©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CaCalmodulin Prevents Myristoylated Alanine-rich Kinase C Substrate Protein Phosphorylation by Protein Kinase Cs in C6 Rat Glioma Cells (*)

(Received for publication, June 19, 1995; and in revised form, August 7, 1995)

Balu R. Chakravarthy (§) Richard J. Isaacs Paul Morley 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
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 Cabulletcalmodulin 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 Cabulletcalmodulin-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 Cabulletcalmodulin can profoundly affect PKCs' signaling at the substrate level.


INTRODUCTION

Cultured keratinocytes proliferate when the culture medium contains EGF (^1)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 Cabulletcalmodulin 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 Cabulletcalmodulin 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](i) 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](i), and a resulting Cabulletcalmodulin, surge.

In this report we extend our observations, using C6 rat glioma cells and BALB/MK keratinocytes, to provide the first direct evidence for Cabulletcalmodulin 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.


MATERIALS AND METHODS

Chemicals

Bovine calmodulin was from Upstate Biotechnology Inc. (Lake Placid, NY). Calmodulin-binding domain peptide, H-LKKFNARRKLKGAILTTMLA-OH, and ionomycin were from Calbiochem (San Diego, CA). Fura-2-acetoxy methyl ester (Fura-2/AM) was purchased from Molecular Probes Inc. (Eugene, OR). 12-O-Tetradecanoyl phorbol 13-acetate (TPA) was from Sigma. 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 PKCs-selective oligopeptide substrate Ac-FKKSFKL-NH(2) and pseudosubstrate peptide inhibitor PKC-(19-36) were synthesized in this laboratory as described previously (Chakravarthy et al., 1991). C6 rat glioma cells were the generous gift of Dr. H. W. Cook (Atlantic Research Center for Mental Retardation, Dalhousie University, Halifax, Nova Scotia) and 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).

Cell Culture

C6 rat glioma cells were grown as monolayer cultures in 90% high glucose Dulbecco's minimum essential medium (DMEM) and 10% fetal bovine serum as described previously (Byers et al., 1993). The EGF-dependent BALB/MK keratinocytes were cultured in low-Ca (0.05 mM) complete medium consisting of 10% (v/v) dialyzed fetal bovine serum (Life Technologies, Inc.), 20 ng/ml EGF, and 90% (v/v) Eagle's minimum essential medium as described by Weissmann and Aaronson(1983, 1985) and Chakravarthy et al. (1995). All cultures were incubated at 37 °C in an atmosphere of 5% CO(2) and 95% air.

For experiments, stock C6 glioma cells or BALB/MK keratinocytes (3 times 10^5) 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(3), 5 mM MgCl(2), 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 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, and 100 µM phenylmethylsulfonyl fluoride) by triturating with a pipette.

Direct Measurement of Membrane-associated PKC Activity

Membrane-associated PKCs activity was measured directly in isolated membranes (without prior extraction and reconstitution of the enzyme in artificial phopholipid membranes) by determining the incorporation of P into a PKC-selective peptide substrate, Ac-FKKSFKL-NH(2) (Chakravarthy et al., 1991, 1992, 1994, 1995; 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 PKCs, being phosphorylated equally by conventional PKC (cPKC) isoforms, alpha, beta, and (Heemskerk et al., 1993) and novel PKC (nPKC) isoforms such as and , but poorly by atypical PKCs (aPKCs) such as (Fujise et al., 1994). 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-III (Chakravarthy et al., 1991; Blackshear, 1993; Heemserk et al., 1993; Orr et al., 1992; Williams et al., 1992).

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 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 80-85-kDa MARCKS Protein in Intact Cells

C6 rat glioma cells or murine BALB/MK keratinocytes were incubated with [P]orthophosphate (250 µCi/100-mm dish) in serum-free medium for 2 h to label their ATP pool. The labeled cells were then either left untreated or incubated with 3.5 µM ionomycin and/or 1 µM TPA for 10 min. Following the treatment, the culture medium was rapidly removed, and the cells were washed once with ice-cold PBS. The 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 MARCKS Protein in the Post-nuclear Fraction

C6 glioma 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. 100-µl of PNF was incubated with the indicated additions (see figure legends) and [P]ATP (25-µM, 900 counts/min/pmol) for 10 min at 25 °C in a final reaction volume of 200 µl. The MARCKS protein was then extracted, and the level of its phosphorylation determined by autoradiography as described above.

Immunoblotting of PKC

Immunoblotting was performed according to Towbin et al.(1979). Proteins from cytosol and solubilized membranes were separated on 10% SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membranes were then incubated with pan-PKC antibody (UBI, Lake Placid, NY) at 1:1000 dilution for 2 h at room temperature. PKCbulletantibody complexes were visualized using an alkaline phosphatase-conjugated goat anti-rabbit IgG as described previously (Chakravarthy et al., 1994).

Measurement of [Ca](i)

For the determination of [Ca](i), glioma cells were plated on glass coverslips (Canadawide Scientific, Ottawa, Canada) in Dulbecco's minimum essential medium with 10% fetal bovine serum. 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; Chakravarthy et al., 1995).

Protein Measurement

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


RESULTS

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-NH(2)in vitro (Fig. 1A). This increase in membrane-associated Ac-FKKSFKL-NH(2) 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(2) 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(2) 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(2) 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 (bullet, 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: Cabulletcalmodulin 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(2) and 1 µM TPA (2), 100 µM CaCl(2), 1 µM TPA and 3.5 µM ionomycin (3), 100 µM CaCl(2), 1 µM TPA and 2 µg of calmodulin (4), 100 µM CaCl(2), 1 µM TPA, and 5 µg of calmodulin (5), 100 µM CaCl(2), 1 µM TPA, and 10 µM calmodulin (6), 100 µM CaCl(2), 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](i) 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](i) surge in C6 glioma cells. [Ca](i) 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 Cabulletcalmodulin 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 Cabulletcalmodulin (Fig. 4). But, the question remained as to whether Cabulletcalmodulin 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 Cabulletcalmodulin 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(2)SO for 15 min followed by 3.5 µM ionomycin for 10 min; 4) treated with 25 µM calmidazolium in 0 .1% Me(2)SO for 15 min followed by 3.5 µM ionomycin for 10 min; 5) 50 µM calmidazolium in 0 .1% Me(2)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 Cabulletcalmodulin-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.''




DISCUSSION

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](i) 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](i) 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](i) 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](i), 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 Cabulletcalmodulin as the intracellular mediator of the interference with PKCs' substrate phosphorylation by Ca. Since a [Ca](i) surge produced by ionomycin or triggered by activated surface Ca receptors/sensors or growth factor receptors produces Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin 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](i) 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](i) 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 (^2)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](i) 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, Cabulletcalmodulin appears to be a physiological modulator of MARCKS phosphorylation by PKCs.

The ability of Cabulletcalmodulin to modulate the phosphorylation and function of PKCs'substrate does not appear to be restricted to the actin-binding MARCKS protein alone. Cabulletcalmodulin 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 Cabulletcalmodulin 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 Cabulletcalmodulin (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.


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.

§
To whom correspondence should be addressed: Institute for Biological Sciences, Bldg. M-54, Montreal Road Campus, National Research Council of Canada, Ottawa K1A 0R6, Canada. Tel.: 613-990-0899; Fax: 613-941-4475.

(^1)
The abbreviations used are: EGF, epidermal growth factor; MARCKS, myristoylated alanine-rich kinase C substrate; PKC, protein kinase C; TPA, 12-O-tetradecanoyl phorbol 13-acetate; [Ca], intracellular calcium concentration; 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) and Stephen Beazley for preparing the illustrations.


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