©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Protein Kinase C during Ca-induced Keratinocyte Differentiation
SELECTIVE BLOCKADE OF MARCKS PHOSPHORYLATION BY CALMODULIN (*)

(Received for publication, May 4, 1994; and in revised form, October 13, 1994)

Balu R. Chakravarthy Richard J. Isaacs Paul Morley Jon P. Durkin James F. Whitfield

From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2), 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 CabulletCaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca. Cabulletcalmodulin 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 Cabulletcalmodulin complexes, formed by the internal Ca surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.


INTRODUCTION

BALB/MK mouse keratinocytes, like primary human and mouse keratinocytes, proliferate in medium containing EGF (^1)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(3) and Ins(1,3,4)P(3)), sn-1,2-diacylglycerols, and an alkylether glyceride (Moscat et al., 1989). The Ins(1,4,5)P(3) 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 Cabulletcalmodulin complexes rather than due to dephosphorylation of PKCs' phosphorylated substrate by a Ca-stimulated phosphatase.


MATERIALS AND METHODS

Chemicals

EGF was from Upstate Biotechnology Inc. (Lake Placid, NY). Bovine calmodulin and TPA were from Sigma. Bisindolylmaleimide GF 109203X (BIS), calphostin C, and calmodulin-binding domain peptide H-LKKFNARRKLKGAILTTMLA-OH were from Calbiochem (San Diego, CA). Fura-2-acetoxy methyl ester (Fura-2/AM) was purchased from Molecular Probes Inc. (Eugene, OR). Protein reagent was from Bio-Rad. [-P]ATP (6000 Ci/mmol) and [P]orthophosphoric acid (9000 Ci/mmol) were from New England Nuclear (Du Pont Canada Inc., Mississauga, Ontario). Cyclosporin A (Sandoz Pharmaceuticals) was a generous gift of Dr. J. P. MacManus of this institute. The PKC-selective oligopeptide substrate Ac-FKKSFKL-NH(2) was synthesized in this laboratory as described previously (Chakravarthy et al., 1991). Heat-treated cytosol containing the 85-kDa PKC substrate was prepared from S49 T-lymphoma cells as described by Chakravarthy et al.(1989). The Ca-responsive, differentiation competent BALB/MK keratinocytes were the generous gift of Dr. S. A. Aaronson (Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD), and were the same as the keratinocytes used by Moscat et al.(1989).

Cell Culture

The EGF-dependent BALB/MK keratinocytes were cultured as described by Weissmann and Aaronson(1983, 1985) and Whitfield et al.(1992). Briefly, the cells were grown in low Ca (0.05 mM) complete medium consisting of 10% (v/v) dialyzed fetal bovine serum (Life Technologies, Inc.) and 90% (v/v) Eagle's minimum essential medium (E-MEM, Biofluids, Rockville, MD) with 2 mM glutamine, 0.02 mM Ca-pantothenate, 20 ng EGF/ml, and 100 units of penicillin-G/ml. The cultures were incubated at 37 °C in an atmosphere of 5% CO(2) and 95% air.

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 times 10^6 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(3), 5 mM MgCl(2), 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 times g for 5 min, and membranes in the post-nuclear fractions (PNF) were sedimented at 100,000 times g for 10 min. The 100,000 times 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(2), 1 µM CaCl(2), 100 µM sodium vanadate, 100 µM sodium pyrophosphate, 1 mM sodium fluoride, 100 µM phenylmethylsulfonyl fluoride) by triturating with a pipette.

Direct Measurement of Membrane-associated PKC Activity

Membrane-associated PKC activity was measured directly in isolated membranes (without prior extraction and reconstitution of the enzyme in artificial phospholipid membranes) by determining the incorporation of P into a PKC-selective peptide substrate, Ac-FKKSFKL-NH(2) (Chakravarthy et al., 1991, 1992, 1994; Whitfield et al., 1992). This peptide corresponds to residues 160-166 of the phosphorylation site domain (151-175) of the MARCKS protein, a specific cellular substrate of PKC (Aderem, 1992; Blackshear, 1993). It and the MARCKS protein's phosphorylation site domain to which it belongs are highly selective substrates for PKC. Thus, their phosphorylation is not significantly catalyzed by other protein kinases such as cyclic AMP- and cyclic GMP-dependent protein kinases or calmodulin-dependent protein kinases I, II, III (Chakravarthy et al., 1991; Blackshear, 1993; Heemskerk et al., 1993; Orr et al., 1992; Williams et al., 1992). Moreover, the substrate peptide is phosphorylated equally by the several PKC isoforms (Heemskerk et al., 1993).

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 times 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.

Measurement of Cytosolic PKC Activity

Cytosolic PKC activity was determined with a mixed micellar assay using Ac-FKKSFKL-NH(2) peptide substrate as described previously (Chakravarthy et al., 1991).

Phosphorylation of the Endogenous 85-kDa MARCKS Protein in Intact BALB/MK Keratinocytes

BALB/MK cells were incubated with [P]orthophosphate (250 µCi/100-mm dish) in serum-free, low Ca medium for 2 h to label their ATP pool. The labeled cells were then either left untreated or incubated with 1.8 mM Ca and/or 1 µM TPA for 10 min. At 10 min, the culture medium was rapidly removed, and the cells were washed once with ice-cold PBS. The 85-kDa MARCKS protein was extracted with 40% acetic acid as described by Robinson et al.(1993) with slight modification. Briefly, the cells were hypotonically lysed by suspending them in ice-cold 1 mM NaHCO(3) solution containing 5 mM MgCl(2), 100 µM sodium vanadate, 100 µM sodium pyrophosphate, 1 mM sodium fluoride, 100 µM phenylmethylsulfonyl fluoride and vortexing vigorously. Nuclei and unlysed cells were removed by centrifugation at 600 times g for 5 min, and the post-nuclear supernatant was solubilized in electrophoresis buffer (Laemmli, 1970). The solubilized samples were adjusted to contain 40% acetic acid, left on ice for 1 h, and the precipitated proteins were removed by microfuging at top speed for 5 min. Acetic acid extract containing the MARCKS protein was processed for SDS-polyacrylamide gel electrophoresis as described by Robinson et al.(1993), and the phosphorylation of MARCKS protein was visualized by autoradiography using Kodak XAR film.

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 times 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 times 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(2) 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 times 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.

Phosphorylation of the 85-kDa MARCKS Protein in the Post-nuclear Fraction

BALB/MK cells were hypotonically lysed, and PNF was prepared as described above. The PNF was adjusted to contain 50 mM Tris-HCl buffer, pH 7.5, 5 mM MgCl(2), 1 µM CaCl(2), 100 µM sodium vanadate, 100 µM sodium pyrophosphate, 1 mM sodium fluoride, 100 µM phenylmethylsulfonyl fluoride. 160 µl of PNF was incubated with the indicated additions and [P]ATP (25 µM, 900 counts/min/pmol) for 10 min at 25 °C in a final reaction volume of 200 µl. The 85-kDa MARCKS protein was then extracted and the level of its phosphorylation determined by autoradiography as described above for the phosphorylation of exogenous 85-kDa protein.

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.

Measurement of [Ca](i)

For the determination of [Ca](i), keratinocytes were plated on glass coverslips (Canadawide Scientific, Ottawa, Canada) in low Ca (0.05 mM) E-MEM with 10% fetal bovine serum and 20 ng/ml EGF. The medium was changed on day 3 and [Ca](i) responses determined on day 5.

Changes in [Ca](i) 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.

Protein Measurement

Protein contents were measured according to the method of Bradford(1976).


RESULTS AND DISCUSSION

Raising the [Ca](i) 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(2) 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(2) 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(2) 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-NHphosphorylating 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(2) 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 Cabulletcalmodulin-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 Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin (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(2) (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(2) peptide, could have been due to a direct interference by Cabulletcalmodulin 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: Cabulletcalmodulin 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(2) 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 Cabullet 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(2) on ice for 3 min, and membranes were sedimented at 100,000 times 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(2) (B), 100 µM CaCl(2) and 10 µg of calmodulin (C), 100 µM CaCl(2), 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 Cabulletcalmodulin 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 Cabulletcalmodulin-induced ciliary beating. To try to demonstrate an in vivo interaction of MARCKS and Cabulletcalmodulin 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 Cabulletcalmodulin, 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](i) 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 MARCKSbulletcalmodulin 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 MARCKSbulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin's inhibition of MARCKS phosphorylation is unclear, but it could affect different functions in these and other cells. Cabulletcalmodulin's interference with the PKCsmediated phosphorylation is not limited to MARCKS protein and may have broad significance. For example, in a separate study (^2)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 Cabulletcalmodulin and that this blockade is overcome by the calmodulin-binding domain peptide. This inhibition of phosphorylation and hence down-regulation of keratinocyte EGF/TGF-alpha receptors by PKCs would enhance the response to EGF and TGF-alpha. 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 Cabulletcalmodulin 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 Cabulletcalmodulin.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: EGF, epidermal growth factor; TGF, transforming growth factor; MARCKS, myristoylated alanine-rich kinase C substrate; PKC, protein kinase C; TPA, 12-O-tetradecanoyl phorbol 13-acetate; [Ca](i), intracellular calcium concentration; E-MEM, Eagle's minimum essential medium; PNF, post-nuclear fractions.

(^2)
B. R. Chakravarthy, R. J. Isaacs, P. Morley, and J. F. Whitfield, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. R. E. Williams for synthesizing PKC-specific peptide substrate Ac-FKKSFKL-NH(2).


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