(Received for publication, May 4, 1994; and in revised form, October 13, 1994)
From the
Raising the external Ca concentration from
0.05 to 1.8 mM stimulated membrane-associated protein kinase
Cs (PKCs) activity as strongly as the specific PKCs activator,
12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse
keratinocytes. This was indicated by the increased phosphorylation of a
PKC-selective peptide substrate, Ac-FKKSFKL-NH
, by
membranes isolated from the Ca
- or TPA-stimulated
keratinocytes. Raising the external Ca
concentration
to 1.8 mM also triggered a 4-fold rise in the intracellular
free Ca
concentration. As reported elsewhere (Moscat,
J. Fleming, T. P., Molloy, C. J. LopezBarahona, M., and Aaronson, S.
A.(1989) J. Biol. Chem. 264, 11228-11235), TPA
stimulated the phosphorylation of the PKCs substrate, the 85-kDa
myristoylated alanine-rich kinase C substrate (MARCKS) protein, in
intact keratinocytes, but Ca
did not. Furthermore,
Ca
-pretreatment reduced the TPA-induced
phosphorylation of the 85-kDa protein in intact cells. There was no
significant increase in MARCKS phosphorylation when keratinocytes were
treated with a Ca
CaM-dependent phosphatase
inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca
. Ca
calmodulin
suppressed the ability of isolated membranes to phosphorylate the
85-kDa MARCKS holoprotein in vitro in the presence of
phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate,
and this inhibition was overcome by a calmodulin antagonist, the
calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca
to strongly stimulate the membrane PKCs
activity without stimulating the phosphorylation of the MARCKS protein
in keratinocytes is consistent with the possibility of
Ca
calmodulin complexes, formed by the internal
Ca
surge, binding to, and blocking the
phosphorylation of, this PKC protein substrate.
BALB/MK mouse keratinocytes, like primary human and mouse
keratinocytes, proliferate in medium containing EGF ()and a
low concentration of Ca
(e.g. 0.05
mM), but they stop cycling and start differentiating (e.g. form cornified envelopes) when the Ca
concentration in the medium is raised above 1.0 mM (Falco et al., 1988; Moscat et al., 1989;
Weissmann and Aaronson, 1983, 1985; Whitfield et al., 1992).
The Ca
-activated receptors on the BALB/MK
cells' surfaces stimulate phospholipase C which catalyzes the
breakdown of polyphosphoinositides and the generation of inositol
trisphosphates (Ins(1,4,5)P
and Ins(1,3,4)P
), sn-1,2-diacylglycerols, and an alkylether glyceride (Moscat et al., 1989). The Ins(1,4,5)P
triggers a
Ca
surge, and the diacylglycerols would be expected
to stimulate the PKCs which are widely believed to be principal
mediators of keratinocyte differentiation (Dlugosz et al.,
1992; Yuspa, 1993; Yuspa et al., 1988, 1991). Surprisingly,
however, raising the external Ca
concentration does
not appear to stimulate PKCs as judged by the lack of down-regulation
of the EGF receptor, a PKCs target, and the lack of phosphorylation of
80-85-kDa MARCKS protein (Moscat et al., 1989), a
ubiquitous principal substrate of cellular PKCs and a widely used
indicator of PKCs activation (Aderem, 1992; Blackshear, 1993; Nairn and
Aderem, 1992). This unexpected observation has cast considerable doubt
on the PKCs having a role in the Ca
-induced
keratinocyte differentiation program (Brown, 1991; Moscat et
al., 1989).
Here we show that raising the Ca concentration in the medium from 0.05 to 1.8 mM does in
fact stimulate membrane-associated PKCs activity in these
keratinocytes, and it does so as strongly as the potent PKCs activator,
12-O-tetradecanoyl phorbol-13-acetate (TPA). However, we, like
Moscat et al.(1989), have found that Ca
does
not stimulate the phosphorylation of the 85-kDa MARCKS protein in
intact cells as does TPA. Evidently, some
Ca
-dependent process(es) blocks the phosphorylation
of the MARCKS protein, and presumably other substrates such as the EGF
receptor (Mocat et al., 1989) and a 26-kDa protein found in
the present study, by the stimulated PKCs in intact cells. In this
study we focused on the MARCKS protein and will present some evidence
which is consistent with the failure of the activated PKCs in
Ca
-stimulated keratinocytes to phosphorylate their
MARCKS protein substrate being due to a direct blockage of the
substrate's phosphorylation site domain by
Ca
-induced Ca
calmodulin
complexes rather than due to dephosphorylation of PKCs'
phosphorylated substrate by a Ca
-stimulated
phosphatase.
Stock BALB/MK
cells were plated in 100-mm dishes in 10 ml of complete medium, and 3
days later the spent medium was replaced with 12.5 ml of fresh medium.
On the fifth day, when the cultures contained 5-6
10
cells, the medium was discarded, the cell monolayers
washed twice with phosphate-buffered saline, and 5 ml of fresh
serum-free E-MEM containing 0.05 mM Ca
was
added. Two h later, the monolayers were either left untreated or
exposed to either 1.8 mM Ca
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 cell membranes
prepared as described previously (Chakravarthy et al., 1991).
Briefly, the washed cells were covered with 1.2 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 then the swollen cells were
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, 100 µM phenylmethylsulfonyl fluoride) by triturating with a pipette.
The 100-µl reaction mixture contained 10 µg
of membrane protein, 75 µM PKC-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.
Phosphorylation of exogenous 85-kDa MARCKS holoprotein (prepared
from S49 T-lymphoma cytosol) by isolated keratinocyte membranes was
determined as described previously (Chakravarthy et al.,
1989). Briefly, the cells were hypotonically lysed, and the cell lysate
was centrifuged at 100,000 g for 10 min. Supernatant
cytosolic fraction was collected and heated in boiling water for 5 min.
The precipitated proteins were removed by centrifugation at 16,000
g for 5 min, and the supernatant containing the MARCKS
protein was lyophilized. For experiments, the lyophilized samples were
solubilized in PKC assay buffer and used for phosphorylation studies.
The assay conditions were as described above for the direct assay
except that heat-treated lymphoma cytosol (10 µg) containing the
85-kDa MARCKS holoprotein replaced the Ac-FKKSFKL-NH
peptide and the [
P]ATP concentration was
25 µM (900 counts/min/pmol) instead of 50 µM (220 counts/min/pmol). The reaction was carried out in the
presence and absence of calmodulin and/or calmodulin-binding domain
peptide (Payne et al., 1988) and was stopped by adding equal
volumes of 2
electrophoresis buffer (Laemmli, 1970). The 85-kDa
protein was extracted with 40% acetic acid, resolved on a 10%
SDS-polyacrylamide gel, and its phosphorylation visualized by
autoradiography using Kodak XAR film as described above.
In some experiments, heat-treated cytosol from
untreated and 1.8 mM Ca-treated BALB
keratinocytes were prepared, and the MARCKS protein in the heated
cytosol (10 µg/100 µl reaction volume) was phosphorylated in
the presence and absence of calmodulin-binding domain peptide by PKCs
in membranes isolated from untreated keratinocytes as described above
for the phosphorylation of exogenous MARCKS protein.
Changes in
[Ca]
were monitored using the
Ca
-sensitive fluroprobe, fura-2, as described in
detail previously (Grynkiewicz et al., 1985; Morley and
Whitfield (1993). Briefly, the cells were incubated for 30 min at 37
°C in low Ca
, serum-free E-MEM containing 2.5
µM fura-2AM. The cells were washed twice, and the
fluorescence measurements were performed on small groups of 8-10
cells in the monolayer cultures using a variable diameter aperture. The
figure shown is a fluorometric tracing of the ratio of the fura-2
fluorescence intensities at 505 nm following excitation at 350 and 380
nm.
Raising the [Ca]
concentration in the medium from 0.05 mM to 1.8 mM did not significantly stimulate the phosphorylation of the
endogenous 85-kDa MARCKS protein in BALB/MK cells whose ATP pools had
been prelabeled with [
P] orthophosphate (Fig. 1). Densitometric analysis of the autoradiogram revealed
that the increase in MARCKS phosphorylation in
Ca
-stimulated cells was only 10-15% over
control unstimulated cells. Thus, as reported by Moscat et
al.(1989), Ca
appeared not to stimulate PKCs in
BALB/MK keratinocytes. By contrast, adding a potent PKC stimulator, TPA
(1 µM), to the low Ca
(0.05 mM)
medium caused a 2-fold increase in the phosphorylation of the 85-kDa
protein (Fig. 1). However, this TPA-induced phosphorylation was
suppressed by 80% when BALB/MK cells were exposed to 1.8 mM Ca
for 2 min before adding TPA (Fig. 1).
This suggested that Ca
induced a blocker of
PKC-mediated MARCKS protein phosphorylation.
Figure 1:
The effect of
Ca and TPA on the phosphorylation
of 85-kDa MARCKS protein in BALB/MK keratinocytes. BALB/MK
cells were prelabeled with [
P]orthophosphate in
low Ca
(0.05 mM) medium for 2 h, and the
labeled cells were then (lane A) left untreated; laneB, incubated for 10 min with 1 µM TPA; lane C, incubated for 10 min with 1.8 mM Ca
; or lane D, incubated for 2 min with
1.8 mM Ca
followed by 1 µM TPA
for 10 min. The phosphorylation of the 85-kDa protein was determined by
autoradiography as described under ``Materials and
Methods.''
As expected from these
results, adding TPA (1 µM) for 10 min to the low
Ca (0.05 mM) medium more than doubled the
ability of membranes isolated from the treated cells to phosphorylate
the Ac-FKKSFKL-NH
PKC substrate peptide corresponding to a
part of the MARCKS protein's phosphorylation site domain (Fig. 2A). However, although raising the external
Ca
concentration from 0.05 to 1.8 mM did not
stimulate the phosphorylation of the 85-kDa holoprotein in intact
keratinocytes (Fig. 1), it, like TPA, more than doubled the
ability of membranes from the stimulated cells to phosphorylate the
MARCKS-derived Ac-FKKSFKL-NH
peptide in vitro (Fig. 2A). This phosphorylating activity was
mainly that of PKCs because, as has been shown previously (Chakravarthy et al., 1991; Whitfield et al., 1992), more than 95%
of it was inhibited by adding the PKC-selective pseudosubstrate peptide
inhibitor PKC-(19-36) (House and Kemp, 1987) to the reaction
mixture. This was further confirmed by the fact that depleting cellular
PKCs by prolonged treatment with 300 nM TPA for 20 h
(Chakravarthy et al., 1989; Chida et al., 1986)
substantially reduced the ability of isolated keratinocyte membranes to
phosphorylate the peptide substrate and completely abolished both
Ca
- and TPA-induced increases in the
membrane-associated peptide-phosphorylating activity (Fig. 3).
Furthermore, bisindolylmaleimide and calphostin C, believed to be among
the most specific of PKCs inhibitors (Toullec et al.; 1991,
Kobayashi et al., 1989), also almost completely inhibited the
basal as well as Ca
- and TPA-induced increases in the
membrane-associated peptide-phosphorylating activity (Fig. 4).
Therefore, the Ca
-induced increase in the
phosphorylation of AcFKKSFKL-NH
must have been due to
activated PKCs. The increase in membrane-associated PKCs activity
appeared to be due to the translocation of cytosolic PKCs to membranes
because both Ca
and TPA significantly reduced the
cytosolic PKCs activity (Fig. 2B). Thus, although
Ca
stimulated membrane-associated PKCs activity as
strongly as TPA in BALB/MK keratinocytes, the stimulated PKCs could not
phosphorylate their 85-kDa MARCKS substrate.
Figure 2:
Raising extracellular
Ca from 0.05 mM to 1.8
mM increases membrane-associated PKCs in BALB/MK cells. BALB/MK cells in low Ca
, serum-free medium
were left untreated or were exposed to either 1.8 mM Ca
or 1 µM TPA for 10 min. Membrane (A) and cytosolic (B) fractions were prepared and PKC
activity determined as described under ``Materials and
Methods.'' Results are expressed as mean ± S.E. of
triplicate determinations of 30 independent experiments for membranes
and six for cytosol.
Figure 3:
Prolonged TPA-pretreatment of BALB/MK
cells abolishes Ca-induced increase
in membrane-associated AcFKKSFKL-NH
phosphorylating activity. BALB/MK cells were incubated with (hatched bars) or without (open bars) 300 nM TPA for 20 h before being exposed to either 1 µM TPA
or 1.8 mM CaCl
for 10 min. Protein kinase activity
in the subsequently isolated membrane fractions was determined as
described under ``Materials and
Methods.''
Figure 4:
Inhibition of membrane-associated
Ac-FKKSFKL-NH phosphorylating activity by
the PKCs inhibitors bisindolylmaleimide and calphostin C.Protein kinase activity in membranes isolated from untreated and
TPA- and Ca
-treated cells was measured in the
presence (hatched bars) and absence (open bars) of 10
µM bisindolylmaleimide or 5 µM calphostin C
in the reaction mixture as described under ``Materials and
Methods.''
The apparent lack of
substrate phosphorylation by the stimulated PKCs in
Ca-treated cells was not confined to the MARCKS
protein because, as shown in Fig. 5A (lane C),
an as yet unidentified PKCs substrate protein of apparent molecular
mass of 26 kDa was also not phosphorylated in the
Ca
-treated cells. This protein appeared to be a PKC
substrate because TPA strongly stimulated its phosphorylation (Fig. 5A, lane B). Moreover, Ca
treatment not only failed to stimulate the phosphorylation of
this protein but also suppressed its TPA-induced phosphorylation (Fig. 5A, lane D). Furthermore, as mentioned
in the introduction, Moscat et al.(1989) found that
Ca
stimulation did not down-regulate the EGF
receptor, which should have happened if PKCs had phosphorylated the
receptor. This failure of stimulated PKCs in
Ca
-treated cells to phosphorylate at least three
substrates which were phosphorylated by the stimulated PKCs in
TPA-treated cells could have been the result of a general
dephosphorylation of the substrates due to the activation of a
Ca
calmodulin-dependent phosphatase such as
PP2B/calcineurin. To assess the role of this phosphatase in the failure
of MARCKS protein phosphorylation in Ca
-stimulated
keratinocytes, the cells were treated for 15 min with 1 µM cyclosporin A (believed to be a highly specific inhibitor of
calmodulin-dependent phosphatase, PP2B/calcineurin (Groblewski, et
al., 1994)) before stimulation with Ca
. As can
be seen in Fig. 5B, cyclosporin A did not significantly
affect the phosphorylation of the MARCKS protein either in unstimulated
keratinocytes (lanes A and C) or in
Ca
- stimulated keratinocytes (lanes B and D) which suggested that the apparent failure of PKCs to
phosphorylate the MARCKS protein in Ca
-stimulated
cells was not due to rapid dephosphorylation by calmodulin-dependent
phosphatase. This is consistent with the recent report (Clarke et
al., 1993) that neither the MARCKS protein nor a peptide
substrate derived from it was a significant substrate for the
calmodulin-dependent phosphatase calcineurin. Since the failure to
phosphorylate MARCKS was not attributable to
Ca
-dependent phosphatase activity, it must have been
due to something else, perhaps the direct binding of
Ca
calmodulin complexes to MARCKS which is known
to occur in vitro (Aderem, 1992; Blackshear, 1993; Nairn and
Aderem, 1992) and believed to occur in vivo (Hinrichen and
Blackshear, 1993). Thus, we attempted to find out whether
Ca
calmodulin might prevent PKCs from
phosphorylating MARCKS protein by binding to the protein and blocking
its phosphorylation site domain.
Figure 5:
The effect of
Ca and TPA on the phosphorylation
of a 26-kDa protein (A) and the effect of cyclosporin A on the
phosphorylation of the 85-kDa MARCKS protein in BALB/MK keratinocytes (B). A, BALB/MK cells were prelabeled with
[
P]orthophosphate in low Ca
(0.05 mM) medium for 2 h and the labeled cells were then
left untreated (lane A); incubated for 10 min with 1
µM TPA (lane B); incubated for 10 min with 1.8
mM Ca
(lane C); or incubated for 2
min with 1.8 mM Ca
followed by 1 µM TPA for 10 min (lane D). The phosphorylation of 26-kDa
protein was determined by autoradiography as described for 85-kDa
phosphorylation under ``Materials and Methods.'' B,
BALB/MK cells were prelabeled with
[
P]orthophosphate in low Ca
(0.05 mM) medium for 2 h and incubated for an additional
15 min with (lanes C and D) or without (lanes A and B) 1 µM cyclosporin A. The labeled cells
were then left untreated (lanes A and C) or incubated
for 10 min with 1.8 mM Ca
(lanes B and D). The phosphorylation of the 85-kDa protein was
determined by autoradiography as described under ``Materials and
Methods.''
For
Cacalmodulin complexes to prevent PKCs from
gaining access to the MARCKS protein's phosphorylation site
domain, they must be produced before the PKCs are activated. Raising
the Ca
concentration in the medium from 0.05 to 1.8
mM triggered a Ca
surge (Fig. 6)
which peaked by 3 min at a value (394 ± 66 nM; n = 5) which was 4.6 ± 0.9-fold higher than the basal
value (85 ± 16 nM; n = 5). This
Ca
surge clearly preceded the PKCs activation which
peaked at 10 min (data not shown). Such a rapid and large internal
Ca
surge would generate
Ca
calmodulin complexes, which are known to be
able to bind to the MARCKS protein and modulate its activities (Aderem,
1992; Albert et al., 1984; Blackshear, 1993; Nairn and Aderem,
1992), well before PKCs activation. To find out whether the interaction
of MARCKS protein with Ca
calmodulin could in
fact block its phosphorylation by PKCs, the ability of membranes from
unstimulated keratinocytes to phosphorylate the 85-kDa MARCKS
holoprotein in vitro was determined in the presence of various
concentrations of exogenous calmodulin in a reaction mixture containing
1 µM added Ca
. Since the contaminating
Ca
in the assay medium was 4 µM (as
determined by atomic emission spectrometry, Chakravarthy et
al., 1994), the total Ca
concentration in the
reaction mixture was 5 µM. It should also be noted that
the reaction mixture contained three phosphatase inhibitors, sodium
vanadate, sodium pyrophosphate, and sodium fluoride. It is apparent
from Fig. 7A that Ca
calmodulin
blocked the phosphorylation of the MARCKS holoprotein in a
dose-dependent manner with the maximally effective concentration being
6 µg/100 µl (4 µM) which is within the
physiological range of cellular calmodulin concentration (Blackshear,
1993; Klee and Vanaman, 1982). This inhibition of MARCKS
phosphorylation (Fig. 7B, lane B) was
completely overcome by a calmodulin antagonist, the calmodulin-binding
domain peptide, H-LKKFNARRKLKGAILTTMLA-OH (Payne et al., 1988)
which would compete with MARCKS for
Ca
calmodulin (Fig. 7B, lanes C and F). It should be noted that the
calmodulin-binding domain peptide itself did not significantly affect
the phosphorylation of the 85-kDa protein (Fig. 7B, lanes D and E) or Ac-FKKSFKL-NH
(data not
shown). Thus, the failure of Ca
to stimulate the
phosphorylation of the 85-kDa MARCKS protein in the BALB/MK
keratinocytes, despite a large increase in the ability of their
membranes to phosphorylate the MARCKS-derived Ac-FKKSFKL-NH
peptide, could have been due to a direct interference by
Ca
calmodulin complexes.
Figure 6:
Raising extracellular
Ca from 0.05 mM to 1.8
mM triggers a
[Ca
]
surge in BALB/MK cells. Intracellular
Ca
was measured as described under ``Materials
and Methods.'' The arrow indicates the time of addition
of 1.8 mM Ca
. The result shown is a typical
tracing from one of five separate
experiments.
Figure 7:
Cacalmodulin
inhibits the phosphorylation of the exogenous 85-kDa protein by
isolated keratinocyte membranes and the inhibition is overcome by the
calmodulin antagonist, H-LKKFNARRKLKGAILTTMLA-OH. A,
phosphorylation of the exogenous 85-kDa protein by membranes isolated
from untreated BALB/MK cells was determined as described under
``Materials and Methods'' in the presence of 0 (A),
2 (B), 4 (C), 6 (D), 8 (E), and 10 (F) µg of calmodulin in the reaction mixture. B,
phosphorylation of the exogenous 85-kDa protein by keratinocyte
membranes was determined as described above. The reaction was carried
out with no additions (A), with 10 µg of calmodulin (B), with 10 µg of calmodulin and 5 µg of calmodulin
antagonist (C), with 5 µg calmodulin antagonist (D), with 10 µg of calmodulin antagonist (E), and
with 10 µg of calmodulin and 10 µg of calmodulin antagonist (F).
To find out whether
the failure of the Ca-induced increase in membrane
PKC activity to stimulate the phosphorylation of the 85-kDa MARCKS
protein occurred only in intact cells, phosphorylation of this protein
was determined in BALB/MK cell lysates. As shown in Fig. 8A, treating a keratinocyte PNF with 100
µM Ca
almost tripled the
membrane-associated PKCs activity as indicated by the increased
phosphorylation of Ac-FKKSFKL-NH
peptide by the
subsequently isolated membranes. Unlike the
Ca
-stimulated PKCs in intact cells, the
Ca
-stimulated PKCs in the post-nuclear fraction were
able to phosphorylate the endogenous MARCKS protein (Fig. 8B), probably because the endogenous calmodulin
concentration had been reduced to an ineffective level by dilution
during the preparation of the fraction. Indeed the MARCKS
phosphorylation was suppressed by adding calmodulin to the reaction
mixture, and this suppression was overcome by the calmodulin-binding
domain peptide (Fig. 8B). These results also eliminated
the possibility that the inability of Ca
to stimulate
MARCKS protein phosphorylation in intact cells was due to the
stimulation of PKC isoform(s) for which this protein was a poor
substrate. Parenthetically, phosphatases could not have affected the
phosphorylation of the MARCKS protein in PNF because of the presence of
three general phosphatase inhibitors used in the experiments of Fig. 7. Thus, the suppression of MARCKS phosphorylation by
calmodulin was probably not due to the stimulation of endogenous
Ca
calmodulin-dependent phosphatase(s).
Figure 8:
Stimulation of protein kinase Cs activity
and the phosphorylation of 85-kDa protein by
Ca in keratinocyte cell lysate.
Untreated BALB/MK keratinocytes were lysed and post-nuclear fraction (PNF) was prepared as described under ``Materials and
Methods.'' A, PNF was incubated with or without 100
µM CaCl
on ice for 3 min, and membranes were
sedimented at 100,000
g. PKC activity associated with
the isolated membranes was measured using peptide substrate as
described under ``Materials and Methods.'' B, the
85-kDa protein phosphorylation in PNF was determined as described under
``Materials and Methods'' in the absence (A), or the
presence of 100 µM CaCl
(B), 100
µM CaCl
and 10 µg of calmodulin (C), 100 µM CaCl
, 10 µg of
calmodulin and 10 µg of calmodulin antagonist peptide (D).
The
results presented so far suggested that the failure of MARCKS protein
to be phosphorylated significantly by the stimulated PKCs in
Ca-treated BALB/MK keratinocytes might be due to the
binding of Ca
calmodulin complexes to the MARCKS
protein. The possibility of a physiologically significant intracellular
association of calmodulin and MARCKS has been strongly suggested by the
results of experiments of Hinrichen and Blackshear(1993) which showed
that microinjection of MARCKS-derived peptide into Paramecium could reverse Ca
calmodulin-induced ciliary
beating. To try to demonstrate an in vivo interaction of
MARCKS and Ca
calmodulin in intact BALB/MK
keratinocytes, cells were treated with 100 µM calmodulin
antagonists, calmidazolium or the naphthalene sulfonamide W-7 for 20
min before stimulation with 1.8 mM Ca
. But
we observed only a marginal increase (about 20%) in the phosphorylation
of MARCKS protein in Ca
-stimulated cells, possibly
because these agents, besides affecting
Ca
calmodulin, also have other effects such as
reducing PKCs activity and/or affecting movement of ions, including
Ca
, across the cell membrane (Busse et al.,
1988; Hegemann and Mahle, 1991). We have also found that 10 µM calmidazolium could by itself induce a 4-fold increase in
[Ca
]
in BALB/MK keratinocytes
(data not shown). Since MARCKS and calmodulin are heat-stable proteins
(Chakravarthy et al., 1989; Sharma and Wang, 1979), an
alternative approach was taken to find out whether calmodulin could
bind MARCKS protein under physiological conditions. Cytosolic fractions
from untreated and Ca
-treated keratinocytes were
first heated in boiling water for 5 min to eliminate enzyme activities
while preserving MARCKS protein and calmodulin.Then the ability of the
PKCs in standard membranes from untreated BALB/MK cells to
phosphorylate the MARCKS protein in the heated cytosol was determined
in the presence and absence of calmodulin-binding peptide. As can be
seen in Fig. 9, adding the calmodulin-binding peptide to the
reaction mixture significantly increased the phosphorylation of MARCKS
protein in the heated cytosol from untreated and
Ca
-treated keratinocytes by the PKCs in intact
keratinocyte membranes. This result is consistent with pre-existence in
the cytosol of MARCKS
calmodulin complexes which could be
dissociated by the calmodulin-binding peptide to render the MARCKS
protein maximally phosphorylatable by PKCs in the test membranes. It is
unlikely that the MARCKS
calmodulin complexes were formed during
cell lysis because of a large dilution of calmodulin in the lysis
medium as discussed for the PNF experiments (Fig. 8). It should
be noted that these results indicate that binding of calmodulin to
MARCKS protein can occur at physiological concentrations of calmodulin
because there was no exogenous calmodulin present during the
preparation of heat-treated cytosol. Although these results provide
strong, albeit indirect, evidence supporting the hypothesis that
calmodulin binding modulates the PKCs-mediated phosphorylation of the
MARCKS protein in BALB/MK keratinocytes, other ways must be found (e.g. use of permeabilized cells to get calmodulin-binding
peptide into the cell) to prove more directly that
Ca
calmodulin complexes can inhibit the
phosphorylation of the MARCKS protein in intact keratinocytes as they
can in broken cell preparations (Fig. 8).
Figure 9:
Phosphorylation of the 85-kDa protein in
the heat-treated cytosol from keratinocytes by membrane-associated
PKCs. Heat-treated cytosol was prepared from unstimulated (lanes A and B) and 1.8 mM Ca-stimulated (lanes C and D)
BALB/MK keratinocytes as described under ``Materials and
Methods.'' The phosphorylation of the 85-kDa MARCKS protein in the
heated cytosol by PKCs in the membranes isolated from unstimulated
keratinocytes was determined in the presence (lanes B and D) and absence (lanes A and C) of 10 µg
of calmodulin-binding peptide H-LKKFNARRKLKGAILTTMLA-OH as described in
the legend for Fig. 7B.
In conclusion, the
Ca-triggered differentiation signal in BALB/MK
keratinocytes includes a large internal Ca
surge
followed by a strong stimulation of membrane-associated PKCs activity.
However, the stimulated PKCs cannot phosphorylate one of their
principal intracellular substrates, the 85-kDa MARCKS protein as well
as others such as a 26-kDa protein (Fig. 5) and EGF receptor
(Moscat et al. 1989). The reason why the
Ca
-stimulated membrane-associated PKCs do not
stimulate the MARCKS protein phosphorylation could be the binding of
Ca
calmodulin complexes (produced by the
intracellular Ca
surge (Fig. 6)) to the
protein's actin-binding site before the stimulation of
membrane-associated PKCs (Aderem, 1992; Blackshear, 1993; Nairn and
Aderem, 1992). Regardless of what the mechanism responsible for
blocking PKCs action turns out to be in BALB/MK cells, it is clear that
the phosphorylation of the ubiquitous 85-kDa MARCKS protein does not
serve as a marker of PKCs activation in some cells, especially if the
agonist should rapidly trigger a large intracellular Ca
surge.
It is apparent that the phosphorylation of the MARCKS
protein by PKCs is not needed for Ca-induced
keratinocyte differentiation. Thus, the physiological significance of
Ca
calmodulin's inhibition of MARCKS
phosphorylation is unclear, but it could affect different functions in
these and other cells. Ca
calmodulin's
interference with the PKCsmediated phosphorylation is not limited to
MARCKS protein and may have broad significance. For example, in a
separate study (
)we have found that the PKCs-mediated
phosphorylation of the EGF receptor in the post-nuclear fractions of
murine BALB/MK keratinocytes and human A431 epidermoid carcinoma cells
is also blocked by Ca
calmodulin and that this
blockade is overcome by the calmodulin-binding domain peptide. This
inhibition of phosphorylation and hence down-regulation of keratinocyte
EGF/TGF-
receptors by PKCs would enhance the response to EGF and
TGF-
. Another example is neuromodulin (also known as GAP-43, F1,
P-57), a neuron-specific calmodulin-binding PKCs substrate whose
phosphorylation by PKCs is also blocked by calmodulin (Chan et
al., 1986; Alexander et al.,1987). It
is likely that the blockade of its phosphorylation by
Ca
calmodulin would profoundly affect neurite
outgrowth and presynaptic neurotransmission. Clearly, further work
needs to be done to better understand the cellular functions of MARCKS
and how it is affected by modulation of its phosphorylation by
Ca
calmodulin.