(Received for publication, June 19, 1995; and in revised form, August 7, 1995)
From the
Ionomycin stimulated membrane-associated protein kinase Cs
(PKCs) activity in C6 rat glioma cells as much as the potent PKCs
stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA).
However, while TPA, as expected, powerfully stimulated the
phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich
protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did
not. Instead, ionomycin reduced the basal MARCKS phosphorylation.
Pretreating the glioma cells with ionomycin prevented TPA-stimulated
PKCs from phosphorylating the MARCKS protein. The stimulation of
membrane PKCs activity and the prevention of MARCKS phosphorylation by
ionomycin required external Ca because they were both
abolished by adding 5 mM EGTA to the culture medium. Recently
(Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and
Whitfield, J. F.(1995) J. Biol. Chem. 270, 1362-1368),
we proposed that Ca
calmodulin complexes block
MARCKS phosphorylation by the activated PKCs in keratinocytes
stimulated by raising the external Ca
concentration.
In the present experiments calmodulin prevented MARCKS phosphorylation
by TPA-stimulated PKCs in glioma cell lysates, and this blockade was
lifted by a calmodulin antagonist, the calmodulin-binding domain
peptide. But, physiologically more significant, pretreating intact
glioma cells with a cell-permeable calmodulin antagonist,
calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation
by PKCs in unstimulated and TPA-stimulated cells. The effect of
ionomycin on MARCKS phosphorylation was not due to the stimulation of
Ca
calmodulin-dependent phosphoprotein
phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of
this phosphatase, did not stop ionomycin from preventing MARCKS
phosphorylation. The ability of ionomycin to prevent TPA-stimulated
PKCs from phosphorylating MARCKS depended on whether ionomycin was
added before, with, or after TPA. Maximum blockade occurred when
ionomycin was added before TPA but was less effective when added with
or after TPA. These results indicate that
Ca
calmodulin can profoundly affect PKCs'
signaling at the substrate level.
Cultured keratinocytes proliferate when the culture medium
contains EGF ()and a low Ca
concentration
such as 0.05 mM, but raising the Ca
concentration above 1 mM stops proliferation and starts
differentiation-related processes such as cornified envelope formation
(Chakravarthy et al., 1995; Falco et al., 1988;
Moscat et al., 1989; Weissmann and Aaroson, 1983, 1985;
Whitfield, 1995; Whitfield et al., 1992, 1995). The signal
triggered by extracellular Ca
in murine BALB/MK
keratinocytes includes an internal Ca
transient and a
large burst of membrane-associated PKCs activity (Chakravarthy et
al., 1995). Despite the large burst of PKCs activity, neither
Moscat et al.(1989) nor we (Chakravarthy et al.,
1995) have observed an increased phosphorylation of the
keratinocytes' 80-85-kDa MARCKS protein, an ubiquitous
substrate of the conventional and novel PKC isoforms (Blackshear, 1993;
Heemskerk et al., 1993; Fujise et al., 1994).
We
have recently presented strong, but indirect, evidence for the failure
of the Ca-treated BALB/MK keratinocyte's
activated PKCs to phosphorylate their MARCKS protein substrate being
due to Ca
calmodulin complexes somehow blocking
access of the PKCs to their substrate's phosphorylation site
domain (Chakravarthy et al., 1995). However, this suggestion
seems to be contradicted a priori by the known abilities of
mitogenic factors such as bradykinin, bombesin, platelet-derived growth
factor and vasopressin to stimulate an internal Ca
transient, membrane-associated PKCs activity, and MARCKS
phosphorylation (Rozengurt, 1986; Issandou and Rozengurt, 1990). The
reason for this apparent contradiction may lie in the relative timing
of the internal Ca
transient (and the
Ca
calmodulin complexes it produces) and the
burst of membrane-associated PKCs activity in the
Ca
-stimulated BALB/MK cells: in mitogen-stimulated
cells the Ca
and PKC signals are coincident
(Rozengurt, 1986; Issandou and Rozengurt, 1990) while in
Ca
-treated BALB/MK keratinocytes PKCs activity peaked
7 min after the [Ca
]
surge (Chakravarthy et al., 1995). This suggested
that the MARCKS substrate can be phosphorylated only when the
membrane-associated PKCs activity peaks before or at the same time as
[Ca
]
, and a resulting
Ca
calmodulin, surge.
In this report we
extend our observations, using C6 rat glioma cells and BALB/MK
keratinocytes, to provide the first direct evidence for
Cacalmodulin actually being the blocker of
PKCs-mediated MARCKS phosphorylation in the cell and to confirm the
suggestion arising from the previous report (Chakravarthy et
al., 1995) that the relative timing of surges of internal
Ca
and PKCs activity determines the extent of MARCKS
protein phosphorylation.
For experiments, stock C6 glioma cells or BALB/MK
keratinocytes (3 10
) were plated in 60-mm dishes in
5 ml of complete medium, and 3-4 days later confluent cultures
were washed twice with phosphate-buffered saline and incubated for 2 h
in 2 ml of fresh serum-free Dulbecco's minimum essential medium
(Eagle's minimum essential medium in the case of keratinocytes).
The cultures were then either left untreated or exposed to either 3.5
µM ionomycin and/or 1 µM TPA for 10 min at 37
°C. The medium was then removed, cells washed once with ice-cold
phosphate-buffered saline, and their membranes prepared as described
previously (Chakravarthy et al., 1991, 1994). Briefly, the
washed cells were covered with 1.0 ml of ice-cold hypotonic lysis
medium (1 mM NaHCO
, 5 mM MgCl
, and 100 µM phenylmethylsulfonyl
fluoride), left on ice for 2 min, and the swollen cells were then
scraped off the dish with a rubber policeman, and lysed by vortexing
vigorously for 2 min. Nuclei and unlysed cells were removed by
centrifugation at 600
g for 5 min, and membranes in
the post-nuclear fractions (PNF) were sedimented at 100,000
g for 10 min. The 100,000
g supernatant was
the cytosol fraction. The sedimented membranes were suspended in 200
µl of the assay buffer (50 mM Tris-HCl, pH 7.5, 5
mM MgCl
, 1 µM CaCl
, 100
µM sodium vanadate, 100 µM sodium
pyrophosphate, 1 mM sodium fluoride, and 100 µM phenylmethylsulfonyl fluoride) by triturating with a pipette.
The 100-µl reaction mixture contained
5-10 µg of membrane protein, 75 µM PKCs-selective peptide substrate, and 50 µM [P]ATP (220 counts/min/pmol) in assay
buffer. After incubation for 10 min at 25 °C, the reaction was
stopped by adding 10 µl of 5% acetic acid and the samples clarified
by centrifugation at 16,000
g for 5 min. A 90-µl
sample of each supernatant was applied to P81 Whatman paper which was
then washed twice for 10 min in 5% acetic acid (10 ml/piece of paper).
The radioactivity bound to the washed paper was determined with a LKB
1217 RACKBETA liquid scintillation spectrometer. To calculate the
amount of radioactivity incorporated specifically into the peptide
substrate, the nonspecific binding of
P to the P81 papers
was determined as above in the absence of the peptide.
Exposing rat C6 glioma cells to 3.5 µM ionomycin
triggered a 4-5-fold increase in membrane-associated PKCs
activity which was as large as that induced by the potent
PKCs-activating phorbol ester TPA as determined by the ability of
membranes isolated from the treated and untreated cells to
phosphorylate the MARCKS-derived PKCs-selective peptide substrate
Ac-FKKSFKL-NHin vitro (Fig. 1A).
This increase in membrane-associated Ac-FKKSFKL-NH
phosphorylating activity began within 30 s of adding ionomycin
and peaked by 2 min (Fig. 1B). We have previously shown
that the Ac-FKKSFKL-NH
phosphorylating activity in
membranes from several types of cells is that of PKCs (Chakravarthy et al., 1991, 1995; Whitfield et al., 1992). The same
was true for C6 glioma cell membrane-associated protein kinase
activity. Thus, more than 90% of the Ac-FKKSFKL-NH
phosphorylating activity associated with the glioma cell
membranes was inhibited by adding the PKCs-selective pseudosubstrate
peptide inhibitor PKC-(19-36) (House and Kemp, 1987) to the assay
mixture (Fig. 1C). Moreover, depleting PKCs by a 20-h
preincubation of cells with 300 nM TPA (Chakravarthy et
al., 1989; Chida et al., 1986) totally abolished both
TPA- and ionomycin-induced increases in the membrane-associated
peptide-phosphorylating activity (Fig. 1D). Therefore,
the ionomycin-induced increase in the phosphorylation of
Ac-FKKSFKL-NH
must have been due to activated PKCs. The
ionomycin-induced increase in membrane PKCs activity was accompanied by
a large drop in cytosolic PKCs activity indicating a redistribution of
these enzymes to membranes (Fig. 1B). This was
corroborated by immunoblot analysis which revealed that ionomycin
treatment increased the amount of membrane-associated PKCs and
decreased the amount in cytosol fraction (see Fig. 1B, inset).
Figure 1:
Ionomycin increases
membrane-associated PKCs activity in C6 rat glioma cells. A,
C6 glioma cells in serum-free medium were left untreated (Cont) or were exposed to either 1 µM TPA or 3.5
µM ionomycin (Iono) for 10 min. Membrane
fractions were prepared, and the PKCs' activity determined as
described under ``Materials and Methods.'' Values are the
means ± S.D. of triplicate determinations from one of three
independent experiments. B, C6 glioma cells in serum-free
medium were exposed to 3.5 µM ionomycin for the indicated
periods of time and PKCs activity in isolated membrane (, MEMB) and cytosolic (
, CYTO) fractions was
determined as described under ``Materials and Methods.''
Values are the means ± S.D. of triplicate determinations from
one of two independent experiments. Inset, membrane (lanes
A and B) and cytosolic (lanes C and D)
fractions from untreated (lanes A and C) and 10-min
ionomycin-treated (lanes B and D) glioma cells were
subjected to immunoblot analysis using pan-PKC antibody as described
under ``Materials and Methods.'' C, protein kinase
activity in membranes isolated from untreated (Cont) and TPA-
(1 µM) and ionomycin- (3.5 µM) treated glioma
cells was measured in the absence (open bars) or presence (hatched bars) of pseudosubstrate peptide inhibitor,
PKC-19-36 (5 µg), in the assay mixture. Values are the means
± S.D. of triplicate determinations from one of two independent
experiments. D, C6 glioma cells were incubated without (open bars) or with (hatched bars) 300 nM TPA for 20 h before being exposed to either 1 µM TPA
or 3.5 µM ionomycin for 10 min. Protein kinase activity in
the subsequently isolated membrane fractions was measured as described
under ``Materials and Methods.'' Values are the means
± S.D. of triplicate determinations from one of two independent
experiments.
Despite inducing a robust increase in membrane PKCs
activity (Fig. 1A, bar 3), which was as large
as that induced by TPA (Fig. 1A, bar 2),
ionomycin, unlike TPA, did not stimulate the phosphorylation of MARCKS
protein in intact C6 glioma cells whose ATP pools had been prelabled
with [P]orthophosphate (Fig. 2, lane
3). In fact, ionomycin actually reduced the basal
phosphorylation of MARCKS in intact cells. Furthermore, a 2-min
exposure of the glioma cells to ionomycin before adding TPA to the
culture medium completely prevented the TPA-induced phosphorylation of
MARCKS protein (Fig. 2, lane 4). This was not due to
ionomycin pretreatment somehow affecting the ability of TPA to
stimulate PKCs activity in these cells because ionomycin and TPA
actually cooperated to produce an enhanced burst of membrane PKCs
activity (Fig. 3A, bar 3). Furthermore,
ionomycin probably did not interfere directly with PKC-mediated MARCKS
phosphorylation in intact cells because it did not affect the
phosphorylation of MARCKS by TPA-stimulated PKCs in broken cell
preparation (Fig. 4, lane 3).
Figure 2:
Ionomycin blocks the phosphorylation of
85-kDa MARCKS protein in C6 glioma cells. C6 glioma cells in serum-free
medium were prelabeled with [P]orthophosphate
for 2 h, and the labeled cells were: 1) left untreated; 2) incubated for 10 min with 1 µM TPA; 3) incubated for 10 min with 3.5 µM ionomycin; 4) incubated for 2 min with 3.5 µM ionomycin
followed by 1 µM TPA for 10 min. The phosphorylation of
the 85-kDa protein was determined autoradiographically as described
under ``Materials and Methods.''
Figure 3:
Chelating external Ca with EGTA abolishes both the ionomycin-induced increase in
membrane-associated PKCs activity and the blockade by ionomycin of
85-kDa MARCKS protein phosphorylation by TPA-activated PKCs in C6
glioma cells. A, C6 glioma cells maintained in serum-free
medium for 2 h were: 1) left untreated; 2) exposed to
1 µM TPA for 10 min; 3) exposed to 3.5
µM ionomycin and 1 µM TPA for 10 min; 4) exposed to 5 mM EGTA and 1 µM TPA for
10 min; 5) exposed to 5 mM EGTA, 3.5 µM
ionomycin, and 1 µM TPA for 10 min; 6) exposed to
3.5 µM ionomycin for 10 min; 7) exposed to 5
mM EGTA and 3.5 µM ionomycin for 10 min. Membrane
fractions were prepared and PKC activity determined as described under
``Materials and Methods.'' It should be noted that the
difference between the PKCs activities in this figure and in Fig. 1A were due to to normal culture variations.
Values are the means ± S.D. of triplicate determinations from
one of three independent experiments. B, C6 glioma cells
prelabeled with [
P]orthophosphate for 2 h were: 1) left untreated; 2) exposed to 1 µM
TPA for 10 min; 3) exposed to 3.5 µM ionomycin
and 1 µM TPA for 10 min; 4) exposed to 5 mM EGTA and 1 µM TPA for 10 min; 5) exposed to
5 mM EGTA, 3.5 µM ionomycin, and 1 µM TPA for 10 min; 6) exposed to 3.5 µM ionomycin for 10 min. The phosphorylation of the 85-kDa protein
was determined autoradiographically as described under ``Materials
and Methods.''
Figure 4:
Cacalmodulin
inhibits the phosphorylation of 85-kDa MARCKS protein in a C6 glioma
cell lysate. C6 glioma cells were lysed, PNF prepared, and the 85-kDa
MARCKS protein phosphorylation in PNF was determimed as described under
``Materials and Methods'' in the absence (1) or the
presence of 100 µM CaCl
and 1 µM TPA (2), 100 µM CaCl
, 1
µM TPA and 3.5 µM ionomycin (3), 100
µM CaCl
, 1 µM TPA and 2 µg of
calmodulin (4), 100 µM CaCl
, 1
µM TPA, and 5 µg of calmodulin (5), 100
µM CaCl
, 1 µM TPA, and 10
µM calmodulin (6), 100 µM CaCl
, 1 µM TPA, 10 µM
calmodulin, and 10 µg of calmodulin antagonist peptide (7).
Both the stimulation
of membrane-associated PKCs and the prevention of these PKCs from
phosphorylating the MARCKS protein by ionomycin was probably due to the
instant large [Ca]
surge
induced by the ionophore (Fig. 5). Thus, ionomycin neither
stimulated membrane-associated PKCs activity (Fig. 3A, bar 7) nor prevented MARCKS phosphorylation by TPA-stimulated
PKCs (Fig. 3B, lane 5) when external
Ca
was chelated with 5 mM EGTA before
exposing the cells to ionomycin and TPA. Removing external
Ca
with EGTA only marginally affected TPA-stimulated
PKCs activity (Fig. 3A, bar 4) and MARCKS
phosphorylation (Fig. 3B, lane 4).
Figure 5:
Ionomycin (IONO)-induced
[Ca]
surge in C6
glioma cells. [Ca
]
was
measured as described under ``Materials and Methods.'' The arrows indicate the time of addition of ionomycin (3.5
µM) or EGTA (10 mM). The result shown is a
typical tracing from one of five separate
experiments.
We
previously suggested that Cacalmodulin
complexes may be the actual blockers of MARCKS protein phosphorylation
by the activated PKCs in Ca
-stimulated keratinocytes
(Chakravarthy et al., 1995). Indeed, in the present study
MARCKS phosphorylation in C6 glioma cell lysates was suppressed by
adding calmodulin to the reaction mixture, and this suppression was
prevented by the calmodulin-binding domain peptide
H-LKKFNARRKLKGAKTTMLA-OH (Payne et al., 1988) which would
compete with MARCKS for Ca
calmodulin (Fig. 4). But, the question remained as to whether
Ca
calmodulin was the blocker of MARCKS
phosphorylation in intact glioma cells. If so, a calmodulin antagonist,
such as calmidazolium, should have been able to prevent ionomycin from
blocking MARCKS phosphorylation by TPA-activated PKCs. Indeed, 25 or 50
µM calmidazolium enabled ionomycin to stimulate MARCKS
phosphorylation in glioma cells (Fig. 6A, lanes 4 and 5) without affecting the ionomycin-induced increase
in membrane-associated PKCs activity (data not shown). Furthermore,
calmidazolium pretreatment also partially prevented ionomycin from
blocking MARCKS phosphorylation by TPA-stimulated PKCs (Fig. 6B, lane 4).
Figure 6:
A Cacalmodulin
blocker, calmidazolium, but not a calcineurin blocker, cyclosporin A,
prevents ionomycin from inhibiting MARCKS phosphorylation in intact C6
glioma cells. A, C6 glioma cells were prelabeled with
[
P]orthophosphate in serum-free medium for 2 h
and were: 1) left untreated; 2) treated with 3.5
µM ionomycin for 10 min; 3) treated with 0.1%
Me
SO for 15 min followed by 3.5 µM ionomycin
for 10 min; 4) treated with 25 µM calmidazolium
in 0 .1% Me
SO for 15 min followed by 3.5 µM ionomycin for 10 min; 5) 50 µM calmidazolium
in 0 .1% Me
SO for 15 min followed by 3.5 µM ionomycin for 10 min. B, C6 glioma cells prelabeled with
[
P]orthophosphate were: 1) left
untreated; 2) treated with 1 µM TPA; 3)
treated with 3.5 µM ionomycin for 2 min followed by 1
µM TPA for 10 min; 4) treated with 50 µM calmidazolium for 15 min followed by 3.5 µM ionomycin
for 2 min followed by 1 µM TPA for 10 min; 5)
treated with 1 µM cyclosporin A for 15 min followed by 3.5
µM ionomycin for 2 min followed by 1 µM TPA
for 10 min. Phosphorylation of the 85-kDa MARCKS protein was determined
autoradiographically as described under ``Materials and
Methods.''
We have previously shown
(Chakravarthy et al., 1995) that the failure of PKCs to
phosphorylate MARCKS protein in Ca-stimulated BALB/MK
keratinocytes is not due to a general dephosphorylation of the
substrate by Ca
calmodulin-dependent phosphatase
such as PP2B/calcineurin. Pretreatment with cyclosporin A (1
µM), a highly specific inhibitor of PP2B/calcineurin
(Groblewski et al., 1994) also did not overcome
ionomycin-induced blockade of TPA-stimulated MARCKS protein in glioma
cells (Fig. 6B, lane 5).
The extent of inhibition of TPA-stimulated phosphorylation of MARCKS in the C6 glioma cells by ionomycin depended on whether the ionophore was added before, with, or after TPA. Thus, while exposing the glial cells to ionomycin 2 min before TPA totally prevented MARCKS phosphorylation by the stimulated PKCs, adding ionomycin along with, or 2 or 3 min after TPA blocked MARCKS phosphorylation less effectively (Fig. 7). Similar timing-dependent effects of ionomycin on membrane-associated PKCs activity and MARCKS phosphorylation were observed with BALB/MK keratinocytes. Thus, while ionomycin slightly enhanced TPA-stimulated membrane-associated PKCs activity in the keratinocytes regardless of whether it was added before or after TPA (Fig. 8A), it completely prevented a TPA-induced increase in MARCKS phosphorylation when added before TPA, but it was less effective in blocking MARCKS phosphorylation when added along with or after TPA (Fig. 8B).
Figure 7:
Prevention of TPA-induced MARCKS
phosphorylation by ionomycin in C6 glioma cells depends on when the
ionophore is added to the culture medium. A, C6 glioma cells
were labeled with [P]orthophosphate in
serum-free medium for 2 h and were: 1) left untreated; 2) treated with 1 µM TPA for 10 min; 3)
treated with 3.5 µM ionomycin for 10 min; 4)
treated with 3.5 µM ionomycin for 2 min followed by 1
µM TPA for 10 min; 5) treated with 1 µM TPA for 10 min with 3.5 µM ionomycin added 3 min
after TPA addition; 6) treated with 1 µM TPA for
10 min with 3.5 µM ionomycin added 2 min after TPA
addition; 7) treated with 1 µM TPA and 3.5
µM ionomycin added together for 10 min. The
phosphorylation of the 85-kDa MARCKS protein was determined
autoradiographically as described under ``Materials and
Methods.''
Figure 8:
Prevention of TPA-induced MARCKS
phosphorylation by ionomycin in BALB/MK keratinocytes also depends on
when the ionophore is added to the culture medium. A, BALB/MK
keratinocytes in serum-free medium were: 1) left untreated; 2) treated with 1 µM TPA for 10 min; 3)
treated with 3.5 µM ionomycin for 10 min; 4)
treated with 3.5 µM ionomycin for 2 min followed by 1
µM TPA for 10 min; 5) treated with 3.5 µM ionomycin and 1 µM TPA added together for 10 min.
Membrane fractions were prepared and PKCs activity determined as
described under ``Materials and Methods.'' Values are the
means ± S.D. of triplicate determinations from one of three
independent experiments. B, BALB/MK keratinocytes were labeled
with [P]orthophosphate in serum-free medium for
2 h and left untreated (1), treated with 1 µM TPA
for 10 min (2), treated with 3.5 µM ionomycin for
10 min (3); treated with 3.5 µM ionomycin for 2
min followed by 1 µM TPA for 10 min (4); treated
with 1 µM TPA for 10 min with 3.5 µM ionomycin added 3 min after TPA addition (5); treated
with 1 µM TPA for 10 min with 3.5 µM ionomycin added 2 min after TPA addition (6); treated
with 1 µM TPA and 3.5 µM ionomycin added
together for 10 min (7). The phosphorylation of the 85-kDa
MARCKS protein was determined autoradiographically as described under
``Materials and Methods.''
Emerging from these and our previous observations on mouse
keratinocytes is a novel regulatory mechanism for PKCs signaling which
operates on the substrate level. It was first seen as an unexpected
stimulation of keratinocyte PKCs by a physiological Ca concentration without phosphorylation of the PKCs' MARCKS
protein substrate (Chakravarthy et al., 1995). In these
Ca
-stimulated keratinocytes, there was an internal
Ca
surge which preceded the burst of
membrane-associated PKCs activity by several minutes (Chakravarthy et al., 1995). In the present study, when we triggered a
[Ca
]
surge in C6 glioma cells
with ionomycin before stimulating PKCs with TPA, the kinases similarly
could not phosphorylate their MARCKS protein substrate. Moreover,
ionomycin, which caused an instant large
[Ca
]
surge and a later
(1-2 min) burst of PKCs activity did not increase MARCKS
phosphorylation in the glioma cells. The results of the timing
experiments in the present study have now established that it is the
relative timing of [Ca
]
and
PKCs activity surges that determines whether PKCs can phosphorylate the
MARCKS protein. Thus, as would be expected from the existence of
coincident surges of [Ca
]
, PKCs
activity, and MARCKS phosphorylation in bradykinin-, bombesin-,
platelet-derived growth factor-, and vasopressin-stimulated Swiss
albino 3T3 mouse cells (Rozengurt, 1986; Issandou and Rozengurt, 1990),
ionomycin became increasingly unable to prevent TPA-induced MARCKS
phosphorylation in glioma cells and keratinocytes when added along
with, or at increasing times after, TPA.
The present results also
establish Cacalmodulin as the intracellular
mediator of the interference with PKCs' substrate phosphorylation
by Ca
. Since a [Ca
]
surge produced by ionomycin or triggered by activated surface
Ca
receptors/sensors or growth factor receptors
produces Ca
calmodulin complexes, and since
calmodulin binds to the MARCKS protein (Aderem, 1992; Blackshear, 1993;
Nairn and Aderem, 1992), we suggested in our previous report that it
was these complexes that mediated Ca
's blocking
action (Chakravarthy et al., 1995). In support of this we
showed that Ca
calmodulin did indeed prevent
MARCKS protein phosphorylation in PNF of BALB/MK keratinocytes. This
suggestion has now been confirmed by the present demonstration of the
ability of a Ca
calmodulin inhibitor,
calmidazolium, to stop an ionomycin-induced Ca
surge
from preventing a subsequent TPA-induced burst of membrane-associated
PKCs activity from phosphorylating MARCKS protein in intact C6 glioma
cells.
Since ionomycin is a non-physiological agent, the question
immediately arises as to whether a
[Ca]
surge triggered by a
natural agonist would similarly prevent PKCs from phosphorylating
MARCKS. The answer appears to be yes, because in our previous study
(Chakravarthy et al., 1995) we demonstrated that an external
Ca
-induced,
Ca
-receptor/sensor-mediated
[Ca
]
surge, which is part of
the physiological trigger of keratinocyte differentiation (Whitfield,
1995; Whitfield et al., 1995), blocked MARCKS phosphorylation
by PKCs, although these enzymes' activity increased by
3-4-fold. Furthermore, in a new study (
)we have
already found that endothelin-1 and ATP, which are known to be
physiological agonists for C6 glioma cells (Lin et al., 1990,
1992, 1993) trigger rapid increases in
[Ca
]
and that pretreating these
cells with 10 nM endothelin-1 or 100 µM ATP 2 min
before adding TPA prevents the TPA-stimulated PKCs from phosphorylating
MARCKS just as did Ca
in the BALB/MK keratinocytes of
our previous study (Chakravarthy et al., 1995) and ionomycin
in the glioma cells of the present study. Thus,
Ca
calmodulin appears to be a physiological
modulator of MARCKS phosphorylation by PKCs.
The ability of
Cacalmodulin to modulate the phosphorylation
and function of PKCs'substrate does not appear to be restricted
to the actin-binding MARCKS protein alone.
Ca
calmodulin complexes appear to prevent PKCs
from phosphorylating and down-regulating EGF receptors (Chakravarthy et al., 1995; Moscat et al., 1989). An interference
with PKCs action by Ca
calmodulin has also been
observed with several other functionally important proteins which,
unlike MARCKS and EGF receptor, have calmodulin-binding
``IQ'' motifs in or near their phosphorylation site domains.
Among these are the ``DEAD-box'' helicases such as p68-kDa
RNA helicase and eIF-4A helicase (Buelt et al., 1994; Schmid
and Linder, 1992), membrane Ca
-ATPase (Hofmann et
al., 1994), neurogranin, and neuromodulin (Alexander et
al., 1987; Apel et al., 1990; Baudier et al.
1991). It has also been shown that phosphorylation of
microtubule-associated tau protein (the component of neurofibrillary
tangles in Alzheimer disease) by PKCs is prevented by
Ca
calmodulin (Baudier et al., 1987).
Although most of these were in vitro studies, they do however,
suggest that the interaction of these two major signalers at the
substrate level can affect EGF receptor signaling (Chakravarthy et
al., 1995; Moscat et al., 1989), Ca
extrusion (Hofmann et al., 1994), eIF-4A's
enhancement of the translatability of mRNA with highly structured
5`-untranslated regions (reviewed by Whitfield, 1995), and perhaps
neuronal functions. The striking timing-dependence of this interaction
observed in glioma cells suggests that it might be particularly
important in brain where slightly different times of arrival of
Ca
and PKCs signals from converging afferents could
greatly affect neuronal responses such as synaptic vesicle mobilization
and neurotransmitter release, long term potentiation, and neurite
outgrowth by affecting the extent of phosphorylation of targets such as
the neuron-specific neurogranin and neuromodulin.