©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carbachol, Substance P, and Phorbol Ester Promote the Tyrosine Phosphorylation of Protein Kinase C in Salivary Gland Epithelial Cells (*)

Stephen P. Soltoff (§) , Alex Toker

From the (1) Department of Medicine, Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The initiation of saliva formation by parotid acinar cells, which comprise the majority of cells in this salivary gland, is initiated by the release of neurotransmitters (acetylcholine, substance P) from parasympathetic nerves. In response to substance P and the muscarinic agonist carbachol, two ligands that activate phospholipase C-linked receptors, which stimulate fluid secretion, PKC was phosphorylated on tyrosine residues. The maximal agonist-dependent tyrosine phosphorylation occurred within seconds of the addition of either agonist and then returned rapidly to a smaller increased level. Phorbol ester also caused a rapid increase in tyrosine phosphorylation, which reached a maximal level 5 min after the addition of phorbol 12-myristate 13-acetate. The increase in tyrosine phosphorylation of PKC was blocked by tyrosine kinase inhibitors genistein and staurosporine. Ionophore-mediated elevation of [Ca] or activation of the -adrenergic receptor, epidermal growth factor receptor, or insulin receptor did not promote the tyrosine phosphorylation of PKC. These results indicate that tyrosine phosphorylation plays a role in early signal transduction events promoted by the activation of muscarinic and substance P receptors and suggests that the tyrosine phosphorylation of PKC has a role in the activation of fluid secretion by neurotransmitters binding to phospholipase C-linked receptors.


INTRODUCTION

The protein kinase C (PKC)() family of proteins currently consists of 12 members that are phospholipid-dependent serine/threonine-specific protein kinases (for review, see Nishizuka(1992) and Hug and Sarre(1993)). The family is subdivided into those members that are calcium-dependent conventional PCKs (, I, II, ), calcium-independent novel PKCs (, , , ), and atypical PKCs ( and /). Another subgroup may include PKCµ, a recent addition to the PKC family. With the exception of the atypical PKC group, all members are activated by phorbol esters, which bind to the site at which the endogenous activator sn-1,2-diacylglycerol binds. In addition to the activation of PKC by phospholipids and/or Ca, other phospholipids, notably phosphoinositides produced by the activation of phosphatidylinositol 3-kinase, have been found to activate members of the conventional PKC and novel PKC family (Singh et al., 1993; Toker et al., 1994; Nakanishi et al., 1993). Different cells have different profiles of PKC isoforms, and recent studies suggest that isoforms may have distinct and different functions in cells (Li et al. (1994b) and Mischak et al. (1993a), and for a review, see Dekker and Parker(1994)). Multiple members of the PKC family of enzymes exist in parotid acinar cells, including PKC (Terzian et al., 1993).()

Parotid acinar cells have been a focus of study for the production of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol, the release of Ca from intracellular stores, and the subsequent entry of Ca across the plasma membrane via capacitative entry mechanisms (Komabayashi et al.(1992) and Takamura and Putney(1989) and for a review, see Baum et al. (1993)). These events lead to the activation of Ca-sensitive channels and the initiation of saliva formation, events that are controlled by the release of neurotransmitters (acetylcholine, substance P) from parasympathetic nerves. While examining the effects of growth factors and other receptor ligands on the tyrosine phosphorylation pattern of parotid acinar cells, we found that the muscarinic agonist carbachol and substance P promoted the tyrosine phosphorylation of an 80-kDa protein and that phorbol ester promoted the tyrosine phosphorylation of the same band, which was identified as the isoform of PKC.

Several studies have overexpressed various PKC isoforms to examine the functions of specific isoforms. These and other studies have reported that PMA promoted the tyrosine phosphorylation of PKC. PKC (and PKC) expression was increased in c-Ha-ras-transformed fibroblasts (Borner et al., 1992b), and PKC was constitutively tyrosine phosphorylated in c-Ha-ras-transformed keratinocytes (Denning et al., 1993). In a myeloid progenitor cell line (32D cells) and in NIH 3T3 cells that overexpressed PKC, phorbol ester stimulated the tyrosine phosphorylation of PKC (Li et al., 1994a). In 32D cells in which PKC and the platelet-derived growth factor receptor were overexpressed, platelet-derived growth factor stimulated the tyrosine phosphorylation of PKC and myeloid differentiation (Li et al., 1994b). In an earlier study using 32D cells, differentiation was promoted in cells in which PKC and - were overexpressed, but not in cells in which PKC II, , , or was overexpressed (Mischak et al., 1993a). In NIH 3T3 cells, overexpression of PKC produced changes in cell morphology and slowed cell growth, while very different changes (increase in growth rate and in cell density at confluence) were obtained by overexpression of PKC in these cells (Mischak et al., 1993b).

The present study utilizes freshly isolated dispersed cells from rat parotid gland. The rapid tyrosine phosphorylation of PKC in the acinar cells suggests that this biochemical event contributes to the normal physiological responses promoted by the release of neurotransmitters from parasympathetic nerves that promote fluid secretion and saliva formation in this exocrine gland.


EXPERIMENTAL PROCEDURES

Chemicals

All chemicals were reagent grade or better. PMA and 4-PMA were obtained from Life Technologies, Inc. (Grand Island, NY). Carbamyl choline (carbachol) and substance P were purchased from Sigma. Anti-phosphotyrosine was a generous gift of Dr. Tom Roberts (Dana Farber, Boston, MA). [P]PO was purchased from DuPont NEN. Anti-protein kinase C antibody was purchased from Santa Cruz Biotechnology. Male Sprague-Dawley rats (Charles River Laboratories, Kingston, NY, or Taconic, Germantown, NY), 200-250 g, were used for all experiments.

Cell Preparation and Solutions

Freshly dispersed parotid acinar cells were prepared as described previously (Soltoff et al., 1989). The cells were suspended at 2 mg/ml in a medium (Solution A) of the following composition: 116.4 mM NaCl, 5.4 mM KCl, 1 mM NaHPO, 25 mM HEPES, 1 mM CaCl, 0.8 mM MgCl, 5 mM butyrate, 5.6 mM glucose, pH 7.4. Cells were kept on ice prior to use. Samples (1.5 ml) of the cell suspension were stirred and equilibrated at 37 °C for 10-15 min prior to use.

Immunoprecipitations and Western Blotting

After the cells were exposed to the agonists or inhibitors, the cells were pelleted by a brief spin in a microcentrifuge (Brinkmann 5414). The supernatant was removed and replaced by 1 ml of ice-cold lysis buffer (137 mM NaCl, 20 mM Tris, 0.2 mM vanadate, 1 mM EGTA, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 4 µg/ml pepstatin, 2 mM dithiothreitol, pH 7.5). The lysates were vortexed and centrifuged at 16,000 g at 4 °C for 15 min. The cleared supernatants were transferred to fresh 1.5-ml microcentrifuge tubes. A portion (5-10% of the volume) of the lysate was removed and combined with an equal volume of 2 sample buffer (62.5 mM Tris, pH 6.8, 10% (v/v) glycerol, 6.25% (v/v) SDS, 0.72 N -mercaptoethanol, bromphenol blue for color). The remainder was incubated with either anti-P-Tyr (6.6 µg/ml) or anti-PKC (1-2 µg/ml) antibodies and protein A-Sepharose (4 mg/ml lysate), and the samples were rocked at 4 °C for 3 h or overnight. At the end of the incubation, the immunoprecipitates were collected by centrifugation, washed 3 times in ice-cold phosphate-buffered saline (PBS) (137 mM NaCl, 15.7 mM NaHPO, 1.47 mM KHPO, 2.68 mM KCl, 1% Nonidet P-40, pH 7.4), two times in 0.1 M Tris (pH 7.5), 0.5 M LiCl, and two times in TNE (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.5). All wash solutions contained 0.2 mM vanadate. The majority of the TNE was removed, the remaining volume was diluted with an equal volume of 2 sample buffer, and the samples were boiled for 5-10 min. The immunoprecipitated proteins and the lysate fractions were subjected to electrophoresis or stored at - 80 °C prior to electrophoresis.

Samples were subjected to electrophoresis on a sodium dodecyl sulfate (SDS)-7% polyacrylamide separating gel with a 3% stacking gel. Proteins were transferred to 0.2 µm pore-size nitrocellulose filters, and the filters were blocked with TBS (20 mM Tris (pH 7.6), 137 mM NaCl), 2% (w/v) BSA for 1 h. The filters were washed in TTBS (TBS, 0.2% (v/v) Tween 20) three times. The nitrocellulose filters were exposed to anti-PKC antibody (0.2 µg/ml) or anti-P-Tyr antibody (1 µg/ml) in TTBS/1% BSA for 16 h at 4 °C. The filters were washed three times in TTBS, and exposed to anti-rabbit (for anti-PKC) or anti-mouse (for anti-P-Tyr) horseradish peroxidase (Boehringer-Mannheim) at a 1:10,000 dilution in TTBS/1% BSA for 1 h. All washes and exposure to the secondary antibody were performed at room temperature. Filters were washed three times with TTBS and twice with TBS, and were visualized on x-ray film (Kodak) using a chemiluminescence system (Amersham or DuPont NEN). In some experiments, the filters were stripped by exposing them to 62.5 mM Tris (pH 6.8), -mercaptoethanol (0.1 M), 2% (w/v) SDS at 70 °C for 40 min. The stripped filters were washed several times in TTBS, once in TBS, blocked with TBS, 2% BSA for one hour, and reprobed with antibody for 16 h. Blots were then treated as described above.

In Vivo Labeling and Phosphoamino Acid Analysis

In vivo phosphorylation studies were performed by labeling the cells with [P]PO. Cells were suspended in solution A, except NaHPO4 was omitted. A suspension of cells was incubated at 37 °C and exposed to [P]PO (2-3 mCi/ml cells) for 1-2 h, after which they were resuspended in an identical pre-warmed medium in the absence of [P]PO. Cells were exposed to PMA, carbachol, or vehicle (MeSO). The cells from each experimental condition were collected and pelleted, the supernatant was removed and the tube was rinsed with phosphate-buffered saline without dislodging the pellet. The cell pellet was lysed in ice-cold lysis buffer, to which anti-PKC (2-3 µg/ml) antibody was added. The antibody was collected using protein A-Sepharose and washed as described above. The proteins were transferred to 0.45 µm pore size Immobilon-P (Millipore) using a Bio-Rad Trans-Blot system and a transfer buffer of the following composition: 25 mM Tris base, 192 mM glycine in 15% (v/v) methanol, pH 8.2-8.3.

In some cases the immobilized phosphorylated PKC proteins were cut out and subjected to acid hydrolysis for two-dimensional phosphoamino acid analysis as described previously (Boyle et al., 1991).

Cell Fractionation (Translocation) Experiments

Fractionation experiments were conducted with modifications of the protocol outlined in Olivier and Parker(1994). Cells were suspended and stirred at 2 mg of protein/ml and equilibrated at 37 °C for 10 min prior to use. Upon treatment, cells were quickly pelleted, the physiological solution was replaced with Buffer A (25 mM Tris-HCl, pH 7.5, 250 mM sucrose, 2.5 mM magnesium acetate, 10 mM sodium fluoride, 2 mM dithiothreitol, 10 mM benzamidine), and this was removed and replaced with 2 ml of homogenization buffer (Buffer A plus 5 mM EGTA, 5 mM EDTA, 4 µg/ml of aprotinin, leupeptin, and pepstatin; 1 mM phenylmethylsufonyl fluoride). The cells were resuspended, and the suspension was homogenized for 40 strokes in a tight Dounce homogenizer followed by a 5-s sonication (Branson Ultrasonics, Danbury, CT; setting 7) repeated 3 times. The homogenate was centrifuged for 30 min at 70,000 rpm at 4 °C in a Beckman Optima TLX ultracentrifuge. A portion (5%) of the supernatant (cytosolic fraction) was collected as cytosolic lysate, and an equal volume of 2 sample buffer was added, and the samples were heated at 100 °C for 8 min. The detergent-soluble proteins were solubilized by adding Buffer B (25 mM Tris-HCl, pH 7.5, 10 mM sodium fluoride, 5 mM EDTA, 1% Nonidet P-40 detergent, 4 µg/ml of aprotinin, leupeptin, and pepstatin, 1 mM phenylmethylsufonyl fluoride, 5 mM dithiothreitol, 10 mM benzamidine) to the pellet; the solution was vortexed, transferred to a Dounce homogenizer, and broken apart with 20 strokes. Detergent-soluble proteins were collected by spinning the sample at 16,000 g for 15 min at 4 °C. A portion (5%) of the supernatant was collected as membrane lysate, sample buffer was added, and the samples were heated. In some experiments, the remainder of the supernatants of the cytosolic fraction and membrane fraction were used for immunoprecipitation studies using anti-PKC antibody (2 µg/ml) or anti-P-Tyr antibody (6.6 µg/ml). After addition of protein A-Sepharose as above, the immunoprecipitates were rocked at 4 °C for 3 h or overnight and collected and washed as described above.

Data

All experiments were conducted at least twice with similar results to those shown in the figures.


RESULTS

Carbachol, Substance P, and PMA Stimulate the Tyrosine Phosphorylation of PKC

Carbachol and substance P, two receptor ligands that stimulate the production of diacylglycerol in parotid cells, and PMA, which binds to the diacylglycerol binding site on PKC, all stimulated the tyrosine phosphorylation of PKC. Differences among the stimuli were highlighted in a comparison of their time courses (Fig. 1, A and B). In cells exposed to carbachol or substance P for varying periods of time up to 15 min, the increase in tyrosine phosphorylation of PKC was greatest for cells exposed to these stimuli for 15 s, and then it diminished to a lower level. Within 15 min, the level of tyrosine phosphorylation was close to that found in untreated cells. Previous studies on inositol trisphosphate production and [Ca] elevation in parotid cells demonstrated that the stimulatory effects of substance P rapidly diminished due to homologous receptor desensitization, although the effects of carbachol were maintained for up to 10 min or longer (McMillian etal., 1987; Sugiya etal., 1987). However, the tyrosine phosphorylation of PKC elicited by both carbachol and substance P displayed a very early increase and then a decline. The decrease in tyrosine-phosphorylated PKC (Fig. 1, A and B) without a decrease in the mass of the PKC protein (Fig. 1C) indicates that a protein-tyrosine phosphatase was activated. This suggests that the activation of tyrosine phosphatase activity is also involved in the stimulation of these cells by carbachol and substance P.Effects of PMA were observed at times as early as 15 s (Fig. 1, A and B). However, in contrast to the effects of the two receptor-mediated ligands, the PMA-promoted phosphorylation of PKC continued to increase up to 5 min of exposure. For all three stimuli, similar findings were observed when proteins were immunoprecipitated using anti-PKC antibody and immunoblotted with anti-P-Tyr antibody (Fig. 1A) or when proteins were immunoprecipitated with anti-P-Tyr antibody and immunoblotted with anti-PKC antibody (Fig. 1B). The results suggest that PKC is involved in early signal transduction events subsequent to the activation of the muscarinic and substance P receptors in the parotid acinar cell.


Figure 1: Time dependence of the increase in PKC tyrosine phosphorylation promoted by the exposure of parotid acinar cells to carbachol, substance P, and PMA. Cells were equilibrated at 37 °C and exposed to carbachol (1 10M), substance P (1 10M), or PMA (200 nM) for 0.2, 1, 5, or 15 min. Unstimulated cells (0) were exposed to vehicle (MeSO or water). Cells were lysed, and proteins were immunoprecipitated using anti-PKC antibody (A and C) or anti-P-Tyr antibody (B). Proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with anti-P-Tyr antibody (A) or anti-PKC antibody (B and C) as indicated. Tyrosine-phosphorylated PKC was immunoprecipitated by anti-P-Tyr antibody or by anti-PKC antibody. Proteins were visualized on x-ray film using enhanced chemiluminescence. Molecular mass markers (in kDa) are indicated on the left. The arrow on the right designates the location of the tyrosine phosphorylated form of PKC.



In lysates of parotid acinar cells, PKC appears as a doublet protein at 80 kDa (Fig. 2). Although the doublet appears in both stimulated and unstimulated cells in some experiments, in most experiments the mass of the upper band appears to increase with stimulation. This is most readily detected in cells treated with PMA, but also it is seen in carbachol- or substance P-stimulated cells (Fig. 1C). Anti-P-Tyr antibody preferentially immunoprecipitates and immunoblots the upper form of the enzyme, indicating that this is the tyrosine-phosphorylated form. The anti-PKC antibody immunoprecipitates and immunoblots both forms of enzyme. This is observed easily when proteins immunoprecipitated using anti-PKC antibody are immunoblotted for both PKC and tyrosine phosphorylation, and are compared side by side with lysates (Fig. 2). This also demonstrates that the tyrosine-phosphorylated enzyme accounts for only a portion of the upper band of PKC (Fig. 2). The identification of the upper band as the tyrosine-phosphorylated form is consistent with the stimuli producing a mobility shift to a slower migrating form of the isoenzyme due to an increase in phosphorylation, which consists of both serine and tyrosine phosphorylation (see below).


Figure 2: Identification of tyrosine phosphorylated PKC in anti-PKC immunoprecipitates of carbachol- and PMA-treated parotid acinar cells. Unstimulated cells (-) or cells stimulated with PMA (200 nM) or carbachol (1 10M) for 5 min were lysed. Proteins were immunoprecipitated with anti-PKC, separated by SDS-PAGE, and transferred to nitrocellulose, and the blot was probed with anti-PKC (leftpanel). The nitrocellulose blot was stripped and reprobed with anti-P-Tyr antibody (middlepanel). Lysates (rightpanel) are from a separate experiment using cells treated as described. PKC is visualized as a doublet in cell lysates and in anti-PKC immunoprecipitates. The upper band of the doublet was selectively immunoprecipitated by anti-P-Tyr antibody (Fig. 1B). Proteins were visualized on x-ray film using enhanced chemiluminescence. Molecular mass markers (in kDa) are indicated on the left. The arrow on the right designates the location of the tyrosine phosphorylated form of PKC.



PKC was immunoprecipitated from PMA-treated rat parotid acinar cells in a concentration-dependent manner using anti-P-Tyr antibody (Fig. 3). For cells treated with PMA for 15 min, 20 nM PMA produced a measurable increase in tyrosine phosphorylation. This was increased by 200 nM PMA, and concentrations of 1 µM (Fig. 3) or 2 µM (not shown) did not produce an increase over that promoted by 200 nM. The results obtained using inactive phorbol ester (4-PMA) were similar to those found in cells treated with vehicle (MeSO) alone (not shown).


Figure 3: Concentration dependence of the stimulatory effect of PMA on the tyrosine phosphorylation of PKC. Cells were treated with 20-1000 nM PMA for 15 min or treated with 0.2% MeSO (0), and proteins were immunoprecipitated with anti-P-Tyr and immunoblotted using anti-PKC antibody. Tyrosine phosphorylation is modest but observable at 20 nM PMA and is maximal at 200 nM PMA. Proteins were visualized on x-ray film using enhanced chemiluminescence. Molecular mass markers (in kDa) are indicated on the left. The arrow on the right designates the location of the tyrosine-phosphorylated form of PKC.



Carbachol and PMA Stimulate Phosphorylation of PKC in Vivo

Carbachol and PMA both produced substantial increases in the phosphorylation of PKC in cells that were prelabeled with [P]PO and then exposed to these stimuli. A 2-min exposure to PMA increased the phosphorylation to 6 times that found in unstimulated cells, and carbachol increased it to 4 times the basal level (Fig. 4). Similar increases were observed in cells treated for 5 or 15 min (not shown).


Figure 4: Carbachol and PMA stimulate the in vivo phosphorylation of PKC. Parotid acinar cells were labeled with [P]PO, washed to remove the extracellular isotope, and resuspended (see ``Experimental Procedures''). Cells were treated with 0.2% MeSO (-), PMA (200 nM), or carbachol (1 10M) for 2 min, after which they were lysed. PKC was immunoprecipitated using anti-PKC antibody, subjected to SDS-PAGE, transferred to Immobilon, and exposed to x-ray film. The arrow on the right designates the location of PKC. Similar results were obtained using cells treated with stimuli for 5 or 15 min.



In some experiments the PKC band at 80 kDa was cut out and subjected to two-dimensional phosphoamino acid analysis. Although the in vivo [P]PO labeling was sufficient to demonstrate an increase in serine phosphorylation (not shown) in PMA-treated cells, consistent with the activation of PKC activity by these stimuli, the in vivo labeling was not sufficient to observe the increase in tyrosine phosphorylation. Thus, tyrosine phosphorylation makes up only a small fraction of the enhanced in vivo phosphorylation in the anti-PKC immunoprecipitates shown in Fig. 4. A similar conclusion concerning the relatively small fraction of tyrosine phosphorylated PKC was made from results obtained from anti-P-Tyr immunoblots of anti-PKC immunoprecipitates (Fig. 2).

Tyrosine Phosphorylation of PKC Is Not Promoted by Protein Kinase A-linked Receptors, EGF and Insulin Receptor Tyrosine Kinases, and Calcium Ionophore

Other stimuli were examined for their ability to stimulate the tyrosine phosphorylation of PKC (Fig. 5). Isoproterenol, a -adrenergic agonist that activates protein kinase A in these cells, was examined at concentrations as high as 10M and did not promote the tyrosine phosphorylation of PKC in any significantly detectible manner. Ionomycin, a calcium ionophore, was without effect at 10 or 10M, which are concentrations that cause large increases in [Ca]. EGF (100 ng/ml) produced an increase in tyrosine phosphorylation of multiple proteins in parotid acinar cells, including the epidermal growth factor receptor (not shown), but it did not promote the tyrosine phosphorylation of PKC. Insulin (100 nM) also was without effect on the tyrosine phosphorylation of PKC. These results suggest that the carbachol-, substance P-, and phorbol ester-promoted tyrosine phosphorylation of PKC was not mediated by an increase in [Ca] or an activation of protein kinase A and that the activation of two receptor protein-tyrosine kinases (the EGF and insulin receptors) also was insufficient to promote the tyrosine phosphorylation of PKC in parotid acinar cells. EGF promotes increases in inositol 1,4,5-trisphosphate and diacylglycerol in some cells (Hughes et al., 1991), and its lack of effect on PKC tyrosine phosphorylation in parotid cells may reflect a relatively low density of EGF receptors and the sensitivity of detection of this event.


Figure 5: Comparison of the effects of various stimulatory agents on PKC tyrosine phosphorylation in parotid acinar cells. Cells were treated with vehicle (-) or exposed to the following concentrations of stimuli: PMA (200 nM), carbachol (1 10M), substance P (1 10M), ionomycin (1 10M), isoproterenol (10M), EGF (100 ng/ml), and insulin (100 µM). Cells were exposed for 5 min (PMA, carbachol, isoproterenol, EGF, insulin), 1 min (ionomycin), or both times (substance P). At the end of the exposure period, proteins were immunoprecipitated from lysed cells using anti-P-Tyr antibody, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted using anti-PKC antibody. Proteins were visualized on x-ray film by using enhanced chemiluminescence. Only cells exposed to PMA, carbachol, and substance P demonstrated a measurable increase in tyrosine phosphorylation of PKC. EGF (100 ng/ml), insulin (100 nM), and isoproterenol (10M) also did not produce a significant effect on PKC tyrosine phosphorylation at shorter times of agonist exposure (0.2 and 1 min). The arrow on the right designates the location of the tyrosine phosphorylated form of PKC.



Genistein and Staurosporine Block PKC Tyrosine Phosphorylation

The effects of two tyrosine kinase inhibitors, genistein and staurosporine, were examined on the tyrosine phosphorylation of PKC. Cells were pretreated with the inhibitors for 15 min prior to exposure to carbachol or PMA. The tyrosine phosphorylation promoted by PMA was partially reduced at 0.1 µM staurosporine and was not observable at 1 µM or 5 µM staurosporine (not shown). The effects of PMA or carbachol were partially reduced by 1 and 10 µM genistein, and 100 µM produced an almost complete reduction in the PKC tyrosine phosphorylation (not shown). These experiments indicate that the effects of carbachol and PMA are mediated via staurosporine- and genistein-sensitive tyrosine kinase activity. Although staurosporine also inhibits PKC activity directly (Tamaoki, 1991), presumably its inhibitory effect in these studies is due to its effects in blocking a tyrosine kinase for which PKC is a substrate (see ``Discussion'').

Translocation of PKC to the Membrane by PMA and Carbachol

The effects of PMA and carbachol treatment of the cells on the translocation of PKC to the plasma membrane were investigated by fractionating the cells into cytosolic and membrane components and immunoprecipitating PKC from these individual fractions. Immunoprecipitated proteins and lysates were subjected to SDS-PAGE and immunoblotting using anti-P-Tyr or anti-PKC antibodies. Tyrosine-phosphorylated PKC was present only in the membrane fraction of PMA- or carbachol-treated cells (Fig. 6, leftpanel). Under basal conditions, a substantial portion of PKC was constitutively localized at the membrane in addition to a cytosolic component (Fig. 6, rightpanel). Other investigators also reported that a significant fraction of PKC was associated constitutively with the membrane fraction in the absence of treatment with phorbol ester (Borner et al., 1992a; Mischak et al., 1993a; Olivier and Parker, 1994). Nearly the entire cytosolic portion of PKC disappeared in cells treated for 5 min with phorbol ester (Fig. 6, rightpanel). In cells treated with carbachol, a portion of the cytosolic PKC was diminished, but this occurred to a lesser extent than that produced by PMA treatment. Similar finding were observed in immunoblots of the lysates (not shown).


Figure 6: Carbachol and PMA stimulate the translocation of cytosolic PKC and its tyrosine phosphorylation at the membrane. Parotid acinar cells were treated with vehicle (-), PMA (200 nM), or carbachol (1 10M) for 5 min, lysed, and fractionated into a cytosolic and membrane component. A lysate portion of each component was retained, and the remainder was subjected to immunoprecipitation using anti-PKC antibody. Proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted using anti-P-Tyr antibody (leftpanel). Tyrosine-phosphorylated PKC was detected only in the membrane fraction of treated cells. The nitrocellulose blot was stripped and reprobed with anti-PKC antibody (rightpanel). The cytosolic component of PKC was substantially diminished in cells treated with PMA and carbachol. Proteins were visualized on x-ray film by using enhanced chemiluminescence. The arrow on the right designates the location of the tyrosine phosphorylated form of PKC.



In other experiments (not shown) using anti-P-Tyr as the immunoprecipitating antibody, PKC was immunoprecipitated from the membrane of PMA-treated cells but not from the cytoplasmic fraction, which was similar to recent results obtained using PKC-transfected myeloid cells (Li et al., 1994b). The parotid cell fractionation experiments indicate that carbachol and phorbol ester promote the translocation of PKC to the plasma membrane, and that tyrosine phosphorylation occurs subsequent to translocation. Consistent with this, neither translocation from the cytosolic fraction nor tyrosine phosphorylation of the membrane fraction of PKC was observed in cells exposed to isoproterenol (10M), EGF (100 ng/ml), and insulin (100 nM) (not shown), suggesting that the two events are linked.


DISCUSSION

The results presented in this study demonstrate that the activation of muscarinic and substance P receptors and phorbol ester stimulated the tyrosine phosphorylation of PKC in rat parotid acinar cells. The tyrosine phosphorylation of PKC reached a maximum within seconds of the addition of the agonists carbachol and substance P, was blocked by genistein and staurosporine, and was not promoted by agonists to other (-adrenergic, EGF, insulin) receptors present on parotid cells. Tyrosine-phosphorylated PKC was immunoprecipitated using anti-P-Tyr antibody or anti-PKC antibody and occurred subsequent to the translocation of cytosolic PKC to the plasma membrane. The rapid decrease in tyrosine phosphorylation after the initial increase suggests the activation of both a protein-tyrosine kinase and tyrosine phosphatase after the addition of carbachol and substance P.

The results presented here, which were obtained using freshly isolated salivary epithelial cells, demonstrate that this biochemical event is one of the signal transduction events that normally occurs in response to acetylcholine and substance P released from parasympathetic nerves at the parotid gland. In this system, neurotransmitters released from parasympathetic nerves initiate fluid secretion, while norepinephrine release from sympathetic nerves promotes protein secretion (exocytosis) via the activation of -adrenergic receptors. Carbachol and substance P mobilize intracellular Ca by binding to receptors that are linked to phospholipase C via GTP-dependent proteins. This promotes the phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol, which elevate the intracellular free Ca concentration ([Ca] ) and activate PKC, respectively (see Nishizuka, 1992). The elevation of [Ca] via the activation of muscarinic and substance P receptors activates Ca-sensitive ion channels that participate in the initiation of saliva formation by the secretion of fluid and electrolytes into the acinar lumen (Baum et al., 1993; Turner, 1993). In contrast, the activation of -adrenergic receptors and the resulting stimulation of cAMP production promote the secretion of amylase but not much water secretion in the salivary system (Quissell, 1993). The lack of effect of isoproterenol, a -adrenergic agonist, on the tyrosine phosphorylation of PKC suggests that this isoform is not involved in the main pathway of activation of exocytosis in these cells. In parotid acinar cells, amylase secretion also is increased by phorbol ester or carbachol, but this stimulation is only small fraction (<50%) of that produced by isoproterenol or cAMP (Takuma and Ichida, 1986; McMillian et al., 1988; Shimomura et al., 1988). Thus, the activation of parotid muscarinic receptors produces a modest effect on amylase release, and this appears to be due to the activation of protein kinase C, although [Ca] may play a modulating role in exocytosis in salivary glands (Tojyo et al., 1992, 1993).

Of interest to the studies presented in this manuscript, staurosporine partially blocked the carbachol-promoted release of amylase but did not block the carbachol-promoted release of K from rat parotid acini (Tojyo et al., 1993). In a study of recombinantly produced PKC isoforms, it was reported that the concentration of staurosporine that inhibits PKC enzyme activity (EC = 500 nM) was about two log orders greater than that required to inhibit PKC (McGlynn et al., 1992). If the inhibition of PKC in parotid cells has a similar sensitivity, then any potential inhibitory effects of staurosporine on physiological processes that may involve PKC could be due to a direct blockade of PKC enzyme activity and/or to a blockade of its tyrosine phosphorylation.

As in many cellular systems, phorbol esters have been reported to have a wide range of effects on various salivary cells, ranging in areas as diverse as promoting the activation of early response genes (Yeh et al., 1992), modulation of Ca-dependent K channels (Moran and Turner, 1993), stimulation of RNA synthesis (Woon et al., 1993), and the phosphorylation of ribosomal protein S6 (Padel and Soling, 1985). Phorbol ester enhanced the -adrenergic receptor-mediated cAMP accumulation in mouse parotid acini (Watson et al., 1993), suggesting that in these cells, as in many others, there is cross-talk between two different signal transduction pathways (e.g. phospholipase C-linked and adenylyl cyclase-linked receptors). Phorbol ester also promoted the differentiation of a neoplastic intercalated salivary duct cell line into one that resembled acinar cells (Hayashi et al., 1987). In this regard, it is interesting that a number of studies have implicated a role for PKC in cell differentiation. As mentioned in the introduction, the tyrosine phosphorylation of PKC appeared to play a role in the platelet-derived growth factor-initiated differentiation of myeloid progenitor cells (Li et al., 1994b). In contrast, staurosporine (acting as a protein kinase inhibitor) reduced the tyrosine phosphorylation of PKC and promoted the differentiation of v-ras-transformed murine keratinocytes that exhibit a constitutively tyrosine-phosphorylated PKC (Denning et al., 1993), which suggested that PKC tyrosine phosphorylation may block differentiation of these cells. Thus, a role for PKC in cellular differentiation may be dependent on the cell type and status, and a specific role in parotid acinar cell differentiation is as yet unrealized.

As in several other recent studies, the protein tyrosine kinase responsible for the tyrosine phosphorylation of PKC remains to be determined. PKC was phosphorylated in vitro by various receptor and cytosolic tyrosine kinases, including Fyn, insulin receptor, and the -platelet-derived growth factor receptor (Li et al., 1994a) and Src (Gschwendt et al., 1994). Two receptor tyrosine kinases on parotid acinar cells, the EGF and insulin receptors, did not promote a detectible tyrosine phosphorylation of PKC in vivo in parotid acinar cells (Fig. 5). In vitro phosphorylation by src occurred only in the presence of PMA, suggesting that a conformational change in PKC may be required prior to its phosphorylation on tyrosine (Gschwendt et al., 1994). This finding is consistent with observations that tyrosine phosphorylation of PKC occurred subsequent to its recruitment to the membrane in phorbol agonist-treated cells (Li et al., 1994b). Tyrosine phosphorylation of PKC has been reported to increase (Li et al., 1994a) or decrease (Denning et al., 1993) its activity.

Our findings that carbachol, substance P, and phorbol ester promote the rapid tyrosine phosphorylation of PKC in parotid acinar cells suggest that the physiological response to this biochemical event is a process that is promoted by these agents due to their common stimulation of PKC activity via sn-1,2-diacylglycerol/phorbol ester binding. As indicated from the summary of secretory events outlined above, potential physiological effects could be related to the major role that these receptors play in fluid secretion, the minor role that they play in protein exocytosis, or to another aspect of cellular physiology or biochemistry. Numerous proteins involved in electrolyte movement have been reported to be substrates for PKC, including the Na,K-ATPase (Beguin et al., 1994) and various ion channels (Dunne, 1994; West et al., 1991). In addition, carbachol produced PKC-mediated changes in cell signaling and transport events in other chloride-secreting epithelia, including promoting the serine phosphorylation of the Na-K-Cl cotransporter in avian salt gland (Torchia et al., 1994) and alterations in receptor-stimulated oscillations in [Ca] in lacrimal cells (Bird et al., 1993).

The increase in tyrosine phosphorylation of PKC that is promoted by carbachol and substance P suggests that the signal transduction pathway promoted by these Ca-mobilizing agonists also involves an increase in tyrosine phosphorylation, a biochemical event that is usually associated with the activation of growth factor receptors. Although the specific effects of PKC tyrosine phosphorylation in parotid acinar cells remains to be determined, the findings outlined in the present report suggest that the tyrosine phosphorylation of a specific isoform of PKC among several that are expressed in this tissue activates an effector downstream of muscarinic and substance P receptors. The fact that the largest effect on tyrosine phosphorylation occurs in the first seconds after the ligands bind to their receptors suggests that PKC participates in the earliest part of the responses mediated by muscarinic and substance P receptors. Thus, PKC tyrosine phosphorylation may be involved in mediating the stimulation of fluid secretion in parotid acinar cells.


FOOTNOTES

*
This work was supported in part by National Institute of Health Grant DE10877 (to S. P. S.) and GM41890 (to A. T., and L. C. Cantley). 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: Div. of Signal Transduction, Harvard Medical School, Alpert Bldg., 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3093; Fax: 617-278-3131; E-mail: ssoltoff@mercury.bih.harvard.edu.

The abbreviations used are: PKC, protein kinase C; anti-P-Tyr, anti-phosphotyrosine; PMSF, phenylmethylsufonyl fluoride; EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.

S. P. Soltoff, unpublished results.


ACKNOWLEDGEMENTS

We thank Margaret Lubkin for excellent technical assistance.


REFERENCES
  1. Baum, B. J., Ambudkar, I. S., and Horn, V. J.(1993) in Biology of the Salivary Glands (Dobrosielksi-Vergona, K., ed) pp. 153-179, CRC Press, Boca Raton, FL
  2. Beguin, P., Beggah, A. T., Chilbalin, A. V., Burgener-Kairuz, P., Jaisser, F., Mathews, P. M., Rossier, B. C., Cotecchia, S., and Geering, K.(1994) J. Biol. Chem. 269, 24437-24445 [Abstract/Free Full Text]
  3. Bird, G. S. J., Rossier, M. F., Obie, J. F., and Putney, J. W., Jr. (1993) J. Biol. Chem. 268, 8425-8428 [Abstract/Free Full Text]
  4. Borner, C., Guadagno, S. N., Fabbro, D., and Weinstein, I. B. (1992a) J. Biol. Chem. 267, 12892-12899 [Abstract/Free Full Text]
  5. Borner, C., Guadagno, S. N., Hsiao, W. W.-L., Fabbro, D., Barr, M., and Weinstein, I. B. (1992b) J. Biol. Chem. 267, 12900-12910 [Abstract/Free Full Text]
  6. Boyle, W. J., van der Geer, P., and Hunter, T.(1991) Methods Enzymol. 201, 110-149 [Medline] [Order article via Infotrieve]
  7. Dekker, L. V., and Parker, P. J.(1994) Trends Biochem. Sci. 19, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  8. Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H.(1993) J. Biol. Chem. 268, 26079-26081 [Abstract/Free Full Text]
  9. Dunne, M. J.(1994) Am. J. Physiol. 267, C501-C506
  10. Gschwendt, M., Keilbassa, K., Kittstein, W., and Marks, F.(1994) FEBS Lett. 347, 85-89 [CrossRef][Medline] [Order article via Infotrieve]
  11. Hayashi, Y., Yoshida, H., Nagamine, S., Yanagawa, T., Yura, Y., Azuma, M., and Sato, M.(1987) Cancer 60, 1000-1008 [Medline] [Order article via Infotrieve]
  12. Hug, H., and Sarre, T. F.(1993) Biochem. J. 291, 329-343 [Medline] [Order article via Infotrieve]
  13. Hughes, A. R., Bird, G. St. J., Obie, J. F., Thastrup, O., and Putney, J. W., Jr.(1991) Mol. Pharmacol. 40, 254-262 [Abstract]
  14. Komabayashi, T., Yakatua, A., Izawa, T., Fujinami, H., Suda, K., and Tsuboi, M.(1992) Eur. J. Pharmacol. 225, 209-216 [Medline] [Order article via Infotrieve]
  15. Li, W., Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. A. (1994a) J. Biol. Chem. 269, 2349-2352 [Abstract/Free Full Text]
  16. Li, W., Yu, J.-C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994b) Mol. Cell. Biol. 14, 6727-6735 [Abstract]
  17. McGlynn, E., Liebetanz, J., Reutener, S., Wood, J., Lydon, N. B., Hofstetter, H., Vanek, M., Meyer, T., and Fabbro, D.(1992) J. Cell. Biochem. 49, 239-250 [Medline] [Order article via Infotrieve]
  18. McMillian, M. K., Soltoff, S. P., and Talamo, B. R.(1987) Biochem. Biophys. Res. Commun. 148, 1017-1024 [Medline] [Order article via Infotrieve]
  19. McMillian, M. K., Soltoff, S. P., Lechleiter, J. D., Cantley, L. C., and Talamo, B. R.(1988) Biochem. J. 255, 291-300 [Medline] [Order article via Infotrieve]
  20. Mischak, H., Goodnight, J., Kolch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M. G., Blumberg, P. M., Pierce, J. H., and Mushinski, J. F. (1993a) J. Biol. Chem. 268, 6090-6096 [Abstract/Free Full Text]
  21. Mischak, H., Pierce, J. H., Goodnight, J., Kazanietz, M. G., Blumberg, P. M., and Mushinski, J. F. (1993b) J. Biol. Chem. 268, 20110-20115 [Abstract/Free Full Text]
  22. Moran, A., and Turner, R. J.(1993) Am. J. Physiol. 265, C1405-C1411
  23. Nakanishi, H., Brewer, K. A., and Exton, J. H.(1993) J. Biol. Chem. 268, 13-16 [Abstract/Free Full Text]
  24. Nishizuka, Y.(1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  25. Olivier, A. R., and Parker, P. J.(1994) J. Biol. Chem. 269, 2758-2763 [Abstract/Free Full Text]
  26. Padel, U., and Soling, H.-D.(1985) Eur. J. Biochem. 151, 1-10 [Abstract]
  27. Quissell, D. O.(1993) in Biology of the Salivary Glands (Dobrosielksi-Vergona, K., ed) pp. 181-200, CRC Press, Boca Raton, FL
  28. Shimomura, H., Terada, A., Hashimoto, Y., and Soderling, T. R.(1988) Biochem. Biophys. Res. Commun. 150, 1309-1314 [Medline] [Order article via Infotrieve]
  29. Singh, S. S., Chauhan, A., Brockerhoff, H., and Chauhan, V. P. S. (1993) Biochem. Biophys. Res. Commun. 195, 104-112 [CrossRef][Medline] [Order article via Infotrieve]
  30. Soltoff, S. P., McMillian, M. K., Cantley, L. C., Cragoe, E. J., Jr., and Talamo, B. R.(1989) J. Gen. Physiol. 93, 285-319 [Abstract]
  31. Sugiya, H., Tennes, K. A., and Putney, J. W., Jr.(1987) Biochem. J. 244, 647-653 [Medline] [Order article via Infotrieve]
  32. Takamura, H., and Putney, J. W., Jr.(1989) Biochem. J. 258, 409-412 [Medline] [Order article via Infotrieve]
  33. Takuma, T., and Ichida, T.(1986) FEBS Lett. 199, 53-56 [CrossRef][Medline] [Order article via Infotrieve]
  34. Tamaoki, T.(1991) Methods Enzymol. 201, 340-347 [Medline] [Order article via Infotrieve]
  35. Terzian, A. R., and Rubin, R. P.(1993) Arch. Oral. Biol. 38, 1051-1056 [Medline] [Order article via Infotrieve]
  36. Tojyo, Y., Tanimura, A., Matsui, S., and Matsumoto, Y.(1992) Cell Struc. Funct. 17, 223-227 [Medline] [Order article via Infotrieve]
  37. Tojyo, Y., Tanimura, A., Matsui, S., and Matsumoto, Y.(1993) Jpn. J. Pharmacol. 63, 439-446 [Medline] [Order article via Infotrieve]
  38. Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M., and Cantley, L. C.(1994) J. Biol. Chem. 269, 32358-32367 [Abstract/Free Full Text]
  39. Torchia, J., Yi, Q., and Sen, A. K.(1994) J. Biol. Chem. 269, 29778-29784 [Abstract/Free Full Text]
  40. Turner, R. J.(1993) in Biology of the Salivary Glands (Dobrosielksi-Vergona, K., ed) pp. 105-127, CRC Press, Boca Raton, FL
  41. Watson, E. L., Jacobson, K., and Meier, K.(1993) Cell. Signal. 5, 583-592 [Medline] [Order article via Infotrieve]
  42. West, J. W., Numann, R., Murphy, B. J., Scheuer, T., and Catterall, W. A.(1991) Science 254, 866-868 [Medline] [Order article via Infotrieve]
  43. Woon, P. Y., Jeyaseelan, K., and Thiyagarajah, P.(1993) Arch. Oral Biol. 38, 1021-1023 [Medline] [Order article via Infotrieve]
  44. Yeh, C.-K., Ambudkar, I. S., and Kousvelari, E.(1992) Am. J. Physiol. 263, G934-G938

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