From the Department of Physiology, Hamamatsu
University School of Medicine, 1-20-1 Handayama, Hamamatsu
431-3192, Japan, the ** Medical Research Council (MRC)
Secretory Control Research Group, Physiological Laboratory, University
of Liverpool, Liverpool L69 3BX, United Kingdom, the ¶ Department
of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma
University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan, and the
Laboratory of Molecular Pharmacology, Biosignal Research Center,
Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan
Received for publication, October 17, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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In electrically excitable cells, membrane
depolarization opens voltage-dependent
Ca2+ channels eliciting Ca2+
influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca2+ influx
alone can activate PKC. The present study was conducted to investigate
the Ca2+ influx-induced activation mechanisms for two
classes of PKC, conventional PKC (cPKC; PKC Since the first molecular cloning and sequencing of a bovine brain
protein kinase C (PKC)1 (1),
PKC has been one of the most extensively studied enzymes in eukaryotic
cells. We now know that PKC plays a pivotal role in a myriad of
cellular functions. Ten isoforms of PKC have been identified so far and
have been classified into three categories based on structural
differences in the regulatory domain: conventional PKC (cPKC; PKC We focused this study on PKC Recent advances in the use of green fluorescent protein (GFP) has
allowed us to investigate PKC activity in intact living cells by
monitoring translocation of GFP-tagged PKC (13, 14). Inactive PKCs are
located in the cytosol. Upon activation, following PIP2
hydrolysis, they translocate from the cytosol to other cellular locations, such as the plasma membrane. By simultaneously measuring the
cytosolic Ca2+ concentration and the translocation of
GFP-PKC To address these questions, we monitored translocation of PKC Plasmid Construction
PKC Cell Culture and Transfection
INS-1 cells (7), insulin-producing cells, were a gift from Dr.
Sekine (Tokyo University).The cells were grown in 100-mm culture dishes
at 37 °C and 5% CO2 in a humidified atmosphere. The
culture medium was RPMI 1640 with 10 mM glucose
supplemented with 10% fetal bovine serum, 1 mM sodium
pyruvate, and 50 µM mercaptoethanol. For fluorescence
imaging, the cells were cultivated on a coverslip at 50% confluency 2 days before transfection. A plasmid of the GFP- or DsRed-tagged
proteins was transfected into the cells by lipofection using
TransITTM-LT1 (Mirus, Madison, WI). The experiments were
performed 2 days after transient transfection.
Solutions
The standard extracellular solution contained 140 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 3 mM glucose, and 10 mM Hepes-NaOH (pH 7.3). The solutions for membrane
depolarization contained 105 mM NaCl, 40 mM
KCl, 1 mM MgCl2, 2.5 mM
CaCl2, 3 mM glucose, and 10 mM
Hepes-NaOH (pH 7.3) or contained 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 20 mM tetraethylammonium
chloride, 3 mM glucose, and 10 mM Hepes-NaOH
(pH 7.3). In some experiments, CaCl2 was not included
(Ca2+-free solution). The cells, placed on a glass
coverslip attached to an open perifusion chamber, were continuously
perifused from a gravity-fed system. The experiments were performed in
the standard extracellular solution at room temperature, unless
otherwise stated.
Materials
Fura2/AM, 12-O-tetradecanoylphorbol-13-acetate (TPA),
1,2-dioctyl-sn-glycerol (DiC8) was purchased
from Sigma. U73122 was obtained from Calbiochem (La Jolla, CA).
Imaging Experiments
Epifluorescence Microscopy--
The fluorescence images were
captured using a Olympus inverted microscope (60×, water immersion
objective, and 60×) equipped with a cooled ( TIRFM (Evanescent Wave)--
To obtain high signal-to-noise
ratio over the conventional epifluorescence microscopy (see
supplemental figure), we installed a TIRFM unit (Olympus) into the same
imaging system mentioned above. The incidental light was introduced
from the objective lens for TIRFM (Olympus NA = 1.45, 60×). GFP
and Fura Red were excited by a 488-nm laser, and each emitted light was
collected through 535/45 and 645/75 nm, respectively. For simultaneous
measurement of relative fluorescence change in intensity of
C12-GFP and [Ca2+]i in the Fura
Red-loaded cells, we used a W-view Optics (Hamamatsu Photonics), a
branching optics that splits the incident light into using a Dichroic
mirror of 550 nm, so that two separate images of the GFP and Fura Red
fluorescence can be produced.
Membrane Depolarization Induces Transient Translocation of PKC Threshold Value of [Ca2+]i for PKC Depolarization-evoked Translocation of PKC
To compare the effects of Ca2+ influx and
IP3-mediated Ca2+ mobilization on PKC PKC Depolarization-evoked Ca2+ Influx Induces Translocation
of PKC Depolarization-evoked Ca2+ Influx
Translocates GFP-tagged Pleckstrin Homology Domain of
PLC In Situ Calibration of Depolarization-evoked Increase in DAG
Content--
We calibrated the depolarization-evoked increase in DAG
concentration in single C12-GFP expressing cells. An
extracellular solution containing the DAG analogue DiC8 was
introduced at the end of each experiment, following 40 mM
K+ stimulation, using TIRFM. Fig.
10 shows a representative experiment where application of three different concentrations of DiC8
(1, 3 and 10 µM) resulted in different quasi-steady state
levels of C12 translocation. Because the concentration of
DiC8 inside the cell, at each level, is thought to
equilibrate with that outside the cell, the depolarization-evoked
increase in DAG concentration was estimated (from the calibration
curves of the three experiments) to be 1.90 ± 0.02 µM (mean ± S.D.).
Microdomains of Elevated [Ca2+] beneath the Plasma
Membrane, but Not Elevation of the Bulk [Ca2+]i,
Are Required for cPKC Translocation--
We have shown that there is a
threshold value of the bulk [Ca2+]i at ~400
nM for PKC
Ca2+ mobilization induced by ACh most likely fails to
translocate PKC Ca2+ Influx through VDCCs Is Both Necessary and
Sufficient for Activation of cPKC--
To fully substantiate that
Ca2+ influx through VDCCs is both necessary and sufficient
for activation of cPKC, we employed the phosphorylation state of MARCKS
as another marker of PKC activity. As seen in Fig. 5B,
plasma membrane-anchored MARCKS is turned into phosphorylated MARCKS,
thereby moving into the cytosol, as soon as PKC Depolarization-evoked Ca2+ Influx through
VDCCs Can Generate DAG and Thereby Activate cPKC and nPKC, Whose
Activation Is Structurally Independent of Ca2+--
nPKC,
which lacks the functional C2 domain for Ca2+ binding, is
activated either by DAG or TPA. This can be seen by the sustained PKC
Our data also have important implications for cPKC activation. If the
amplitude of the nPKC translocation reflects the amount of DAG
synthesized, depolarization-evoked Ca2+ influx could
translocate as well as activate cPKC by generating [Ca2+]sub and DAG. Ca2+ signals
per se, such as action potential-induced Ca2+
oscillations, could function as second messengers as well as operate as
primary activators of cPKC and nPKC in neuronal, endocrine and muscle
cells. In other words, Ca2+ signals and the two PKCs
signals may not be segregated in certain conditions. Therefore, the
roles of these signals in a myriad of cellular functions may overlap.
Short-lived Ca2+ Signals through PKC
Activation Are Transduced into Long-lived Phosphorylated MARCKS;
Ca2+ Oscillation-driven Activation of cPKC and nPKC May
Modulate Long Term Physiological Phenomena--
Our finding that
Ca2+ oscillation-evoked activation of both cPKC and nPKC
can keep MARCKS phosphorylated (Fig. 5) has important implications for
the control of long term physiological phenomena such as insulin
secretion (37), long term potentiation (38), the redox state in the
mitochondria (39), and the control of gene expression (40, 41). We can
envisage that, as long as Ca2+oscillation-driven activation
of a first kinase such as PKC continues in a "sinus-like" manner,
then the first kinase is maintained in the phosphorylated state, which
in turn leads to activation of a second kinase on a time scale of hours
or days. In this way, not only Ca2+ oscillations but also
Ca2+ oscillation-driven activation of both PKCs may
modulate a long term physiological phenomenon. Thus, we should bear in
mind that two classes of PKCs can be activated in conditions where
Ca2+ oscillations take place.
) and novel PKC (nPKC;
PKC
), in insulin-secreting cells. We have demonstrated simultaneous
translocation of both DsRed-tagged PKC
to the plasma membrane and
green fluorescent protein (GFP)-tagged myristoylated alanine-rich C
kinase substrate to the cytosol as a dual marker of PKC activity in
response to depolarization-evoked Ca2+ influx in the
DsRed-tagged PKC
and GFP-tagged myristoylated alanine-rich C kinase
substrate co-expressing cells. The result indicates that
Ca2+ influx can generate diacylglycerol (DAG), because cPKC
is activated by Ca2+ and DAG. We showed this in three
different ways by demonstrating: 1) Ca2+ influx-induced
translocation of GFP-tagged C1 domain of PKC
, 2) Ca2+
influx-induced translocation of GFP-tagged pleckstrin homology domain,
and 3) Ca2+ influx-induced translocation of GFP-tagged
PKC
, as a marker of DAG production and/or nPKC activity. Thus,
Ca2+ influx alone via voltage-dependent
Ca2+ channels can generate DAG, thereby activating cPKC and
nPKC, whose activation is structurally independent of
Ca2+.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
PKC
I, PKC
II, and PKC
), novel PKC (nPKC; PKC
, PKC
,
PKC
, and PKC
), and atypical PKC (PKC
and PKC
) (2, 3). The C1 and C2 regions in the regulatory domain are responsible for
diacylglycerol (DAG) and Ca2+ binding, respectively. DAG
comes mainly from plasma membrane phosphatidylinositol 4,5-bisphosphate
(PIP2) hydrolysis. This is caused by phospholipase C (PLC)
activation, following agonist binding to a G protein-coupled receptor.
In excitable cells, cytosolic Ca2+ signals are generated
either by Ca2+ influx through voltage-dependent
Ca2+ channels (VDCCs) and/or by Ca2+ release
from the endoplasmic reticulum through inositol 1,4,5-trisphosphate (IP3) receptors upon binding of IP3, the other
product of PIP2 hydrolysis (4, 5). DAG and Ca2+
activate the first family of cPKCs that have both the C1 and C2
regions. The second family of the novel PKCs is also activated by DAG,
but in a Ca2+-independent manner because of the absence of
the functional C2 region. The third family of the atypical PKCs can be
activated by phosphoinositide-dependent kinase 1 in a
Ca2+-independent manner (6).
and PKC
as representatives of cPKC
and nPKC, respectively, to probe further into the mechanisms underlying
the Ca2+ signaling-induced activation of cPKC and nPKC. To
this end, we employed INS-1 cells, an insulin-secreting cell line
established from a rat insulinoma (7), as a model system in which VDCCs are the main pathways for the generation of Ca2+ signals.
Key observations from other laboratories have shown that PKC
is
activated within the physiological Ca2+ concentration range
in the presence of both DAG and phosphatidylserine (8) and that
depolarizing K+ concentrations evoke an increase in the
IP3 concentration in rat pancreatic islets, suggesting
PLC-mediated production of DAG (9). Two important questions arise: 1)
Can depolarization-evoked Ca2+ influx through the opening
of VDCCs activate cPKC? and 2) Can Ca2+ influx also
activate nPKC, if the Ca2+ influx results in production of
DAG? Furthermore, in INS-1 cells, as in normal insulin-secreting cells,
glucose-induced oscillations in membrane potential elicit repetitive
openings of VDCCs (10). This is responsible for oscillations in the
cytosolic Ca2+ concentration
([Ca2+]i), which causes pulsatile insulin
secretion (11). Ca2+ oscillations play an essential role in
exocytotic secretion in neuroendocrine cells (12). It is therefore
important to know how depolarization-evoked Ca2+
oscillations are decoded into long term physiological modifications, such as insulin secretion, through PKC activation.
in astrocytes, it has been shown that there is a marked
temporal correlation between glutamate-elicited Ca2+ spikes
and PKC translocation (15). A current model for activation of cPKC (14)
proposes that: 1) the [Ca2+]i elevation recruits
cPKC to the plasma membrane via the C2 region, 2) the site on the
enzyme where the pseudosubstrate inhibitory region in the regulatory
domain is occupied at rest is exposed and becomes available for
substrate binding, and (3) full activation of the enzyme takes place
when DAG tightly tethers the enzyme to the plasma membrane via the C1
region. The sequence of these events suggests that translocation and
activation of cPKC may not always correspond. This raises a final
question: How do we know when the pseudosubstrate inhibitory region is
removed (i.e. when exactly does activation of cPKC take place)?
-GFP,
as markers for cPKC, in response to depolarization-evoked Ca2+ influx through VDCCs in INS-1 cells. The
Ca2+ influx resulted in translocation of PKC
-GFP to the
plasma membrane. We also assessed the phosphorylation state of the PKC
substrate myristoylated alanine-rich C kinase substrate (MARCKS) (16) as another marker of PKC activity, by monitoring translocation of
GFP-tagged MARCKS (MARCKS-GFP) with DsRed-tagged PKC
(PKC
-DsRed). When phosphorylated by PKC, MARCKS translocates from the plasma membrane to the cytosol (17). Translocation of MARCKS-GFP to the
cytosol took place as soon as PKC
-DsRed translocated to the plasma
membrane upon stimulation of Ca2+ influx. These results
indicate that the Ca2+ influx can generate DAG, because
cPKC is activated by Ca2+ and DAG. We showed this in three
different ways by demonstrating: 1) Ca2+ influx-induced
translocation of GFP-tagged C1 domain of PKC
, 2) Ca2+
influx-induced translocation of GFP tagged pleckstrin homology domain
(GFP-PHD), and 3) Ca2+ influx-induced translocation of
PKC
-GFP as a marker of DAG production. The depolarization-evoked
increase in DAG concentration was estimated from in situ
calibration to be 1.90 ± 0.02 µM. We have
demonstrated for the first time that depolarization-evoked
Ca2+ influx can generate DAG, thereby activating cPKC and
nPKC. We also observed that MARCKS remained phosphorylated through PKC activation as long as the depolarization-evoked Ca2+
oscillations continued. Our results show that short-lived
Ca2+ signals can be transduced via PKC activation into long
term phosphorylated MARCKS.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pEGFP, PKC
-pEGFP, pEGFP-N2, and pDsRed1-N1 were
obtained from Clontech Lab, Inc. (Palo Alto, CA).
To attain brighter fluorescence of MARCKS-GFP, the GFP of MARCKS-GFP
(17) was replaced with pEGFP-N2. pEGFP of PKC
-pEGFP was replaced
with pDsRed1-N1. A GFP-tagged C1 region of PKC
(C12-GFP)
was produced from a DNA clone of
CKR
1, which was subcloned into
an expression plasmid for mammalian cells, pTB701 (18). A cDNA
fragment of PKC
for C1 region with an EcoRI site in the
5' terminus and a BglII in the 3' terminus was produced by
PCR using pTB701 as a template. The sense and antisense primers used
were 5'-TTGAATTCGCCATGGTGAAGAGCCACAAGTTCACC-3' and
5'-TTAGATCTGTCCACGCCGCAAAGGGAGGG-3', respectively. A PCR product for C1
region of PKC
was subcloned into the EcoRI site and the BglII site in GFP containing pTB701 (18). The PCR product
was verified by sequencing. A GFP-tagged pleckstrin homology domain of
PLC
1 (GFP-PHD) was donated by Dr. Hirose (Tokyo University, Tokyo,
Japan) (19).
50 °C) coupled charge
device digital camera (ORCA-II and ORCA-ER; Hamamatsu Photonics,
Hamamatsu, Japan) and recorded and analyzed on a Aquacosmos imaging
station (Hamamatsu Photonics). The excitation light source was 150 W
xenon lamp with a Polychrome I monochromator (T.I.L.L. Photonics GmbH.,
Planegg, Germany). GFP fluorescence was excited at 488 nm for high time
resolution of GFP-tagged PKCs digital imaging. We measured the
fluorescence intensity of the GFP (DsRed)-tagged proteins (PKCs,
MARCKS, PHD, and C1 domain) in the cytosol of the cell, excluding the
nucleus, and/or at the plasma membrane, as a marker of translocation.
These values (F) were normalized to each initial value
(F0), so that the relative fluorescence change
was referred to as the "ratio F/F0." For simultaneous
measurements of the relative change in fluorescence intensity of the
GFP-tagged PKCs in the cytosol and [Ca2+]i, GFP
fluorescence was excited at 488 nm, whereas Fura2 was excited at
wavelengths alternating between 340 and 380 nm. We put a short pass
filter of 330-495 nm to reduce background fluorescence in the light
pass between a dichroic mirror of 505 nm and an emission filter of
535/45 nm band pass. The cells transiently expressing GFP-tagged PKCs
were loaded with 2 µM Fura2/AM in the standard
extracellular solution for 30 min at room temperature. The cells were
washed twice and used within 2 h. The Fura2 ratio was calibrated
using exposure to 10 µM ionomycin and 10 mM
Ca2+ or 10 mM EGTA in the Fura2-loaded cells
without transfection of the GFP-tagged PKCs. A dissociation constant of
150 nM for Ca2+ and Fura2 at room temperature
was used. For simultaneous measurement of relative fluorescence change
in intensity of MARCKS-GFP and PKC
-DsRed using a dual band for
fluorescein isothiocyanate and TRITC, GFP fluorescence was excited at
488 nm, whereas PKC
-DsRed fluorescence was excited at 558 nm. To
reduce cross-talk between them, an emission filter wheel was used, and
alternate emission filters of 535/45-nm band pass and 605/50-nm long
pass were synchronously set with excitation filters for GFP and DsRed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Following [Ca2+]i Elevation--
We first
examined the distribution of PKC
-GFP with the help of high time
resolution digital imaging. Fig.
1A shows the rapid and
reversible translocation of PKC
-GFP in response to a depolarizing K+ concentration (40 mM), which evoked
Ca2+ influx through opening of VDCCs. The relative changes
in the fluorescence intensities of PKC
-GFP in the cytosol and at the plasma membrane are plotted in Fig. 1B, as a function of
time. PKC
-GFP translocated from the cytosol to the plasma membrane, and this can be seen by the reciprocal changes in the two parameters (Fig. 1B) (n = 6). Thus, either parameter
can be used as a marker of PKC
-GFP plasma membrane translocation. We
chose to employ the relative fluorescence change in the cytosol as a
marker of translocation. Next, we simultaneously measured
[Ca2+]i and PKC
translocation in Fura2-loaded
and PKC
-GFP-expressing INS-1 cells. As seen in Fig. 1C, a
depolarizing K+ concentration induced a transient
translocation of PKC
-GFP to the plasma membrane following a
transient [Ca2+]i elevation (n = 8). Thus, the temporal profile was similar to that observed when
PKC
-GFP measurements were carried out alone, suggesting successful
dissection of Ca2+ and GFP signals.
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Fig. 1.
Depolarization-evoked Ca2+ influx
induces translocation of PKC in
PKC
-GFP transiently expressing INS-1
cells. A, images of PKC
-GFP translocation induced by
40 mM K+ at high time resolution (sampling
interval, 0.25 s). Panels a-e were taken at the times
indicated by the arrows in B. The bar
represents 10 µm. Maximal translocation was observed at 2 s
(panel c) after stimulation. The subcellular distribution of
PKC
-GFP in panel a was indistinguishable from that in
panel e. B, the entire time course of PKC
-GFP
translocation induced by the 40 mM K+ solution.
The regions of interests were at the plasma membrane (PM)
and in the cytosol (white boxes in A).
C, simultaneous measurements of
[Ca2+]i and PKC
translocation in Fura2-loaded
and PKC
-GFP-expressing INS-1 cells. ratio (PKCcyt)
represents the relative fluorescence change in intensity of PKC
-GFP
in the cytosol. The translocation of PKC
-GFP had ceased (back to the
prestimulation level) before [Ca2+]i returned to
the resting level.
Translocation--
We loaded INS-1 cells with Fura2, without
expressing PKC
-GFP, to accurately estimate
[Ca2+]i. In the standard extracellular solution
containing 2.5 mM CaCl2 and 3 mM
glucose, more than 50% of the Fura2-loaded INS-1 cells displayed
spontaneous cytosolic Ca2+ oscillations (20). The peak
[Ca2+]i was no more than 400 nM. When
the same cells were depolarized by the K+ channel blocker
tetraetheylammonium (TEA) (20 mM), the Ca2+
oscillations became more pronounced (21), and the peak
[Ca2+]i was in the range of 600-800
nM (Fig. 2A)
(n = 10). To avoid cross-talk between Ca2+
and GFP signals, exactly the same protocol as in Fig. 2A was applied to PKC
-GFP-expressing cells without Fura2 loading. No translocation of PKC
-GFP took place in the standard extracellular solution, whereas oscillatory translocations of PKC
-GFP started immediately after introduction of the TEA-containing solution (Fig.
2B) (n = 5), suggesting a threshold value of
[Ca2+]i of more than 400 nM for
PKC
translocation. The temporal profile of the TEA-evoked PKC
-GFP
translocations (Fig. 2, C and D)
(n = 5) was similar to that observed in response to the
membrane depolarization evoked by a high K+ concentration
(Fig. 1B).
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Fig. 2.
Threshold value of
[Ca2+]i for translocation of
PKC in INS-1cells. A,
spontaneous and TEA-evoked Ca2+ oscillations. Three
different cells displayed spontaneous oscillatory changes in
[Ca2+]i under 400 nM. The amplitude
of the Ca2+ oscillations became larger (>400
nM) during TEA stimulation. B, two PKC
-GFP
expressing cells, not loaded with Fura2, showed oscillatory
translocations of PKC
-GFP in the presence of TEA, but not in the
standard solution. C, images of PKC
-GFP translocation
induced by 20 mM TEA at high time resolution (sampling
interval, 0.25 s). Panels a-e were taken at the times
indicated by the arrows in D. the bar
represents 10 µm. Maximal translocation was observed 2 s
(panel d) after stimulation. The subcellular distribution of
PKC
-GFP in panel a was indistinguishable from that in
panel e. D, the entire time course of PKC
-GFP
translocation. The regions of interests were at the plasma membrane
(PM) and in the cytosol (white boxes in
C).
Depends on
Ca2+ Influx but Not on Ca2+
Mobilization--
Fig. 3A
shows that upon removal of external Ca2+, the TEA-induced
Ca2+ oscillations and the PKC
-GFP translocations were
abolished (n = 5), indicating that both are totally
dependent on Ca2+ influx. We then tested whether PKC
can
be fully activated in the presence of DAG at physiological
Ca2+ concentrations. Using PKC
-GFP translocation to the
plasma membrane as a marker of activation, short exposure to a
combination of TEA and the diacylglycerol analogue DiC8
(100 µM) induced sustained PKC
activation, despite the
fact that [Ca2+]i quickly returned to the steady
resting level upon removal of the stimulation (Fig. 3B)
(n = 8). This suggests that even a single TEA-evoked
Ca2+ spike (within the physiological Ca2+
concentration range) can fully activate PKC
in the presence of a
sufficient amount of DAG (8).
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Fig. 3.
Translocation of PKC
is dependent on Ca2+ influx through VDCCs.
Simultaneous monitoring of the translocation of PKC
-GFP and
[Ca2+]i in Fura2-loaded cells with expression of
PKC
-GFP is shown. ratio (PKCcyt) represents relative
fluorescence change in intensity of PKC
-GFP in the cytosol.
A, repetitive translocations of PKC
-GFP were synchronous
with Ca2+ oscillations in the presence of TEA. Both were
abolished upon removal of external Ca2+. B,
combined application of 100 µM DiC8 and TEA
resulted in sustained translocation of PKC
-GFP, although
[Ca2+]i had returned to the prestimulation level
upon removal of the stimulation.
-GFP
translocation, we tested, in the same cells, the actions of TEA and
acetylcholine (ACh) (using a supramaximal concentration (100 µM), which might produce sufficient DAG to activate
PKC
(see Figs. 7B and 9A)). As shown in Fig.
4A, the TEA-evoked
translocation of PKC
-GFP was much more substantial than that induced
by ACh, although the bulk [Ca2+]i elevations
produced by the two agents were nearly equal (n = 11).
For more accurate evaluation of the cytosolic Ca2+
elevation caused specifically by IP3-induced store release,
we removed the Ca2+ influx component due to capacitative
Ca2+ entry through store-operated Ca2+ channels
in the plasma membrane (22, 23). This was simply done by removing
external Ca2+ during stimulation with ACh (100 µM) (Fig. 4B). The result was similar
(n = 5) to that shown in Fig. 4A, suggesting
that there was little effect of store-operated Ca2+ influx
on PKC
translocation. Reversing the sequence of events (applying TEA
first and then subsequently ACh) gave similar results to those shown in
Fig. 4 (data not shown).
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Fig. 4.
Ca2+ influx through VDCCs, but
not Ca2+ mobilization induced by ACh, is important for
translocation of PKC . Simultaneous
monitoring of the translocation of PKC
-GFP and
[Ca2+]i in Fura2-loaded cells with expression of
PKC
-GFP is shown. A, a supramaximal concentration of ACh
(100 µM) induced a small translocation of PKC
-GFP,
whereas TEA-evoked depolarization induced a marked PKC
-GFP
translocation, although the magnitude of the
[Ca2+]i elevation induced by ACh was virtually
the same as that elicited by TEA and lasted longer. B, the
same concentration of ACh (100 µM) in the absence of
external Ca2+ also induced a very small translocation. It
should be noted that the third TEA-evoked translocation was much larger
than the second ACh-elicited translocation.
, Activated by Depolarization-evoked Ca2+ Influx,
Can Phosphorylate Its Substrate, MARCKS--
One line of evidence has
shown that [Ca2+]i elevations drive translocation
of cPKC to the plasma membrane (13, 14). However, we do not know
whether cPKC can be activated by depolarization-evoked Ca2+
influx alone. To answer this question, we employed a GFP-tagged MARCKS
as another marker of PKC activity (17), which is a putative and direct
substrate for PKC (16), as well as PKC
-DsRed. We co-transfected both
of them into INS-1 cells. When activated PKC phosphorylates the plasma
membrane-anchored MARCKS, then phosphorylated MARCKS translocates from
the plasma membrane to the cytosol. Thus, simultaneous monitoring of
PKC
-DsRed and MARCKS-GFP allows us to test whether
depolarization-evoked Ca2+ influx can activate cPKC. Fig.
5 (A and B) shows
translocations of MARCKS-GFP and PKC
-DsRed induced by TEA,
indicating that depolarization-evoked Ca2+ influx can
activate PKC
. Phosphorylated MARCKS only slowly and gradually
returned to the plasma membrane (~2.5 min) in contrast to the rapid
temporal profile of PKC
translocation (~30 s) (Fig. 5B)
(n = 6). Ca2+ oscillation-driven
translocations of PKC
kept MARCKS phosphorylated in the cytosol
until termination of the repetitive PKC
translocations (Fig.
5C) (n = 8).
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Fig. 5.
Ca2+ oscillation-driven
translocation of PKC -DsRed results in
phosphorylation of MARCKS-GFP. Simultaneous monitoring of the
translocations of PKC
-DsRed and MARCKS-GFP is shown. A,
superimposed panels a-f were taken at the times indicated
by the arrows in B. The bar represents
10 µm. The regions of interests were at the plasma membrane and in
the cytosol (white box). Panel a, MARCKS-GFP
(green) anchored at the plasma membrane, whereas
PKC
-DsRed (red) was in the cytosol. A short exposure to
TEA resulted in a dramatic reverse change in the red and
green colors; MARCKS that had been phosphorylated was
translocated to the cytosol as soon as PKC
translocation to the
plasma membrane had taken place (panels b and c).
MARCKS was still in the cytosol as merged color of yellow
can be seen in panels d and e. The last image
(panel f) shows the same distribution of PKC
-DsRed and
MARCKS-GFP as in panel a. B, the entire time
course of the translocations of PKC
-DsRed and MARCKS-GFP shown in
A. C, trains of oscillatory PKC
-DsRed
translocations kept MARCKS in the cytosol. The ratio represents
relative fluorescence change in intensity of PKC
-DsRed and
MARCKS-GFP at the same region of interest in the cytosol (B
and C).
, Despite the Absence of the Functional C2 Domain for
Ca2+ Binding--
As seen in Fig. 5 (A and
B), we now know that depolarization-evoked Ca2+
influx can activate PKC
. This observation prompted us to explore whether depolarization-evoked Ca2+ influx can generate DAG,
because Ca2+ and DAG are required for activation of cPKC
(2). It has been shown that K+-induced membrane
depolarization increases IP3 production in
insulin-secreting rat pancreatic islets (9). Taken together, these
observations indicate that there should be production of DAG in
response to depolarization-evoked Ca2+ influx. To test this
hypothesis, we employed PKC
-GFP as a marker of nPKC activity as well
as DAG production because nPKC is activated by DAG alone in a
Ca2+-independent manner (2). TPA caused a rapid and
sustained translocation of PKC
-GFP in the complete absence of
Ca2+ influx (Fig. 6,
A and B) (n = 6). The
simultaneous measurements of PKC
-GFP translocation and cytosolic
Ca2+ concentration, shown in Fig. 6C, confirm
that extracellular Ca2+ is not required for PKC
-GFP
translocation (n = 4). We then applied a depolarizing
K+ concentration to PKC
-GFP-expressing cells. Fig.
7A clearly shows that this
stimulus induced a gradual translocation of PKC
-GFP to the plasma
membrane, which was reversible upon removal of the high K+
solution (n = 12). The amplitude of the translocation
induced by K+-induced depolarization was comparable with
that elicited by ACh (Fig. 7B). It should be noted that ACh
still continued to induce translocation of PKC
-GFP after
[Ca2+]i had returned to the resting level,
verifying that 100 µM ACh generated enough DAG to sustain
the translocation until termination of the stimulus (n = 8) (Figs. 7B and 9B). The amplitude of the
TEA-evoked translocation was also comparable with that induced by ACh,
and the translocation was not synchronous with the TEA-evoked
Ca2+ spikes (Fig. 7C) (n = 8).
View larger version (26K):
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Fig. 6.
PKC -GFP
translocation as a representative of nPKC is induced by TPA in the
absence of Ca2+. A and B, images
of PKC
-GFP translocation induced by 100 nM TPA at high
time resolution (sampling interval, 0.25 s) and the entire time
course of the PKC
-GFP translocation. The regions of interests were
at the plasma membrane (PM) and in the cytosol (white
boxes in B). The panels a-e in B
were taken at the times indicated by the arrows in
A. The bar in B represents 10 µm.
C, simultaneous monitoring of PKC
-GFP translocation and
[Ca2+]i in Fura2-loaded cells with expression of
PKC
-GFP. ratio (PKCcyt) represents relative fluorescence
change in intensity of PKC
-GFP in the cytosol. Translocation of
PKC
-GFP immediately started following application of 100 nM TPA in the absence of external Ca2+, whereas
spontaneous Ca2+ oscillations had no effect on the
translocation. Simultaneous monitoring of PKC
-GFP translocation and
[Ca2+]i in Fura2-loaded cells with expression of
PKC
-GFP (A-C).
View larger version (25K):
[in a new window]
Fig. 7.
Depolarization-evoked Ca2+ influx
and ACh induce PKC -GFP translocation.
Simultaneous monitoring of PKC
-GFP translocation and
[Ca2+]i in Fura2-loaded cells with expression of
PKC
-GFP is shown. ratio (PKCcyt) represents relative
fluorescence change in intensity of PKC
-GFP in the cytosol.
A, relatively slow translocation of PKC
-GFP induced by a
depolarizing K+ concentration (40 mM).
PKC
-GFP continued to be translocated until cessation of the
stimulation. Thereafter, PKC
-GFP started to return to the cytosol.
At the same time, [Ca2+]i started to decrease
toward the prestimulation level. B, ACh gradually and
continuously translocated PKC
-GFP until removal of ACh, when
[Ca2+]i had already returned to the
prestimulation level. C, TEA-evoked repetitive
Ca2+ spikes also induced translocation of PKC
-GFP, with
a temporal profile similar to that induced by ACh.
1 (GFP-PHD) and GFP-tagged C1 Domain of PKC
(C12-GFP)--
To fully corroborate the above evidence
that the Ca2+ influx generates DAG and activates PKC
, we
performed further experiments using GFP-PHD and C12-GFP.
First, GFP-PHD allows us to visualize IP3 production by
translocating from the plasma membrane to the cytosol because of the
20-fold higher affinity for IP3 than for PIP2
(19), such that we can assess indirectly the simultaneous production of
DAG upon PIP2 hydrolysis by a
Ca2+-dependent PLC activation. As shown in Fig.
8A, depolarization-evoked Ca2+ influx resulted in the relatively transient
translocation of PHD-GFP, whereas the translocation was sustained
during ACh stimulation (n = 11), indicating DAG
production. Second, to directly monitor the plasma membrane DAG levels,
we also employed translocation of the C12-GFP as a DAG
sensor (14, 15) using TIRFM. This has a ~10-fold higher
signal-to-noise ratio (see "Experimental Procedures") than
conventional epifluorescence microscopy in terms of the fluorescent
protein translocation near the plasma membrane (for example PKC-GFPs)
(15). We loaded the C12-GFP-expressing cells with Fura Red
to define the relationship between the [Ca2+]i
change and DAG production in response to the Ca2+ influx.
Depolarization-evoked Ca2+ influx clearly translocated
C12-GFP to the membrane (Fig.
9A) after
[Ca2+]i had risen to the peak (Fig.
9B), in contrast to the sustained ACh-induced translocation
of C12-GFP following the transient [Ca2+]i elevation. Stimulation with 40 mM K+ also resulted in a C12
translocation of an undiminished magnitude in 5 µM
thapsigargin-pretreated C12-GFP-expressing cells,
indicating little contribution of either Ca2+- or
IP3-induced Ca2+ release to the C12
translocation (data not shown). As long as both high K+ and
ACh stimulation continued, C12-GFP remained at the plasma membrane. The translocation induced by Ca2+ influx was
inhibited by the PLC inhibitor U73122 (n = 4), suggesting that DAG production is mediated by
Ca2+-dependent PLC activation (Fig.
9D).
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Fig. 8.
Depolarization-evoked Ca2+ influx
and ACh induce GFP-PHD translocation. A, the entire time
course of GFP-PHD translocation induced by the high K+
solution (40 mM) and 100 µM ACh. The regions
of interests were at the plasma membrane (PM) and in the
cytosol (white boxes in B). ratio
(PKCcyt) represents relative fluorescence change in intensity of
GFP-PHD in the cytosol. GFP-PHD translocation was transient with
stimulation of high potassium, whereas ACh continued to
translocate GFP-PHD until the removal of ACh. B,
panels a-d were taken at the times indicated by the
arrows in A. The bar represents 10 µm.
View larger version (24K):
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Fig. 9.
Depolarization-evoked Ca2+ influx
and ACh induce C12-GFP translocation. Shown are
simultaneous measurements of [Ca2+]i and
C12-GFP translocation in Fura Red-loaded and
C12-GFP expressing INS-1 cells using TIRFM. The ratio
represents the relative fluorescence change in intensity of
C12-GFP at the plasma membrane and of
[Ca2+]i. Note that rises in Ca2+ are
reported as a fall in fluorescence. The region of interest for both
parameters was at the plasma membrane (white box in
C). A, the entire time course of simultaneous
measurements of [Ca2+]i and C12-GFP
translocation. C12-GFP translocation to plasma membrane
induced by both high potassium and ACh has biphasic pattern.
B, areas denoted by the dashed box in
A are expanded. Translocation of C1-GFP started 9 s
after the [Ca2+]i elevation. C,
panels a-c were taken at the times indicated by the
arrows in A. The bar represents 10 µm. D, effect of U73122 on C12-GFP
translocation induced by high potassium. The cell was pretreated with
U73122 for 15 min following the control experiment with 40 mM K+ stimulation. U73122 almost completely
inhibited C12-GFP translocation (red) induced by
high potassium stimulation in comparison with the control experiment
(blue).
View larger version (29K):
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Fig. 10.
Calibration of depolarization-evoked
increase in DAG concentration in C12-GFP expressing cells
using TIRFM. A, cells were first treated with 40 mM K+ to establish the magnitude of the
translocation of C12-GFP. Thereafter, the cells were
perfused with the Ca2+-free extracellular solution
containing 0.2 mM EGTA for the first 2 min followed by
consecutive applications of DiC8 1 µM (0.35 µg/ml), 3 µM (1.05 µg/ml), and 10 µM
(3.5 µg/ml). B, panels a-e were taken at the
times indicated by the arrows in A. The region of
interest represented by the dashed box was at the plasma
membrane. The ratio represents relative fluorescence intensity of
C12-GFP. The bar represents 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
translocation in the insulin-secreting INS-1
cells (Fig. 2A), which is consistent with data from other laboratories (24). More importantly, we have also demonstrated that
Ca2+ influx is a much stronger stimulus for PKC
translocation than Ca2+ mobilization from intracellular
stores, even when the amplitudes of the induced bulk
[Ca2+]i elevations are similar (Fig.
4A). This finding suggests that microdomains of elevated
[Ca2+] beneath the plasma membrane
([Ca2+]sub) generated by Ca2+
influx through VDCCs may play a pivotal role in cPKC translocation in
excitable cells rather than the elevated bulk
[Ca2+]i, which is also consistent with a recent
report (25). In other words, there is a threshold value of
[Ca2+]sub for translocation of cPKC. In
neuroendocrine cells, the estimated value of
[Ca2+]sub at the mouth of open VDCCs is
several micromoles/liter (12, 26). This indicates that a local
[Ca2+] of several micromoles/liter may be required for
cPKC translocation.
because the [Ca2+] rise at the
critical sites is insufficient. This could be due to the distance
between the plasma membrane and the IP3 channels in the ER,
combined with the substantial Ca2+ buffering capacity in
the cytosol (27). However, it could be argued that Ca2+
mobilization from the ER should result in store-operated
Ca2+ entry (22, 23) and that ACh stimulation therefore also
could be expected to cause local Ca2+ elevation beneath the
plasma membrane. Nevertheless, it would appear (Fig. 4B)
that the magnitude of Ca2+ influx through store-operated
Ca2+ channels is insufficient to generate the threshold
level of [Ca2+]sub needed. Because the entry
sites from store-operated Ca2+ channels would be very close
to Ca2+ uptake sites into the ER through the powerful
Ca2+ ATPase pumps (23, 28-31), the net delivery of
Ca2+ to the cytosol through store-operated Ca2+
channels may be less than through voltage-gated channels even if both
channels have similar ranges of Ca2+ concentrations at the
mouths of their pores. They could also possibly be separately located.
Our finding that Ca2+ entry through VDCCs is sufficient to
cause cPKC and nPKC translocation may be important in relation to the
control of glucose-elicited insulin secretion, because it has been
shown that the L-type VDCCs and the insulin-containing secretory
granules are co-localized (26). Thus, local Ca2+ entry in
the secretory domains could induce PKC activation important for
stimulation of exocytosis (25).
is translocated to
the plasma membrane by Ca2+ influx through VDCCs. In INS-1
cells, in which PKC
and PKC
are predominantly expressed (32),
endogenous cPKC and nPKC may move to the plasma membrane in the same
manner as the exogenous examples. Thus, we have provided the first
direct evidence showing that single Ca2+ spike-driven
translocations of cPKC, whose duration is just 30 s long, enable
MARCKS to be phosphorylated. The pseudosubstrate inhibitory region of
cPKC has been already removed before the association with MARCKS. The
cessation of MARCKS translocation and the return to the prestimulation
level is very much slower than that of the cPKC translocation, because
of sustained MARCKS phosphorylation. We do not know the exact
mechanism, but it might result from the net effect of several factors
such as diacylglycerol kinase (17), PKC, or a phosphatase that
dephosphorylates MARCKS.
translocation induced by TPA in the absence of external
Ca2+ (Fig. 6B). However, depolarization-evoked
Ca2+ influx through VDCCs can also induce gradual and
continuous nPKC translocation to the plasma membrane during
[Ca2+]i elevation (Fig. 7A). It is
possible that some regions of PKC
other than the C1 domain can be
associated with the plasma membrane. However, two additional
experiments using GFP-PHD and C12-GFP have added further
credence to the observations (Fig. 7). First, the Ca2+
influx-evoked translocation of GFP-PHD (Fig. 8A) indicates
that DAG can be generated upon PIP2 hydrolysis mediated by
a Ca2+-dependent PLC activation (33), although
the amplitude of the PHD translocation may parallel the concentration
not of DAG but of IP3 (19). The translocation of GFP-PHD
induced by depolarization was relatively transient, whereas it was
sustained during ACh stimulation. This suggests a relatively transient
increase in DAG concentration (3). Second, the Ca2+
influx-evoked translocation of C12-GFP (Fig. 9A)
as a DAG sensor, directly supports the view that DAG synthesis is
induced by depolarization-evoked Ca2+ influx through VDCCs.
The simplest explanation for this surprising observation could be that
the Ca2+ influx can initiate DAG generation, by triggering
PLC activation, thereby translocating and activating nPKC. The fact
that the translocation of the C1 domain did not start until the
[Ca2+]i had nearly reached its peak (Fig.
9B), taken together with the observation that sustained
Ca2+ influx induced by 1 µM ionomycin kept
C12-GFP at the plasma membrane (data not shown), suggests
that there may be a threshold value of
[Ca2+]sub for DAG synthesis. Conversely, DAG
synthesis can be detected by monitoring the C1 domain translocation, if
the amplitude of the C1 domain translocation parallels the amount of
DAG synthesis. Therefore, we tried to estimate the increase in DAG
content with in situ calibration (Fig. 10A),
which gave a value of 1.90 ± 0.02 µM (mean ± S.D., n = 3). In a report from another laboratory, using a biochemical assay, it was calculated that the amount of accumulated DAG is 13 pmol/106 cells at 30 s in
platelet-derived growth factor-stimulated Balb/c/3T3 cells (34). Given
a cell volume of ~1 pl, the DAG concentration would be 13 µM, which is comparable with our data. As shown in Fig.
5, a DAG concentration of ~2 µM, induced by
depolarization-evoked Ca2+ influx, may be sufficient to
ensure that activated PKC can phosphorylate MARCKS. Monitoring of
C12-GFP translocation has been the most sensitive way of
detecting DAG synthesis beneath the plasma membrane so far
(14, 15). As shown in Fig. 9A, the C1 domain biphasically translocated to the membrane during high K+ stimulation;
the first phase was transient, and the second phase was sustained. This
indicates continuous production of DAG. DAG synthesis can be mediated
by PLC (Figs. 8A and 9D) (33) and/or phospholipase D activated by the [Ca2+]i
rise and/or PKCs (3, 9, 35, 36). However, neither Ca2+- nor
IP3-induced Ca2+ mobilization from the
Ca2+ stores is important for DAG synthesis, because of the
undiminished magnitude of the depolarization-evoked C12
translocation in the thapsigargin-pretreated cells.
![]() |
ACKNOWLEDGEMENTS |
---|
GFP-PHD was a kind gift from Dr. Hirose (Tokyo University). We thank Dr. Alexei Tepikin for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and grants from Takeda Science Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders, the Naito Foundation, the Nissan Science Foundation, and the Suzuken Memorial Foundation. This work was also supported by a Program Grant from the Medical Research Council (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental figure.
§ To whom correspondence should be addressed. Tel.: 81-53-435-2249; Fax: 81-53-435-7020; E-mail: hmogami@hama-med.ac.jp.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M210653200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; DAG, diacylglycerol; PIP2, phophatidylinositol 4,5-bisphosphate; PLC, phospholipase C; VDCC, voltage-dependent Ca2+ channel; IP3, inositol 1,4,5-trisphosphate; GFP, green fluorescent protein; MARCKS, myristoylated alanine-rich C kinase substrate; -GFP, GFP-tagged; -DsRed, DsRed-tagged; PHD, pleckstrin homology domain; TPA, 12-O-tetradecanoylphorbol-13-acetate; DiC8, 1,2-dioctyl-sn-glycerol; TRITC, tetramethylrhodamine isothiocyanate; TIRFM, total internal reflection fluorescence microscopy; TEA, tetraetheylammonium; Ach, acetylcholine; ER, endoplasmic reticulum.
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