylation. The
stoichiometry of autophosphorylation was approximately 0.3 ±
0.05, 0.8 ± 0.13, and 1.2 ± 0.18 mol of phosphate per mol
of CaM-kinase at 0, 2, and 5 min, respectively (n = 5,
±S.D.). Restricted or limited autophosphorylation in
Mn
, was supported by a maximal stoichiometry of
1, whereas in 10 mM Mg
at 30 °C, the
maximal stoichiometry was
2 mol of phosphate per mol of CaM-kinase
(see Fig. 4). Similar to autophosphorylation at 4 °C,
CaM-kinase autophosphorylated in Mn
retained 90% of
its initial activity after 5 min (Fig. 5B). During
these experiments, we observed that the specific activity of CaM-kinase
measured in the second-stage reaction was influenced by the
concentration of Mn
carried over from the
autophosphorylation reaction (0.4 mM Mn
final second-stage concentration). Although we did not
extensively pursue the influence of Mn
on the
catalytic activity of CaM-kinase, the specific activity observed in the
second-stage reaction was significantly improved if the concentration
of Mn
was decreased from 10 to 1 mM during
the initial autophosphorylation reaction, lowering the final
concentration in the second stage reaction to 0.04 mM.
Autophosphorylation in 1 mM Mn
produced
similar results to the data reported above for 10 mM Mn
, including a similar stoichiometry and no
appreciable changes in the activity or sedimentability of the enzyme
over time (data not shown). As seen in Fig. 5B, the
inactivation associated with autophosphorylation in standard conditions
(10 mM Mg
, 10 µM ATP, pH 6.5,
at 30 °C) was greater than inactivation in the absence of
autophosphorylation, suggesting that additional processes may lead to
further enzyme inactivation. Both inactivation and the formation of
sedimentable enzyme are influenced by autophosphorylation. Restricted
autophosphorylation does not produce sedimentable enzyme nor
significant inactivation and may even prevent or protect the enzyme
from ``thermal inactivation.''
Lou et al. (22) previously demonstrated that autophosphorylation of
CaM-kinase in high (0.5 mM) versus low (0.005
mM) ATP produced a similar stoichiometry of
autophosphorylation associated with different sites of
autophosphorylation. The functional consequence of autophosphorylation
was characteristic of the ATP concentration (i.e. inactivation
with little autonomous activity was associated with low ATP, and
autonomous activity with little inactivation was associated with high
ATP). Fig. 6demonstrates the effects of ATP concentration
(0.01, 0.1, and 1 mM) on the sedimentability of CaM-kinase
autophosphorylated at pH 6.5. After 2 min of autophosphorylation in
0.01 mM ATP, the enzyme was completely in the pellet. However,
CaM-kinase autophosphorylated at 0.1 mM ATP remained partially
in the supernatant after 2 min, and enzyme autophosphorylated for 2 min
in 1 mM ATP remained completely in the supernatant. Fig. 6also illustrates that ATP concentration influenced the
formation of the 54-kDa form of the 50-kDa subunit. In 1 mM ATP, the formation of the 54-kDa form was not observed. However,
the formation of the 54-kDa form was enhanced with decreased ATP
concentration. Autophosphorylation in 1 mM ATP produced
changes in the mobility of the 60-kDa subunit without altering the
mobility of the 50-kDa subunit. This is reminiscent of mobility changes
observed when the enzyme is autophosphorylated in 0.01 mM ATP
in the presence of Mn (see Fig. 5A).
After 2 min of autophosphorylation in 0.01, 0.1, or 1 mM ATP,
the percent activity remaining was 53 ± 7.2, 83 ± 6.4,
and 103 ± 13.8, respectively (n = 3,
±S.D.). The percent
Ca
/calmodulin-independent-activity remaining after 2
min of autophosphorylation in 0.01 and 1 mM ATP paralleled
changes in the Ca
/calmodulin-dependent activity; 65
± 6.4 and 122 ± 2.5, respectively (n = 3,
±S.D.). The generation of independent activity in the 0.01 and
1.0 mM ATP conditions was similar, 53.3 ± 0.6 and 53.3
± 3.6 (n = 3, ±S.D.), respectively.
Autophosphorylation for 2 min in 0.01 mM and 1 mM ATP
produced a stoichiometry of 1.2 ± 0.23 and 1.1 ± 0.32 mol
of
PO
per mol of CaM-kinase, respectively (n = 3, ±S.D.). These data indicate that both
inactivation and the formation of sedimentable enzyme are not a
consequence of the total
PO
incorporated into
CaM-kinase during autophosphorylation. In addition, these data
demonstrate that inactivation of the enzyme and the formation of
sedimentable CaM-kinase at pH 6.5 are prevented at high concentrations
of ATP, while low ATP concentrations appear to promote this process.
Figure 6: Western blot demonstrating that ATP concentration influences the sedimentability of CaM-kinase autophosphorylated at pH 6.5. Alterations in the sedimentability of CaM-kinase at 0 and 2 min following autophosphorylation in standard reaction conditions with 0.01 mM, 0.1 mM, and 1.0 mM ATP. Aliquots of enzyme were processed for sedimentation analysis and Western blotting as described in Fig. 2. Supernatant (S) and pellet (P) fractions are indicated at each time point. Arrows indicate the 50- and 60-kDa subunits of CaM-kinase. The specific activity of the enzyme at time 0 was 31.2 ± 4.2, 28.1 ± 0.8, and 22.9 ± 2.5 µmol/min/mg for 0.01, 0.1, and 1.0 mM ATP, respectively.
The formation of sedimentable CaM-kinase suggested dramatic
alterations in the physical characteristics of the soluble enzyme.
Enzyme autophosphorylated in conditions that either promote (0.01
mM ATP at pH 6.5) or prevent (1 mM ATP at pH 6.5) the
formation of sedimentable enzyme was visualized using TEM. CaM-kinase
autophosphorylated in 0.01 mM ATP for 5 min produced distinct
uranyl acetate-staining structures that were uniformly distributed over
the grid (Fig. 7A, 5000). At a higher
magnification (Fig. 7B,
33,000), particles
ranging from 100 nm to 300 nm in diameter were evident and appeared to
interconnect and associate to form branched structures, termed enzyme
complexes. Under these conditions (0.01 mM ATP at pH 6.5)
enzyme complexes were not observed after pre-autophosphorylation on ice
(time 0), yet were evident after 2 min of autophosphorylation at 30
°C (data not shown). In contrast, after 5 min of
autophosphorylation in 1 mM ATP at pH 6.5, the grid was void
of uranyl acetate-staining structures, and increased magnification
revealed only the sizing beads (data not shown). The TEM conditions
utilized in these experiments did not generate sufficient contrast to
expose the 20-30-nm structure of individual CaM-kinase
holoenzymes(34) . In addition, we directly analyzed the
sedimented form of CaM-kinase following centrifugation (15,000
g for 30 min) by TEM and as expected, the enzyme complexes
observed following centrifugation and resuspension were more tightly
packed than enzyme complexes deposited directly to the grid (data not
shown).
Figure 7:
Electron micrographs of sedimentable
CaM-kinase and SDS-PAGE analysis of sedimented enzyme. A and B, CaM-kinase autophosphorylated in standard reaction
conditions was processed for TEM as described under ``Experimental
Procedures.'' A, a field of uranyl acetate-staining
structures at 5000 following autophosphorylation in 0.01
mMATP for 5 min at pH 6.5. B, the same field as in A but at a higher magnification (
33,000). Arrows indicate polystyrene sizing beads 90 nm in diameter. Grid fields
at high and low magnification are representative of the entire grid. C, analysis of sedimentable enzyme by SDS-PAGE and Coomassie
Blue staining was performed on enzyme autophosphorylated in standard
reaction conditions, with the exception of a decrease in reaction
volume (50 µl) and an increase in the amount of enzyme (2.0
µg). Complete reactions were terminated at 0 and 5 min with EDTA
(100 mM final). Centrifugation and processing of
autophosphorylated enzyme was performed as described in Fig. 2through SDS-PAGE. The gel was processed as described under
``Experimental Procedures.'' Arrows indicate the 50-
and 60-kDa subunits of CaM-kinase. Supernatant (S) and pellet
fractions (P) fractions are indicated. Positions of molecular
mass standards are indicated on the right.
Throughout this study we have used immunostaining to
identify the soluble and sedimentable enzyme. Fig. 7C demonstrates the Coomassie Blue staining pattern of enzyme
autophosphorylated in standard conditions for 0 or 5 min. The majority
of the non-CaM-kinase-staining protein remains in the supernatant after
5 min of autophosphorylation. The 50- and 60-kDa subunits of CaM-kinase
were the major constituents of the sedimentable protein following
autophosphorylation. These data indicate that the formation of enzyme
complexes is due primarily to interactions between soluble holenzymes,
presumably through self-association; however, a minor constituent of
the sedimentable protein was detected at approximately 75 kDa, and it
remains possible that proteins that co-purify with CaM-kinase could
influence the formation and/or associate with sedimentable enzyme. We
feel that this is an unlikely possibility because the formation of
sedimentable enzyme during autophosphorylation also was observed using
a recombinant preparation of the alpha subunit of CaM-kinase that is
greater than 95% pure by Coomassie staining of SDS-PAGE analyzed
protein. ()
In this study we observed that the conditions of
autophosphorylation influence both the formation of sedimentable
CaM-kinase and a time-dependent loss of substrate phosphorylation.
Autophosphorylation at 4 °C or in Mn at 30 °C
did not result in a significant loss of enzyme activity (less than 10%)
nor in the formation of sedimentable enzyme, indicating that limited
autophosphorylation either prevents or is not sufficient for these
changes. Lou and Schulman demonstrated that these conditions restrict
the sites of autophosphorylation and maintain the autonomous form of
the enzyme without its inactivation(27) . Furthermore, the
substitution of Mn
for Mg
was
reported to increase the affinity of the kinase for ATP(27) .
The K
for Mg
/ATP is reported to
be within the range of 1.6-20 µM for both substrate
phosphorylation and
autophosphorylation(4, 15, 18) . The low ATP
concentration used in this study, 10 µM, is within the
range of the calculated K
for ATP and, therefore,
may not be saturating. The potential consequence or role of an
unoccupied ATP binding site on the inactivation and formation of
sedimentable CaM-kinase is currently under investigation.
We
observed that ATP concentration influenced both enzyme inactivation and
the formation of sedimentable enzyme. The inactivation associated with
autophosphorylation in low ATP (10 µM) was further
influenced by pH, with maximal inactivation and formation of
sedimentable enzyme occurring at pH 6.5, whereas autophosphorylation in
high ATP (1 mM) prevented these changes. The stoichiometry of
autophosphorylation was comparable between low and high ATP conditions,
indicating that formation of sedimentable enzyme and/or inactivation
were not due to the extent of PO
incorporation. Lou et al. (22) also reported a
similar ATP sensitivity to the inactivation of CaM-kinase associated
with autophosphorylation and similar stoichiometries of
autophosphorylation in high (500 µm) and low (5 µm) ATP. These
authors also reported that tryptic mapping of CaM-kinase
autophosphorylated in low ATP produced unique phosphopeptides compared
to high ATP(22) . In addition, Hanson et al. (9) reported that Thr-253 on CaM-kinase was autophosphorylated
preferentially at low ATP. Site specific autophosphorylation-associated
alterations in the properties of CaM-kinase are complex and well
documented (e.g. Thr-286 phosphorylation and autonomous
activity); however, it is currently unknown whether the differential
autophosphorylation associated with low ATP induces the inactivation
and/or formation of sedimentable enzyme or is merely a consequence of
these processes.
Inactivation and the formation of sedimentable enzyme during autophosphorylation in low ATP were influenced by pH. Autophosphorylation at pH 6.5 in low ATP produced robust inactivation and formation of sedimentable enzyme. Autophosphorylation at pH 7.5 was the only condition observed that produced enzyme inactivation, yet was not accompanied by the formation of sedimentable enzyme, indicating that inactivation and the formation of sedimentable enzyme may occur by distinct processes. Enzyme inactivation could proceed at elevated pH, yet potential protein-protein associations necessary for the formation of sedimentable enzyme may be unstable or may not form. A potential limitation of this interpretation is that smaller complexes may still form during autophosphorylation at pH 7.5 but are simply not detected by the sedimentation criteria used in this study. Interestingly, we also observed that increased pH slowed the generation of maximal independent-activity in low ATP. Maximal independent activity at higher pH values (pH 7.5) required increasing the time period of preautophosphorylation on ice.
Lai et al. (21) attributed the loss of enzyme activity associated with
CaM-kinase autophosphorylation to increased thermal lability. However,
consistent with previous data(22, 27) , we observed
that both autophosphorylation in low ATP with Mn and
in high ATP with Mg
prevented the inactivation of
CaM-kinase associated with autophosphorylation at 30 °C, indicating
that autophosphorylated enzyme is differentially susceptible to thermal
inactivation. The time course of inactivation following
autophosphorylation in low ATP at pH 6.5 was correlated temporally with
the incorporation of
PO
into the enzyme (see Fig. 4). Interpreting the role of autophosphorylation in the
inactivation of CaM-kinase is complicated by a loss of enzyme activity
in the presence of Ca
/CaM and in the absence of
autophosphorylation (see Fig. 5A). This process is
temperature-sensitive and also is attributed to thermal
lability(21, 23) . Ishida et al. (35) recently reported that this mode of inactivation may be
produced via Ca
/CaM interaction with a low affinity
CaM-binding domain. The relationship of this
autophosphorylation-independent inactivation to the inactivation
observed during autophosphorylation is unclear. Determining the role of
autophosphorylation in enzyme inactivation is further complicated by
potential alterations in the structure of CaM-kinase associated with
the formation of sedimentable enzyme. Stearic constraints due to enzyme
aggregation may also contribute to the loss of substrate
phosphorylation observed.
Interestingly, the enzyme complexes formed during autophosphorylation of purified CaM-kinase in our study (Fig. 7B) appeared similar in ultrastructure to preparations of isolated cytoskeletal complexes enriched in CaM-kinase previously described(36) . The formation of the enzyme complexes in our study is apparently due to the self-association of soluble CaM-kinase holoenzymes. The results of our study were consistent with multiple CaM-kinase preparations, with enzyme purities ranging from 70-90% of the total protein, and although potential interactions and associations with proteins that co-purify with CaM-kinase cannot be excluded completely, they do not significantly co-sediment with sedimentable enzyme (Fig. 7C). Whether CaM-kinase self-association has any role in the formation of cytoskeletal specializations such as postsynaptic densities or the cytoskeletal complexes described by Sahyoun et al. (36) is unknown.
The enzyme complexes shown in the micrographs (Fig. 7, A and B) are representative of fields throughout the entire grid and were observed only in autophosphorylation conditions that produced sedimentable enzyme. However, what percentage of the sedimentable enzyme has adopted this macromolecular structure is difficult to ascertain. As suggested by the reviewer, we attempted to quantify these structures by modeling the volume for both holoenzymes and the enzyme complexes and determining the theoretical number of holoenzymes required to construct the enzyme complexes observed in Fig. 7. Based on the assumptions and analysis provided under ``Appendix,'' we estimated that 7% of the holoenzymes in the reaction were recovered as enzyme complexes. Of the many assumptions required to complete the analysis, the 7% estimation was highly dependent upon the assumed volume of the holoenzyme. This includes the values chosen for determining the volume of holoenzyme as well as potential alterations in the holoenzyme's volume associated with formation of enzyme complexes (i.e. packing and compression due to aggregation). For example, if the packing of the holoenzyme causes a decrease in their apparent diameter from 30 to 20 nm, the enzyme recovered increased to 15%, whereas an additional decrease in the diameter to 10 nm produces a recovery of over 60%. One interpretation from our calculations is that the formation of the enzyme complexes could be associated with significant packing and condensation of the holoenzymes, and structural alterations of the enzyme over time could represent an important contribution to the time-dependent losses in the enzyme's activity.
Although the role of autophosphorylation in
the formation of sedimentable enzyme is unknown, this phenomena was not
observed in the absence of autophosphorylation in the reaction
conditions of this study. However, in the absence of 10% glycerol, we
observed that 20-30% of the enzyme sedimented when incubated at
30 °C in the presence of Ca/CaM at pH 6.5 for 5
or 10 min (data not shown). The formation of sedimentable enzyme in the
absence of autophosphorylation was not as rapid nor as complete as the
formation of sedimentable enzyme observed with autophosphorylation, and
was not detected in the standard assay conditions containing 10%
glycerol. Glycerol and ethylene glycol are used routinely to stabilize
purified CaM-kinase and one possibility suggested from these data is
that these agents decrease enzyme aggregation.
CaM-kinase is isolated from both cytosolic and particulate fractions (36, 37, 38, 39) . How CaM-kinase interacts or associates within these subcellular compartments, and whether the enzyme's distribution is regulated is unknown. The changes in the sedimentability of purified CaM-kinase observed during conditional autophosphorylation in this study may reflect conformational changes in the enzyme that enable or promote protein interactions with itself and/or other proteins. Subcellular translocation of particulate to soluble CaM-kinase during autophosphorylation has been reported in the Aplysia ganglia (6) , and the redistribution of the cytosolic enzyme into the particulate fraction has been reported during neuronal activation (40, 41) and following epileptic (42) and ischemic conditions(25, 26, 43) . Cerebral ischemia is one well documented example in which dramatic and rapid fluctuations occur to both pH and ATP. Silver and Erecinska (44) demonstrated that, within minutes, an ischemic insult altered the intracellular pH in hippocampal neurons from approximately pH 7.4 to pH 6.2. Folbergrova et al. (45) reported that the physiological ATP concentration is approximately 2.8 mmol/kg of cortical tissue, and that during ischemic insult, ATP stores are virtually depleted within minutes. It is currently unknown whether autophosphorylation plays a role in the inactivation and redistribution of soluble CaM-kinase into the particulate fraction during ischemia.
Our in vitro data predict that both a decrease in pH and ATP concentration would result in alterations in the physical and catalytic properties of CaM-kinase analogous to the changes observed in vivo and in situ during an ischemic insult(25, 26, 43) . Our in vitro data further predict that activation of cellular CaM-kinase in conditions of neutral pH and high ATP would result in the production of a predominantly soluble enzyme with little corresponding inactivation. In contrast, the activation of CaM-kinase in conditions of neutral pH and low ATP ultimately would result in the production of a predominantly inactive soluble enzyme. The data presented in this report suggest that different intracellular environments will modify the functional output of CaM-kinase through autophosphorylation-associated changes in the activity and/or association of the enzyme with itself and potentially other proteins. The reversibility of autophosphorylation-associated inactivation and/or self-association is unknown. Bidirectional regulation is a hallmark of physiological regulatory mechanisms and the potential to yield active, soluble enzyme from sedimentable enzyme possibly through dephosphorylation and/or other conditions warrants future investigation.
In an attempt to estimate the number of holoenzymes recovered as enzyme complexes (see Fig. 7, A and B), the micrographs were digitized and the electron dense particles quantified using Image software (version 1.44, Wayne Rasband at NIMH). The steps in analysis are given in Table 1. Several assumptions were required to complete the calculations.