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Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2506-2513
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
In Situ Ca Dependence for Activation of
Ca/Calmodulin- dependent Protein Kinase II in
Vascular Smooth Muscle Cells (*)
(Received for publication, May 11,
1995; and in revised form, October 6, 1995)
S. Thomas
Abraham
,
Holly
Benscoter
,
Charles M.
Schworer
,
Harold A.
Singer (§)
From the Sigfried and Janet Weis Center for Research,
Geisinger Clinic, Danville, Pennsylvania 17822
 ABSTRACT
- INTRODUCTION
- EXPERIMENTAL PROCEDURES
- RESULTS
- DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
Activation of Ca /calmodulin (CaM)-dependent
protein kinase II (CaM kinase II) and development of the
Ca/CaM-independent (autonomous) form of the kinase
was investigated in cultured vascular smooth muscle (VSM) cells. Within
15 s of ionomycin (1 µM) exposure 52.7 ± 4.4% of
the kinase became autonomous, a response that was partially maintained
for at least 10 min. This correlated with  P
phosphorylation of CaM kinase II  -subunits in situ and
was abolished by pretreatment with the CaM kinase II inhibitor KN-93.
The in situ Ca dependence for generating
autonomous CaM kinase II was determined in cells selectively
permeabilized to Ca and depleted of sarcoplasmic
reticulum Ca by pretreatment with thapsigargin.
Analysis of the resulting curve revealed an EC (concentration producing 50% of maximal response) of 692 ±
28 nM [Ca] , a
maximum of 68 ± 2% of the total activity becoming autonomous
reflecting nearly complete activation of CaM kinase II and a Hill slope
of 3, indicating a highly cooperative process. Based on this dependence
and measured [Ca] responses in intact cells, increases in autonomous
activity stimulated by angiotensin II, vasopressin and platelet-derived
growth factor-BB (4.6-, 2-, and 1.7-fold, respectively) were
unexpectedly high. In intact cells stimulated by ionomycin, the
correlation between autonomous activity and
[Ca] resulted in a
parallel curve with an EC of 304 ± 23 nM [Ca]. This
apparent increase in Ca sensitivity for generating
autonomous activity in intact VSM cells was eliminated by thapsigargin
pretreatment. We conclude that alteration of
[Ca] over a
physiological range activates CaM kinase II in VSM and that this
process is facilitated by release of Ca from
intracellular pools which initiates cooperative autophosphorylation and
consequent generation of autonomous CaM kinase II activity.
INTRODUCTION
Mobilization of intracellular free calcium
([Ca]) ( )following agonist-stimulated phospholipid hydrolysis is a
major signal transduction pathway in vascular smooth muscle (VSM)
cells(1) . [Ca] controls contractile activity in smooth muscle (2) as well as signaling pathways related to cell growth and
differentiation (3, 4) and
replication(5, 6) . Calcium-mediated activation of a
number of cellular enzymes requires the ubiquitous calcium-binding
protein, calmodulin (CaM)(7) . One such enzyme is
Ca/CaM-dependent protein kinase II (CaM kinase II),
which is expressed in high amounts in the brain and in lesser amounts
in most peripheral tissues(8, 9) . CaM kinase II has
been implicated in diverse Ca-mediated processes,
including neurotransmitter production and
release(10, 11) , regulation of smooth muscle myosin
light chain kinase activity(12) , VSM cell
migration(13) , oocyte fertilization(14) , and gene
expression(15, 16) . CaM kinase II is a large
multimer ( 600 kDa) composed of 8-10 individual kinase
subunits of 54-60 kDa size. Four distinct kinase subunits ( ,
 , ,  ) and a number of variants arising by alternative
splicing have been cloned to
date(9, 17, 18) . A distinguishing feature of
CaM kinase II is that it undergoes autophosphorylation in the presence
of Ca/CaM on a specific conserved threonine residue
(Thr in the -subunit) which results in the
generation of Ca/CaM-independent (or
``autonomous'') kinase activity (9) . 70-80% of
the total Ca/CaM-dependent kinase activity may become
autonomous in vitro under optimal autophosphorylation
conditions(19) . Autophosphorylation on Thr has
also been reported to result in a 1000-fold increase in the affinity of
the kinase subunits for calmodulin(20) . Thr autophosphorylation is predicted to be cooperative, since it has
been shown to occur by an intersubunit intraholoenzyme reaction which
requires that both the phosphorylating subunit and the substrate
subunit have bound Ca/CaM(21) . Autophosphorylation-dependent generation of autonomous activity and
``calmodulin trapping'' could result in CaM kinase II
activity which in vivo would outlast transient increases in
[Ca] and enable the
kinase to respond in a frequency-dependent manner to repetitive
transient increases in
[Ca](20, 21) .
However, previous in vitro studies indicated that at
saturating calmodulin concentrations, CaM kinase II activation and
phosphorylation of exogenous substrates required a relatively high
concentration of free Ca (K > 700 nM)(22, 23) . Given that:
1) inactive CaM kinase II has a low affinity for calmodulin, 2) CaM
kinase II autophosphorylation is predicted to be a cooperative process
requiring at least two activated subunits per holoenzyme, and 3) intact
cells have phosphatase activities that are capable of reversing
autophosphorylation; activation of CaM kinase II in vivo and
subsequent autophosphorylation with generation of autonomous activity
may be relatively insensitive to gradual or sustained small increases
in activator Ca which would be expected in response
to many physiological stimuli. CaM kinase II is present in cultured
rat aortic VSM cells (about th the activity found in comparable brain
extracts) and is comprised mainly of the  -subunit
variant(18) . In the present study, we assessed the ability of
Ca-mobilizing stimuli to activate CaM kinase II in
VSM, resulting in the autophosphorylation-dependent generation of the
autonomous form of the kinase. The Ca dependence for
generating autonomous CaM kinase II activity was determined in cells
that were made selectively permeable to Ca. Despite a
relatively low sensitivity for [Ca] in the
development of autonomous CaM kinase II activity under these conditions
(EC (concentration producing 50% of maximal response)
= 692 nM), all of the Ca-mobilizing
agents tested were able to significantly stimulate the development of
autonomous CaM kinase II activity in intact cells indicating in situ activation of CaM kinase II. Our findings further
suggest an important role for intracellular pools of Ca in the activation of CaM kinase II and the subsequent initiation
of cooperative autophosphorylation and generation of autonomous
activity.
EXPERIMENTAL PROCEDURES
MaterialsAngiotensin II and arginine
vasopressin were obtained from Bachem California (Torrance, CA).
Ionomycin, thapsigargin, and Fura-2/AM were from Calbiochem (La Jolla,
CA). KN-93 was purchased from Seikagaku America (Rockville, MD);
[-P]adenosine triphosphate,
[P]phosphoric acid, and  I-calmodulin were from DuPont NEN. All other materials
were from standard commercial sources.
Cell CultureVSM cells were dispersed from the
medial layer of the thoracic aorta of Sprague-Dawley rats
(150-250 g) using collagenase and elastase (Worthington)
according to the method of Geisterfer et al.(24) . The
cells were grown in an equal mixture of Dulbecco's modified
Eagle's medium and Ham's F-12 medium containing
antibiotic/antimycotic (Life Technologies, Inc.) and 10% fetal calf
serum (Hyclone, Logan, UT) under standard culture conditions. Cells
were subcultured weekly into 60- and 100-mm dishes and
grown to confluence before use in the experiments. VSM cells from the
third to tenth passage were used in this study.
Kinase AssayCells were stimulated with various
Ca-mobilizing agents at 37 °C in HEPES-buffered
Hanks' balanced salt solution (HBSS, pH 7.4) while still attached
to the culture dish. At appropriate times the reaction was stopped by
aspiration of the media and addition of ice-cold lysis buffer
containing an equal mixture of
Ca,Mg-free HBSS (Life Technologies,
Inc.) and Buffer A. Buffer A was composed of 50 mM MOPS (pH
7.4), 2 mM EGTA, 100 mM sodium fluoride, 100 mM sodium pyrophosphate, 2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.4 unit/ml aprotinin and 1%
Nonidet P-40. After 5-10 min on ice, crude lysates were aspirated
and centrifuged at 17,000  g for 10 min. The resulting
supernatant was kept on ice until the kinase assay was performed.
Protein content in the lysates was determined using the Bio-Rad Protein
reagent with bovine serum albumin as the standard. Total CaM kinase II
activity in the lysate was assayed in 25 µl containing 10 mM MOPS (pH 7.4), 10 mM magnesium chloride, 3 mM EGTA, 4 mM calcium chloride, 400 nM calmodulin,
0.2 mM [-P]ATP (400-1000
cpm/pmol), 20 µM autocamtide-2 (KKALRRQETVDAL, (19) ) as substrate, and 0.5-2.5 µg of lysate
protein. To determine the Ca/CaM-independent
(autonomous) activity in the same lysates CaCl and CaM were
omitted from the kinase assay mixture. The reactions were carried out
at 30 °C in a shaking incubator for 3 min and terminated by
precipitation of the phosphorylated peptide on Whatman P-81 paper
(Whatman). The papers were rinsed thoroughly in 75 mM phosphoric acid, and the adherent radioactivity was quantified in
ReadySafe LS mixture (Amersham Corp.) by liquid scintillation counting.
Kinase activity was expressed as nanomoles of P transferred
to the substrate/min/mg lysate protein. Autonomous
(Ca/CaM-independent) kinase activity was expressed as
a percent of total Ca/CaM-dependent activity from the
same samples.
[P]PO Labeling and Immunoprecipitation of CaM Kinase
IICellular ATP pools were labeled for 16 h by addition of
100-200 µCi/ml [P]orthophosphoric acid
in phosphate-free medium under standard culture conditions. Cells were
equilibrated at 37 °C in HEPES-buffered HBSS for 5 min before
stimulation with 1 µM ionomycin for up to 10 min, at which
time the medium was aspirated and cells were disrupted with ice-cold
lysis buffer. Equivalent amounts of lysate protein were incubated with
3 µl/ml CK2-DELTA antiserum precomplexed to protein A-agarose beads (18) for 2 h at 4 °C unless indicated otherwise.
Bead-immune complexes were washed twice with 500 µl of lysis buffer
and solubilized in SDS sample buffer. Proteins were resolved
electrophoretically on 8% SDS-polyacrylamide gels, transferred
electrophoretically onto Immobilon membranes (Millipore), and
visualized by autoradiography.
I-CaM
OverlayImmunoprecipitated lysate proteins were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to Immobilon
membranes as indicated above. Membranes were incubated for 1 h with I-CaM (1 µCi/5 ml) at room temperature in a buffer
containing 50 mM Tris (pH 7.4), 150 mM NaCl
(Tris-buffered saline), 1 mM CaCl and 5% non-fat
dry milk. Following thorough rinsing of membranes with
Ca-containing Tris-buffered saline,
calmodulin-binding proteins in immunoprecipitated samples were
identified by exposing membranes to x-ray film for up to 72 h. Control
overlay procedures were performed under identical conditions except
that 1 mM CaCl was replaced with 1 mM EGTA.
[Ca] Measurements Using Fura-2VSM cells were exposed to
Fura-2/AM for 1 h at 37 °C in HBSS containing 1 mg/ml bovine serum
albumin and 2 mg/ml dextrose to load cells with the dye. The cells were
washed with HBSS to remove unincorporated Fura-2, suspended in HBSS
using 0.25 mg/ml trypsin (Life Technologies, Inc.), and then washed
once with HBSS containing 1 mg/ml soybean trypsin inhibitor.
Intracellular Fura-2 fluorescence was monitored in suspended cells
maintained at 37 °C using a SPEX DM3000 Fluorolog spectrometer
(SPEX Industries, Camden, NJ). Free
[Ca] was calculated according
to the method of Grynkiewicz et al.(25) using 15
mM digitonin and 15 mM EGTA to obtain the maximal and
minimal intracellular fluorescence values, respectively.
Intracellular CaDependence for
Generating Autonomous CaM Kinase IITo study the
relationship between cytoplasmic [Ca] and the activation of CaM kinase II, cells loaded with Fura-2
were suspended in Ca-deficient HBSS containing 1
mM EGTA and then depleted of intracellular Ca by treating with 1 µM thapsigargin and 1 µM ionomycin for 10 min. Cumulative additions of 0.01-5 mM CaCl were used to increase
[Ca] in a stepped manner and at
each steady-state a 40 µl aliquot of cell suspension was quickly
removed from the cuvette and disrupted in an equal volume of lysis
buffer (4 °C). Cell lysates were processed, and total and
Ca/CaM-independent kinase activities were determined
as indicated above. Measured [Ca] was plotted against the Ca/CaM-independent
activity (expressed as a percent of total
Ca/CaM-dependent activity) to construct an
intracellular Ca dependence curve for in situ activation and generation of the autonomous form of the kinase. In a separate protocol, Fura-2-loaded cells were suspended in normal
HBSS (1.8 mM CaCl), and
[Ca] was elevated using single,
noncumulative ionomycin additions (0.01-10 µM)
followed by rapid sampling of the cell suspension at various time
points and kinase assay. In a third protocol, intracellular pools of
calcium were first depleted with 10-min treatment of 1 µM thapsigargin and then cells were challenged with different
concentrations of ionomycin before cell lysis and kinase assay. Total
and Ca/CaM-independent kinase activity was then
plotted against measured [Ca] to produce in situ Ca dependence
curves for generation of autonomous CaM kinase II activity in cells
with or without intact intracellular Ca pools.
Data AnalysisGrouped data represent the mean
± S.E. of three or more experiments, and statistical comparisons
between means were performed using one-way analysis of variance
followed by Bonferroni's post hoc test using GraphPad
Instat software (San Diego, CA). The Ca dependence
plots were subjected to nonlinear regression analyses using GraphPad
Prism software (San Diego, CA). Statistical comparisons among curves
were performed according to Motulsky and Ransnas,(26) .
Briefly, two curve fits were considered statistically different if they
described the data better than a single curve fitted to the pooled data
from both sets. This was achieved by carrying out an F-test to compare
the variance of the pooled data set with the sum of the variance from
the individual data sets. In all comparisons p < 0.05 was
taken to indicate statistical significance.
RESULTS
CaM Kinase II Activation and Autophosphorylation in
SituThe synthetic peptide autocamtide-2 has been reported to be
a relatively specific substrate for CaM kinase II compared with other
multifunctional kinases such as protein kinase C and cAMP-dependent
protein kinase(19) . In representative experiments, total
Ca/CaM-dependent autocamtide-2 kinase activity in
cell lysates from unstimulated VSM cells was found to be 8.81 ±
0.74 nmol/min/mg protein, while Ca/CaM-independent
activity in the same samples was 0.87 ± 0.14 nmol/min/mg of
protein (n = 10). A hallmark of CaM kinase II is
activation-dependent autophosphorylation leading to development of
Ca/CaM- independent or autonomous kinase
activity(9) . Exposure of intact VSM cells to the
Ca ionophore ionomycin (1 µM) resulted
in a rapid increase in the level of
Ca/CaM-independent autocamtide-2 kinase activity
assayed in vitro, from 11.4 ± 1.9% of the total
(Ca/CaM-dependent) kinase activity at rest to a peak
value (15 s) of 52.7 ± 4.4% of total activity, a response which
was partly sustained for the duration of the 10 min exposure to the
ionophore (Fig. 1A).
Figure 1:
A, time course for the activation of
CaM kinase II by 1 µM ionomycin. Each point is the mean
± S.E. of five independent determinations. B, time
course of CaM kinase II phosphorylation in cells labeled with
[P]PO, exposed to ionomycin and
immunoprecipitated with CK2-DELTA antibody. Radiolabeled proteins were
separated by 8% SDS-polyacrylamide gel electrophoresis, and molecular
mass marker positions are indicated to the left. The 52-kDa position
marks the major CaM kinase II subunit immunoprecipitated in VSM cells.
``Overlay'' lane indicates cell proteins that were
immunoprecipitated with CK2-DELTA antibody, separated by
electrophoresis and identified by an I-CaM overlay
technique (see ``Experimental
Procedures'').
Experiments were carried out to
determine if these increases in Ca/CaM-independent
autocamtide-2 kinase activity correlated with phosphorylation of CaM
kinase II subunits and to confirm that the activity measured was in
fact due to CaM kinase II. Ionomycin stimulation of cells with P-labeled ATP pools resulted in incorporation of P into a 52-53-kDa protein which was isolated from
the lysates by immunoprecipitation with an antipeptide antibody
(CK2-DELTA) specific for the carboxyl terminus of the CaM kinase II
-subunit (Fig. 1B). The CK2-DELTA and another CaM
kinase II subunit nonselective antibody have previously been shown to
detect a band of this size on immunoblots of rat aortic VSM
fractions(18) . In the present study, the identity of this band
as a CaM kinase II subunit was confirmed by overlay with I-calmodulin (Fig. 1B, rightmost lane)
and by immunoblotting with a different subunit nonselective anti-CaM
kinase II antibody (obtained from K. Smith and R. Colbran, Vanderbilt
University; data not shown). Phosphorylation of CaM kinase II subunits
was maximal within 15-30 s of ionomycin addition, corresponding
with peak increases in Ca/CaM-independent kinase
activity, and persisted for the duration of the stimulation (Fig. 1B). In addition to the 53-kDa band a high
molecular weight protein (>140 kDa), and proteins with estimated
sizes of 56 and 60 kDa in the immunoprecipitates were also
phosphorylated. The 56- and 60-kDa bands may be minor CaM kinase II
subunits, since corresponding bands could be detected with longer
exposure of the I-CaM overlay. Immunoprecipitation
with CK2-DELTA depleted Ca/CaM-dependent and
-independent autocamtide-2 activity by 80-90% from lysates of
ionomycin-stimulated cells, confirming the specificity of the
autocamtide-2 substrate based assay for CaM kinase II and indicating
that the stimulated Ca/CaM-independent activity was
due to autonomous CaM kinase II (Fig. 2). While
Ca/CaM-independent autocamtide-2 kinase activity was
low in resting cells, the CK2-DELTA antibody also immunoprecipitated
60% of this activity, consistent with a small amount of CaM kinase II
autophosphorylation which was observed in unstimulated cells (Fig. 1B). The residual
Ca/CaM-dependent activity remaining following
immunoprecipitation could be due to other kinases such as CaM kinase IV
which do not undergo autophosphorylation-dependent transition to a
Ca/CaM-independent form(27) . Further
confirmation that Ca/CaM-independent autocamtide-2
kinase activity in the VSM cell lysates was due to autonomous CaM
kinase II was obtained by pretreating the cells with KN-93, a CaM
kinase II inhibitor which interferes with calmodulin binding to the
kinase subunit(10) . In the experiments shown in Fig. 3,
stimulation of VSM cells for 15 s with ionomycin (1 µM)
resulted in a 7.9-fold increase in Ca/CaM-independent
activity without altering total CaM kinase II activity in cells.
Pretreatment of cells with 30 µM KN-93 completely
prevented the ionomycin-induced increase in
Ca/CaM-independent kinase activity. Based on the
experiments shown in Fig. 1Fig. 2Fig. 3we
concluded that autocamtide-2 kinase activity was indicative of CaM
kinase II in the VSM cell lysates and that
Ca/CaM-independent kinase activity was due to the
autophosphorylated or autonomous form of the kinase.
Figure 2:
Immunodepletion of autocamtide-2 kinase
activity from VSM cell lysates with the CK2-DELTA CaM kinase II
antibody. Ca/CaM-independent kinase activity (Indep. Activity) was measured in lysates from unstimulated
cells (Rest; n = 3) and cells stimulated with
ionomycin (1 µM for 15 s; Iono; n = 3). Ca/CaM-dependent kinase activity (Total Activity; n = 6) was measured in
lysates from Rest and Iono-stimulated cells. Lysates
were divided and incubated with preimmune serum (left panel)
or CK2-DELTA antiserum (right panel) for 8 h at 4 °C prior
to clearing of the immune complexes with protein A-agarose beads.
Values are mean ± S.E.
Figure 3:
Effect of KN-93 on CaM kinase II activity
from ionomycin-stimulated VSM cells. Cells were treated with vehicle or
30 µM KN-93 for 1 h before exposure to 1 µM ionomycin for 15 s. Open and closed bars indicate mean ± S.E. (three experiments) of total and
Ca/CaM-independent kinase activity in cell lysates,
respectively.** indicates independent activity that is statistically
different from the independent activity in control cells (p < 0.001).
In Situ [Ca] Dependence for Generating Autonomous CaM Kinase
IIVascular smooth muscle cells were loaded with the fluorescent
Ca indicator Fura-2, depleted of Ca by incubating with thapsigargin (1 µM) media in
Ca-free HBSS containing 2 mM EGTA, and
selectively permeabilized to Ca by addition of
ionomycin (1 µM). Stepped increases in free
[Ca] were obtained by
cumulative additions of CaCl to the media (Fig. 4, inset) and quantified using the Fura-2 fluorescence. After
each step change in [Ca] a
40-µl aliquot of the cell suspension was removed (indicated by the vertical deflections in the tracing) and assayed for
Ca/CaM-dependent and -independent CaM kinase II
activity. The resulting data established the in situ Ca dependence for the generation of autonomous
CaM kinase II activity (Fig. 4). Nonlinear curve fitting to a
sigmoidal (logistic) function resulted in an EC of 692
± 28 nM [Ca] with 68 ± 2% of the total activity being autonomous at
maximal [Ca] (goodness of fit r = 0.915). The calculated Hill slope for
the curve was 3, consistent with a highly cooperative process for
autophosphorylation of the kinase subunits by an intraholoenzyme
intersubunit reaction(9, 28) . Minimum autonomous CaM
kinase II activity was 3 ± 1% of total activity at
[Ca] less than 100 nM.
The total kinase activity assayed in the presence of
Ca/CaM was not significantly altered at any
[Ca] achieved in the procedure.
Figure 4:
Autonomous CaM kinase II activity as a
function of free intracellular Ca
([Ca]) in VSM cells.
Fura-2 loaded cells were depleted of intracellular Ca and permeabilized with ionomycin before increasing
[Ca] by addition of
CaCl to the suspension (inset). After each
CaCl addition the cell suspension in the cuvette was
sampled (vertical deflections) and cell lysates were prepared and
assayed for kinase activity. The scatter plot of open triangles is from 12 independent experiments and the sigmoidal (logistic
function) curve was obtained by nonlinear regression analysis (r = 0.915, see ``Experimental
Procedures''). The dashed lines above and below the curve
mark the 95% confidence interval for the
curve.
Autonomous CaM Kinase II Activity Stimulated by
Physiological Agonists in VSM CellsSeveral physiological
stimuli were evaluated for their ability to increase
[Ca] and autonomous CaM kinase
II activity in intact VSM cells. The average resting
[Ca] in the intact cells was
determined to be 100-150 nM. Angiotensin II (0.1
µM) characteristically stimulated rapid transient
increases in [Ca] of about
200-300 nM as depicted in Fig. 5A.
Vasopressin (0.1 µM) also stimulated rapid increases in
[Ca] (Fig. 5B),
although in these cells the response was generally smaller than that
obtained with angiotensin II. Platelet-derived growth factor (PDGF-BB;
40 ng/ml) stimulated slower and relatively small increases in average
[Ca] (Fig. 5C),
whereas 1 µM ionomycin elicited a rapid somewhat transient
increase in [Ca] which was
partially sustained (Fig. 5D). By quantitatively
comparing these [Ca] responses
with the [Ca] dependence for generating
autonomous CaM kinase II shown in Fig. 4, it would not be
expected that resting levels of [Ca] would support significant autonomous CaM kinase II activity or
that addition of these receptor agonists would substantially increase
autonomous activity. In contrast to this prediction, resting levels of
autonomous activity in the intact VSM cells ranged from 4.4 to 12.5% of
total activity, and each stimulus significantly increased autonomous
CaM kinase II activity with kinetics which paralleled increases in
[Ca] (Fig. 5). In the
case of angiotensin II or vasopressin, transient increases in
autonomous CaM kinase II activity to 41 ± 4% (n = 5) and 22.4 ± 2.2% (n = 3) of
total activity, respectively, were reached within 15 s (Fig. 5, A and B). PDGF also increased autonomous CaM kinase
II activity, but at a slower rate and to a lesser extent (14.7 ±
1.1% autonomous activity at 60 s), as compared with angiotensin II and
vasopressin (Fig. 5C). Angiotensin II-induced increases
in autonomous CaM kinase II activity were concentration-dependent with
an estimated EC of 6 nM (data not shown), which
is similar to the reported dissociation constant of the agonist for its
receptor (29, 30) .
Figure 5:
Increases in free intracellular
Ca ([Ca])
and autonomous CaM kinase II activity (percent of total) in VSM cells
stimulated with 100 nM angiotensin II (AII) (A), 100 nM vasopressin (AVP) (B),
40 ng/ml PDGF-BB (C), and 1 µM ionomycin (IONO) (D). Arrows indicate application of
the stimulatory agent.
Role of Intracellular CaPools in
the Generation of Autonomous ActivityIncreasing
[Ca] by stimulating intact
Fura-2-loaded VSM cells with various concentrations of ionomycin (10
nM to 10 µM) in normal
Ca-containing HBSS (Fig. 6A, inset)
resulted in a second apparent Ca dependence for the
generation of autonomous CaM kinase II activity in situ (Fig. 6A, open circles, n = 8). Curve
fitting analysis predicted a sigmoidal relationship for
Ca-induced activation with an EC of 304
± 23 nM and 67 ± 5% of the kinase becoming
autonomous at saturating [Ca] (goodness of fit r = 0.849). Analysis
of variance predicted that the EC obtained with this
protocol was statistically different from that of the curve in Fig. 3(F = 60.600, p < 0.001). The apparent Ca dependence obtained
with this protocol was quantitatively consistent with the increases in
[Ca] and autonomous CaM kinase
II activity observed in intact VSM cells in response to addition of the
physiological stimuli shown in Fig. 5.
Figure 6:
A, correlation between autonomous CaM
kinase II activity and free intracellular Ca ([Ca]) in Fura-2
loaded VSM cells with intact Ca pools and stimulated
with ionomycin. Inset: representative fluorescence output from
Fura-2-loaded cells in normal Ca medium and treated
with a single concentration of ionomycin to elevate
[Ca], during which
time the cell suspension was sampled (deflections in the Ca tracing) for kinase assay. The scatter plot of open circles is the resulting autonomous activity at measured
[Ca] from 8
experiments (r = 0.849) with the 95%
confidence interval indicated by the dotted lines. The dashed curve is of the data from Fig. 3, which is
included to facilitate comparison, and the dotted lines are
95% confidence intervals for the curves. B, the same
experiment was repeated in cells pretreated with 1 µM thapsigargin (to deplete intracellular Ca pools)
for 10 min prior to stimulation with ionomycin. The dotted lines are the 95% confidence interval for the curve, and the dashed
curve is of the open circles from A above. The
data are from eight independent experiments and goodness of fit r = 0.940 for the
curve.
In order to explain
the apparent higher Ca sensitivity for generating
autonomous CaM kinase II in intact VSM cells, we considered the
possibility that average [Ca] calculated using the Fura-2 technique did not reflect locally
high [Ca], produced in response
to ionomycin and peptide agonists that release intracellular pools of
Ca(3, 30, 31) , which
could support the cooperative autophosphorylation of CaM kinase II. To
test this hypothesis, intact cells in normal Ca HBSS
were pretreated with 1 µM thapsigargin to deplete
intracellular Ca pools and the protocol shown in Fig. 6A was repeated using ionomycin as a stimulus. The
resulting apparent Ca dependence (Fig. 6B; goodness of fit r = 0.940) was significantly to the right of the curve
obtained from VSM cells with intact intracellular pools of
Ca (EC = 616 ± 37
nM; F = 29.278, p < 0.001, n = 8), although the maximal increase
in autonomous activity, Hill slope, and the total
Ca/CaM-dependent kinase activity were not
significantly altered by thapsigargin treatment (Fig. 6B). The apparent
[Ca] dependence for generating
autonomous CaM kinase II activity described by this data set was not
significantly different from that obtained using the Ca step protocol shown in Fig. 4. A 10-min treatment of VSM
cells with 1 µM thapsigargin also abolished a 3-fold
increase in autonomous activity elicited by 0.1 µM angiotensin II (n = 3, data not shown). These
results are consistent with the contribution of intracellular
Ca pools in the activation of CaM kinase II and the
cooperative generation of autonomous kinase activity.
DISCUSSION
The present study was undertaken as a first approach toward
assessing the activation of CaM kinase II in intact VSM cells.
Ca/CaM-independent CaM kinase II activity (autonomous
activity) was measured in VSM cell lysates for two reasons: 1)
generation of autonomous activity provides an index of CaM kinase II
activation in the intact cell prior to lysis, and 2) the appearance of
autonomous CaM kinase II activity in situ has been
hypothesized to be of functional significance. Evidence that the
ionomycin-induced Ca/CaM-independent autocamtide-2
kinase activity was due to autophosphorylation-dependent generation of
autonomous CaM kinase II includes: 1) the activity was efficiently
immunoprecipitated with a CaM kinase II -subunit-specific
antibody; 2) the onset correlated temporally with increased in situP phosphorylation of CaM kinase II; and 3) formation
of autonomous activity was inhibited with KN-93, a reported CaM kinase
II inhibitor which acts by preventing Ca/CaM binding
to the subunit. While the ionomycin-induced increase in autonomous CaM
kinase II was somewhat transient with a peak at 15 s, phosphorylation
of CaM kinase II -subunits subunits was maximal within 15-30
s and sustained for the duration of the stimulus. This apparent
dissociation between subunit phosphorylation and autonomous activity at
later time points may be due to phosphorylation on additional
serine/threonine sites (9) accompanied by dephosphorylation of
Thr. The functional significance of these additional
phosphorylation events is largely unknown. It was previously
estimated that the free [Ca] for
half-maximal activation of CaM kinase II in vitro under
conditions of saturating CaM was relatively high, in the range of
700-2000 nM(22, 23) . Because generation of
autonomous CaM kinase II activity results from intersubunit
phosphorylation between activated (Ca/CaM
bound) subunits(9, 27) , a similar Ca dependence would be predicted for generation of autonomous
activity in vivo. Other factors in intact cells could act to
further decrease the apparent Ca sensitivity for CaM
kinase II activation and generation of autonomous activity, such as
limiting concentrations of free calmodulin and/or protein phosphatase
activities that are capable of reversing CaM kinase II
autophosphorylation. The calculated EC of 692 nM Ca in selectively permeabilized VSM cells is the
first direct estimation of the in situ Ca dependence for generating autonomous CaM kinase II activity.
Significantly, a positive Hill slope of 3 for the relationship
indicates that Ca-dependent generation of autonomous
CaM kinase II activity in situ is a highly cooperative
process, consistent with in vitro data indicating
cooperativity in the intersubunit intraholoenzyme autophosphorylation
reaction(21) . This value also provides an estimate of the
Ca sensitivity for activation of CaM kinase II, which
is a prerequisite for generating autonomous activity. Tansey et
al. (32) reported a similar half-maximal
[Ca] (500 nM) for
phosphorylation and desensitization of myosin light chain kinase in
permeabilized bovine tracheal smooth muscle cells, a process thought to
be mediated by CaM kinase II. Under optimal autophosphorylation
conditions in vitro, we have found that autonomous activity of
the CaM kinase II holoenzyme composed of recombinant
-subunits could reach as much as 70% of the total
Ca/CaM-dependent activity. ()Similar
maximal levels of independent activity have been observed by others
using various purified and recombinant forms of CaM kinase
II(19) . To our knowledge, it is not known to what extent the
difference in maximal autonomous activity compared with total activity
reflects the stoichiometry of holoenzyme autophosphorylation,
specifically on Thr in the subunits, and/or whether autophosphorylated kinase has a lower activity for
substrates than Ca/CaM-activated kinase. Therefore,
as a marker of Ca/CaM-dependent activation of CaM
kinase II in vivo, the level of autonomous activity may
actually underestimate the full extent of CaM kinase II activation.
Similar maximal levels of autonomous activity were also generated in
the selectively permeabilized VSM cells (Fig. 4), or transiently
in cells with intact Ca pools in response to
ionomycin stimulation (Fig. 1A and Fig. 6A), indicating a near complete activation of
kinase subunits in situ. This suggests that in VSM cells under
these conditions, free CaM is not a limiting factor and protein
phosphatases do not significantly antagonize the maximal development of
autonomous activity. Comparable increases in autonomous CaM kinase II
activity in situ were reported previously only in
KCl-depolarized PC12 cells(33) . In most other studies where
this approach has been used to assess CaM kinase II activation, small
increases in autonomous activity (2-fold or less) were reported in
response to stimuli which raise
[Ca](34, 35) ,
indicating either lower levels of CaM kinase II activation and/or
incomplete control of CaM kinase II subunit dephosphorylation during
cell lysis. Average resting VSM cell
[Ca] was found to be between
100 and 150 nM, and the physiological agonists angiotensin II,
vasopressin, and PDGF produced transient increases in
[Ca] to values which were
typically in a range of 200-400 nM. These results are
similar both qualitatively and quantitatively to previously published
data obtained in VSM cells(31) . Based on the quantitative
relationship between [Ca] and generation of
autonomous CaM kinase II activity (Fig. 4), these levels of
[Ca] should have produced
minimal autonomous CaM kinase II activity. However, a small amount of
Ca/CaM-independent activity was measurable in lysates
from unstimulated intact cells (about 3-12% of the total activity
of which about 60% was immunoprecipitated with the CaM kinase II
antibody), and all of the stimuli tested produced significant transient
increases in autonomous CaM kinase II activity, with angiotensin II
producing the largest increases, to as much as 40% of the total kinase
activity. A significant factor in interpreting these data was that
the in situ Ca dependence curve for
generating autonomous CaM kinase II resulted from experiments using
cells which were depleted of intracellular Ca and
which required Ca influx from the extracellular
medium to activate the kinase. In the case of experiments examining
agonist-stimulated increases in CaM kinase II activity, intracellular
pools of Ca were intact and the stimuli used
(angiotensin II, vasopressin, and PDGF) are known to mobilize
Ca from inositol trisphosphate-sensitive
intracellular pools(36, 37, 38) . Ionomycin
was useful in determining the effect of intracellular Ca pools on the apparent Ca dependence for
generating autonomous CaM kinase II activity, since it is able to
mobilize Ca from both intracellular and extracellular
pools to elevate
[Ca](3) . The
relationship between ionomycin-induced increases in
[Ca] and autonomous CaM kinase
II activity in cells with intact [Ca] pools predicted an apparent EC for
[Ca] of 304 nM for the
generation of autonomous kinase activity. This apparent Ca sensitivity was consistent with the physiological agonist-induced
increases in [Ca] and
autonomous kinase activity. Prior depletion of intracellular
Ca pools with thapsigargin markedly decreased the
apparent Ca sensitivity for ionomycin-induced
generation of autonomous CaM kinase II activity and blocked angiotensin
II-induced transients in [Ca] and generation of autonomous activity. At least three factors
could contribute to the observed differences in Ca sensitivities for generating autonomous CaM kinase II in cells
with or without intact pools of intracellular Ca. 1)
In the protocol using ionomycin-permeabilized and
Ca-depleted cells, Ca added to the
extracellular medium might be expected to equilibrate uniformly through
the cytoplasm allowing Fura-2 to faithfully report
[Ca]. This protocol should then
result in a reasonable estimation of the Ca sensitivity for activation of CaM kinase II and generation of
autonomous activity. In cells with intact intracellular Ca pools, locally high [Ca] resulting from sarcoplasmic reticulum release in response to
ionomycin or physiological stimuli may contribute to an average [Ca] reported by Fura-2.
Several studies have documented spatial inhomogeneities in
[Ca] using microscopic imaging
of single cell Ca transients(39, 40) . Models of Ca signaling in VSM, which take into account restricted diffusional
spaces underlying the plasma membrane and surrounding sarcoplasmic
reticulum, suggest that micromolar concentrations of
[Ca] could develop and persist
for short periods of time in these spaces in response to
Ca-mobilizing stimuli(41) . 2.) Assuming that
locally high concentrations of Ca are produced in
intact cells, a second factor contributing to the observed variable
Ca sensitivity is the cooperative nature of the
process for generating autonomous CaM kinase II activity. 3) CaM kinase
II itself could be localized in discrete subcellular regions in close
proximity to the sarcoplasmic reticulum and released
Ca. In this case, the effects of locally high
[Ca] and a cooperative process
for generating autonomous activity would be maximized by CaM kinase II
holoenzymes optimally positioned for activation in response to stimuli
that mobilize intracellular calcium. The fact that Fura-2 reports
average [Ca] levels in intact
cells and that inhomogeneities in
[Ca] could also exist in
unstimulated cells may partially explain the small amount of
Ca/CaM-independent autocamtide-2 kinase activity
which could be attributed to autonomous CaM kinase II in resting VSM
cells. These inhomogeneities in [Ca] could
exist within all cells such that a small fraction of the kinase within
each cell became active (reflected by the resting autonomous activity)
and/or within a small subset of ``leaky'' cells where
[Ca] is maximally elevated and
essentially all of the CaM kinase II is active and autophosphorylated. These arguments are supported indirectly by reports of localization
and/or association of CaM kinase II with the sarcoplasmic reticulum of
cardiac and skeletal muscle, where it is proposed to play a role in
regulation of cytosolic Ca by modulating
Ca-ATPase activity(42, 43) . In
contrast, MacNicol and Schulman (44) demonstrated greater
activation of CaM kinase II in PC12 cells by K -induced
depolarization, which produced influx of Ca, than by
ionomycin, which releases Ca from endoplasmic
reticulum pools, even though the two agents raised average
[Ca] to a similar extent. Thus,
unlike in VSM cells, CaM kinase II in PC12 cells appears to be more
sensitive to activation by Ca from extracellular
sources, which is consistent with a role for the enzyme in
neurotransmitter metabolism and secretion during depolarization-induced
Ca influx (10, 11) . Discrete
localization and activation of CaM kinase II in subcellular
compartments may also be a mechanism for regulating the function of
this enzyme which otherwise shows little substrate specificity in
vitro(8, 9) . The physiological significance
of the autonomous form of CaM kinase II is not known. Development of
the autonomous form of CaM kinase II has been proposed as a mechanism
by which activity of CaM kinase II could be maintained even after
[Ca] returns to resting
values(45) . However, in VSM cells the
Ca/CaM-independent activity did not appear to
significantly outlast the Ca transient generated by
physiological agonists, indicating that once activator Ca falls, phosphatases rapidly dephosphorylate and inactivate CaM
kinase II subunits. This doesn't negate the possible importance
of this mechanism in maintaining kinase activity and trapping
calmodulin on a shorter time scale such as might be encountered in
depolarizing nerve terminals(20) . Another possible role for
autonomous CaM kinase II is that it may phosphorylate specific
substrates(8) . In summary, this study demonstrates that
activation of CaM kinase II and generation of autonomous CaM kinase II
activity occurs in situ over a range of
[Ca] between 0.2 and 2 µM.
Generation of autonomous CaM kinase II activity in situ is
highly cooperative, a reflection of the unique structure of the CaM
kinase II holoenzyme and the requirement for intersubunit
autophosphorylation on Thr to generate autonomous
activity. Locally high concentrations of
[Ca] in intact VSM cells,
produced by agonists that release Ca from
intracellular pools, result in the cooperative generation of autonomous
CaM kinase II activity over a range of average cell
[Ca] of 0.1-1
µM.
FOOTNOTES
- *
- This work was supported by
National Institutes of Health Grant HL49426 (to H. A. S.) and American
Heart Association (Pennsylvania affiliate) Fellowship (to S. T. A.).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore by hereby marked
``advertisement'' in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
- §
- To whom correspondence should be addressed:
Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave.,
Danville, PA 17822-2612.
- ()
- The abbreviations used
are: [Ca], free
intracellular Ca concentration; CaM kinase II,
Ca/calmodulin-dependent protein kinase II; VSM,
vascular smooth muscle; HBSS, Hanks' balanced salt solution;
MOPS, 4-morpholinepropanesulfonic acid; PDGF, platelet-derived growth
factor.
- ()
- S. T. Abraham, H. Benscoter, C. M.
Schworer, and H. A. Singer, unpublished observations.
ACKNOWLEDGEMENTS
We thank Drs. D. Dostal and H. Morgan for critical
review of the manuscript and Drs. K. Smith and R. Colbran for providing
the nonselective CaM kinase II antibody. The expert cell culture work
of D. Cooney is also gratefully acknowledged.
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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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