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STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes
FEMS Microbiology Letters 204 (2001) 65^69
www.fems-microbiology.org
STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes Uma Ganguly
a;b
, Alok Ghosh Chaudhury a , Arindam Basu b , Parimal C. Sen
b
b;
*
a National Institute of Cholera and Enteric Diseases, Kolkata 700010, India Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Kolkata 700009, India
Received 9 April 2001; received in revised form 1 August 2001; accepted 2 August 2001 First published online 14 September 2001
Abstract Escherichia coli heat stable enterotoxin (STa) binds to isolated rat intestinal epithelial cells and triggers a cascade reaction including increase of intracellular calcium levels ([Ca2 ]i ) and membrane bound protein kinase C (PKC) activity. In response to STa, the cytosolic PKC activity falls from 110 to 35 nmol with increase of membrane bound PKC activity from 15 to 78 nmol. Furthermore, the increase of PKC activity induced by STa treatment was always preceded by an increase in [Ca2 ]i . Cytosolic [Ca2 ]i was significantly higher (161 nM) in STa treated cells as compared to untreated cells (51.3 nM). In addition, immunoblot performed on extracts of STa treated rat enterocytes with a monoclonal antibody against PKC K showed a prominent band of PKC K. Translocation of PKC K could be blocked by dantrolene, a drug which inhibits the mobilisation of [Ca2 ]i from the intracellular store. Our results, therefore, provide evidence for the role of [Ca2 ]i in STa treated cells for the translocation of PKC K from cytosol to membrane. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Protein kinase C; Rat enterocyte ; Heat stable enterotoxin
1. Introduction Heat stable enterotoxin (STa) produced by Escherichia coli binds to its receptor guanylyl cyclase type C (GC-C) leading to the activation of the enzyme GC-C resulting in a rise of cGMP in intestinal epithelial cells [1]. Besides cGMP, another signal transduction pathway has been implicated in the induction of intestinal secretion by STa enterotoxin. Previous reports from our laboratory [2^4] and others [5,6] suggest that STa action involves a chain reaction associated with a rise of cytosolic Ca2 and stimulation of diacylglycerol (DG) and phospholipid dependent membrane bound protein kinase C (PKC) activity. PKC is present mainly in the cytosol of resting cells. Activation of PKC by Ca2 and DG or phorbol ester appears to involve the redistribution of enzyme from cytosol to a membrane associated site during stimulation [7,8]. A rise in cytosolic Ca2 alone can bring about redistribution but activation of PKC requires a second interaction with
* Corresponding author : Tel. : +91 (33) 3506619; Fax: +91 (33) 350-6790. E-mail address : [email protected] (P.C. Sen).
phosphatidylserine (PS) and DG. There is abundant evidence that PKC activation in the intestine triggers a secretion of ions and £uid [9]. The PKC activation in response to STa and phosphorylation of GC-C is well established [10] ; however, the physiological relevance of these interactions has not yet been explained. In the present communication, we demonstrate a striking association between inositol trisphosphate (IP3 ) induced elevation of cytosolic Ca2 and the physical translocation of PKC and provide evidence that PKC K is the major translocatable PKC which may have a potential role in the activation of particulate GC-C in STa treated rat enterocytes. 2. Materials and methods 2.1. Reagents Puri¢ed STa and PS, diolein, histone IIIs, dithiothreitol (DTT), phenylmethylsulfonyl £uoride (PMSF), leupeptin and protein kinase C K antibody were obtained from Sigma Chemicals (St. Louis, MO, USA). [Q-32 P]ATP (speci¢c activity 4000 cpm mol31 ) was purchased from Bhabha
0378-1097 / 01 / $20.00 ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 7 3 - 1
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Atomic Research Centre, Hyderabad, India. All other chemicals were of analytical grade and obtained from SRL, India. 2.2. Preparation of rat epithelial cells Rat intestinal epithelial cells were prepared from jejunum as described earlier [11]. The prepared cells were suspended in balanced salt solution (BSS) containing 135 mM NaCl, 4.5 mM KCl, 5.6 mM glucose, 0.5 mM MgCl2 , 10 mM HEPES and 1 mM CaCl2 , pH 7.4 plus 10 Wg ml31 leupeptin. Cell numbers and viability were determined with a haemocytometer and trypan blue exclusion, respectively. 2.3. Preparation of cytosolic and of membrane bound PKC and assay of enzyme activity Epithelial cells (4U106 cells ml31 ) were treated with 6 ng STa and incubated at 37³C for 45 s, 1 min and 2 min. The reaction was stopped by addition of ice cold BSS and the membrane was prepared as described earlier [3]. After centrifugation at 100 000Ug for 60 min, the supernatant was separated from the pellet and used for measuring cytosolic PKC, while the pellet was resuspended in 20 mM Tris^HCl (pH 7.5) and homogenised in the presence of 1% Triton X-100 to solubilise the membrane bound proteins and recentrifuged at 100 000Ug. Supernatant collected after centrifugation was used as the source of membrane PKC. The enzyme was assayed with histone IIIs and pseudosubstrates (PKC substrate based on the classical PKC pseudosubstrate sequence [Ser25 ] PKC fragment 19^31 (RFARKGSLRQKNV) and another substrate containing Thr as phosphate acceptor (VRKRTLRRL) were used for the assay) [12] in the presence of Ca2 , PS and diolein as described previously [3]. 2.4. Detection of PKC by immunoblotting [13] Epithelial cells treated with STa (6 ng) were harvested and suspended in 20 mM Tris bu¡er (pH 7.5), containing
0.25 M sucrose, 10 mM EGTA, 1 mM EDTA, 1 mM PMSF, 10 Wg leupeptin ml31 and 2 mM DTT, homogenised and centrifuged as before at 4³C. The fractions (50 Wg protein) were mixed with one-fourth volume of 5ULaemmli sodium dodecyl sulfate sample bu¡er [14], boiled and subjected to 10% SDS^PAGE, transferred to nitrocellulose membranes and the paper was blocked with 3% bovine albumin overnight. The nitrocellulose blot was incubated with a 1:5000 diluted rabbit monoclonal antisera against PKC K for 2 h at room temperature. Rabbit anti-mouse antibody conjugated to alkaline phosphatase at a dilution of 1:3000 in 2% bovine albumin was used to recognise the band on nitrocellulose membrane. Immunoreactive bands were visualised with NBT-BCIP reagent as the chromogenic substrate. 2.5. Measurement of intracellular [Ca2+] The £uorescent dye fura-2 AM was used as a probe for trapping intracellular free calcium ([Ca2 ]i ). The method was followed as described earlier [4]. In short, the epithelial cells were loaded with the fura-2 AM. Fluorescence was measured at 37³C with excitation at 340 nm and emission at 495 nm. 3. Results and discussion In our earlier studies [3], we reported that membrane bound PKC activity was increased ¢ve-fold in STa treated rat enterocytes compared to normal. STa had no direct role on PKC activity. We also studied the subcellular distribution of PKC activity in both treated and untreated cells [14]. It was observed that at time zero (control), more than 90% of the total PKC activity was found in the cytosolic fraction and decreased after 45 s of STa treatment. Membrane bound activity of PKC was increased and reached a maximum after 1 min of STa incubation. We have further extended the study by measuring cytosolic and membrane bound PKC and [Ca2 ]i under di¡erent conditions.
Table 1 Correlation between accumulation of [Ca2 ]i and translocation of PKC from cytosol to membrane in rat enterocytes under di¡erent condition Condition
No addition STa Dantrolene Dantrolene+STa EGTA EGTA+STa
Concentration
^ 3 ng 6 ng 50 WM 50 WM+6 ng 5 mM 5 mM+6 ng
[Ca2 ]i (nM) 51.3 þ 2.2 105.0 þ 5.8 165.0 þ 14.0 49.7 þ 4.0 65.0 þ 3.5 34.0 þ 4.0 109.0 þ 7.0
PKC activity (nmol (mg protein)31 min31 ) Membrane
Cytosol
15.0 þ 3.2 50.6 þ 8.2 98.4 þ 9.4 18.8 þ 1.2 29.0 þ 2.8 12.0 þ 2.8 51.0 þ 6.5
110 þ 8.5 78 þ 5.2 35 þ 2.6 102 þ 7.6 85 þ 3.7 105 þ 7.0 80 þ 3.5
Cells (4U106 ml31 ) were preincubated with STa for 1 min or with either dantrolene or EGTA for 15 min and 10 min respectively at 37³C, followed by treatment with 6 ng STa for further 1 min. The [Ca2 ]i and PKC activity were measured as described in Section 2. Data represent the mean þ S.E.M. (n = 3).
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In the untreated cells, PKC activity was higher in the cytosolic fraction than in membrane fraction (Table 1). In response to STa, the cytosolic PKC activity fell from 110 to 35 nmol with a simultaneous increase of membrane bound PKC activity from 15 to 98 nmol. Furthermore, the decrease of cytosolic PKC was preceded by an increase in [Ca2 ]i . [Ca2 ]i was signi¢cantly higher (165.0 þ 14.0 nM) in STa treated cells as compared to untreated cells (51.3 þ 2.2 nM) which was also reported earlier [4]. The increase of [Ca2 ]i in the enterocytes appears to be linked to a decrease of cytosolic PKC and concomitant increase of membrane bound PKC activity (Table 1). In order to investigate the functional role of [Ca2 ]i in signalling, the translocation of PKC from cytosol to membrane, the en-
zyme activity was measured in both control and STa treated cells in the presence and absence of dantrolene, a drug which inhibits the mobilisation of [Ca2 ]i from some IP3 sensitive intracellular store, and EGTA (5 mM) which chelates the extracellular Ca2 (1 mM) present in the cell suspension bu¡er resulting in no in£ux of Ca2 . It was found that EGTA reduced both the basal and the STa treated rise in [Ca2 ]i from 51 þ 3 to 34 þ 4 nM and from 165 þ 14 to 109 þ 7 nM, respectively. It was reported earlier that the sharp rise of [Ca2 ]i within 1 min after treatment of STa was followed by rapid decay in the presence of dantrolene whereas in the presence of EGTA the sustained rise of [Ca2 ]i was abolished [4]. A signi¢cant inhibition of both [Ca2 ]i (165 vs 65 nM) and PKC (98 vs 29 nmol) activity in membrane was also noted in STa treated cells on addition of dantrolene, although no direct e¡ect on basal [Ca2 ]i and the activity of PKC was observed. The e¡ect of dantrolene on translocation of K PKC was more pronounced than that of EGTA. The increased activity of PKC in STa treated cells as compared to that in control re£ects the role of released endogenous Ca2 which might act as an activator of PKC. Dose related e¡ects of STa on [Ca2 ]i and PKC are also shown in Table 1. The [Ca2 ]i was increased signi¢cantly from a basal concentration of 51 þ 3 nM to 105 þ 3 nM and 165 þ 14 nM within 1 min of treatment with 3 and 6 ng of STa, respectively. The [Ca2 ]i increased as early as 45 s and reached the maximal level within 1 min of STa incubation (6 ng) followed by a sustained phase up to 5 min at least (Fig. 1). At this period the cytosolic PKC activity decreased rapidly (within 1 min) after STa treatment and remained low after that (Fig. 4, top).
Fig. 2. E¡ect of Ca2 on membrane bound PKC activity in rat enterocytes. Cells (4U106 ml31 ) were treated with 6 ng STa for 1 min and the enzyme was assayed in the presence of di¡erent concentrations of Ca2 as mentioned above. Hanes^Woolf plot. Data represent the mean of three separate experiments.
Fig. 3. E¡ect of diolein on membrane bound PKC activity in rat enterocytes. Cells (4U106 ml31 ) were treated with 6 ng STa for 1 min and the enzyme was assayed in the presence of di¡erent concentrations of diolein as mentioned above. Hanes^Woolf plot. Data represent the mean of three separate experiments.
Fig. 1. Time course of STa (6 ng) treatment on intracellular calcium in rat enterocytes. Cells (4U106 ml31 ) were loaded with 5 WM fura-2 AM for 40 min followed by treatment with STa. The arrow indicates the time of addition of STa. The result shown here is a representative of three separate experiments.
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The studies were further elaborated with di¡erent concentrations of Ca2 (Fig. 2) and diolein (Fig. 3) in the presence of a known amount of PS. In Figs. 2 and 3, the negative intercept on the horizontal axis of each Hanes^Woolf plot gives rise to the Ka value for Ca2 and diolein respectively. In control and STa treated cells, the Ka for Ca2 averaged 0.34 mM and 0.10 mM, respectively, while the corresponding value for diolein was 25 WM and 6.25 WM. The particulate protein kinase, which we measured, was not a¡ected either in control or in STa treated cells by addition of cGMP (2 WM), cAMP (1 WM) and calmodulin (60 Wg ml31 ) to the assay system. The change appeared to be speci¢c for PKC activity as kinetic studies showed the enzyme was Ca2 , PS and diolein dependent. Ka values for Ca2 and diolein were much lower in STa treated cells. The results further con¢rm our previous studies [3] showing a considerable enzyme activity in the absence of PS and diolein suggesting that [Ca2 ]i and DG remained high in the STa treated cells. Fig. 4 (bottom) shows the translocation of PKC into epithelial cell membranes with PKC K monoclonal antibody. The 82-kDa PKC K, the predominant isotype (lane 1) in control cells (zero time), followed by decreased PKC K immunological cross reactivity in the cytosol (lanes 2^4) was noted. In contrast, at zero time (lane 5), the PKC K level was found to be minimal in the membrane and increased to a maximum at 1 min (lane 7). These ¢ndings were corroborated by the PKC activity at di¡erent times of STa treatment (Fig. 4, top). The decreased activity
Fig. 4. Top: Time course of STa treatment on PKC activity in rat enterocytes. Bottom: Immunoblot showing the translocation of PKC K from cytosol to membrane following STa treatment at di¡erent times. Cytosolic and membrane fractions from control and treated enterocytes were subjected to 10% SDS^PAGE. After the run was over, proteins were transferred to nitrocellulose membrane and immunoblotting was done as described in Section 2. The data represent one of the three experiments. Lane 1, control cytosol; lanes 2^4, at 45 s, 1 min and 2 min respectively after STa (6 ng) treatment ; lanes 5^8, the corresponding membrane fractions.
Table 2 E¡ect of di¡erent substrates on membrane bound PKC activity in rat enterocytes Substrate
Histone IIIs 25 Ser pseudosubstratea Threonine pseudosubstrateb
PKC activity (nmol (mg protein)31 min31 ) Normal
STa treated
15.0 þ 3.2 25.0 þ 2.0 20.0 þ 2.5
78.4 þ 9.4 212.6 þ 5.8 106.3 þ 3.5
Cells (4U106 ml31 ) were treated with 6 ng STa for 1 min and PKC activity was assayed with di¡erent substrates as described in Section 2. PKC substrates based on the classical PKC pseudosubstrate sequence [Ser25 ]a PKC fragment 19^31 (RFARKGSLRQKNV) and another substrate containing Thrb as phosphate acceptor (VRKRTLRRL) were used for the assay.
shown in this ¢gure (2 min, top and bottom) is probably due to downregulated cleavage of the enzyme. PKC catalyses the phosphorylation of seryl and threonyl residues in many proteins [7] including the GC-C receptor for STa [16]. Histone HI and HIIIs are routinely used to assay this enzyme activity. However, phosphorylation of HIII histone is very non-speci¢c. To overcome this, we have used some pseudosubstrates speci¢c for PKC and it was observed that synthetic peptides were phosphorylated by PKC in the presence of Ca2 , DG and PS in both control and STa treated cells (Table 2). It has already been shown that inhibition of PKC with speci¢c inhibitors [15] reduces the activity of STa induced GC-C suggesting that the translocatable PKC K plays an important role in the activation of GC-C. It was reported previously that PKC phosphorylates the Ser 1029 residue of the STa receptor GC-C in vitro and in vivo causing activation of the enzyme GC-C [17]. Considerable rises in PKC activity (approximately three-fold) with the 25 Ser pseudosubstrate and one-fold with the Thr pseudosubstrate compared to histone were obtained (Table 2). The time course of phosphorylation of the 25 Ser pseudosubstrate by STa treated membrane proceeded linearly and the Km is considerably low (7 WM) (results not shown). The involvement of PKC K in STa mediated action was further con¢rmed in this study as the Km values for PKC LI and LII were much higher than that for PKC K and this pseudosubstrate is known to be a poor substrate for the N, O and P subspecies [12]. PKC translocation to the plasma membrane has been shown to be related to the rise of Ca2 . STa induced translocation of PKC K to the plasma membrane has been implicated in the mobilisation of Ca2 and DG which is the hydrolysed product of phosphatidylinositol bisphosphate by phospholipase C [2,4]. The activation of PKC activity by STa required its translocation from the cytoplasm to the plasma membrane where it is activated through a complex mechanism. Though the question as to precisely where the events occur at the plasma membrane remains unresolved, it is clear that the translocation of PKC is crucial.
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Acknowledgements The work was supported by the Indian Council of Medical Research in the form of awarding an Emeritus Scientistship to Dr. Uma Ganguly [No. 74/6/96-Pers.(EMS)]. We are thankful to Dr. Ratna Ray, USA for supplying protein kinase C K antibody. References [1] Sears, C.L. and Kaper, B.J. (1996) Enteric bacterial toxins: Mechanisms of action and linkage to intestinal secretion. Microbiol. Rev. 60, 167^215. [2] Banik, N.D. and Ganguly, U. (1989) Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells: E¡ect of E. coli heat-stable toxin. FEBS Lett. 250, 201^204. [3] Ghosh Chaudhury, A., Sen, P.C. and Ganguly, U. (1993) Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli. FEMS Microbiol. Lett. 110, 185^190. [4] Ghosh Chaudhury, A. and Ganguly, U. (1995) Evidence for stimulation of the inositol triphosphate- Ca2 signalling system in rat enterocytes by heat-stable enterotoxin of E. coli. Biochim. Biophys. Acta 1267, 131^133. [5] Knoop, F.C., Martig, R.J. and Boetem, W.J. (1990) The e¡ect of Escherichia coli heat-stable enterotoxin on protein kinase activity. Toxicon 28, 493^500. [6] Weikel, C.S., Spann, C.L., Chambers, C.P., Crane, J.K., Linden, J. and Hewlett, E.L. (1990) Phorbol esters enhance the cyclic GMP response of T84 cells to the heat-stable enterotoxin of E. coli (STa). Infect. Immun. 58, 1402^1407.
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[7] Kikkawa, U., Kishimoto, A. and Nishizuka, Y. (1989) The protein kinase C family : Heterogeneity and its implications. Annu. Rev. Biochem. 58, 31^44. [8] Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607^ 614. [9] Fondacaro, J.D. and Henderson, L.S. (1985) Evidence for protein kinase C as a regulator of intestinal electrolyte transport. Am. J. Physiol. 249, C356^C361. [10] Crane, J.K., Weliner, M.S., Bolen, E.J., Sando, J.J., Linden, J., Gnerrant, R.L. and Sears, C.L. (1992) Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of E. coli (STa) and protein kinase C. Infect. Immun. 60, 5004^5012. [11] Banik, N. and Ganguly, U. (1988) Stimulation of phosphoinositides breakdown by the heat-stable E. coli enterotoxin in rat intestinal epithelial cells. FEBS Lett. 236, 489^492. [12] Kemp, B.F. and Pearson, B.R. (1991) Design and use of peptide substrates for protein kinases. Methods Enzymol. 200, 121^130. [13] Towbin, J., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350^ 4354. [14] Laemmili, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680^685. [15] Ganguly, U. and Ghosh Chaudhury, A. (1996) in: Cytokines, Cholera and the Gut (Keersch, G.T. and Kawakami, M., Eds.), pp. 281^ 288. IOS Press. [16] Danko, S., Kim, D.H., Sreter, F.A. and Ikemoto, N. (1985) Inhibitors of Ca2 -release from the isolated sarcoplasmic reticulum. II. The e¡ect of dantrolene on Ca2 -release induced by ca¡eine, Ca2 and depolarization. Biochim. Biophys. Acta 816, 18^24. [17] Crane, J.K. and Sharks, K.L. (1996) Phosphorylation and activation of the intestinal guanylyl cyclase receptor for E. coli heat-stable toxin by protein kinase. Mol. Cell. Biochem. 165, 111^120.
FEMSLE 10121 17-10-01
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STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes Uma Ganguly
a;b
, Alok Ghosh Chaudhury a , Arindam Basu b , Parimal C. Sen
b
b;
*
a National Institute of Cholera and Enteric Diseases, Kolkata 700010, India Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Kolkata 700009, India
Received 9 April 2001; received in revised form 1 August 2001; accepted 2 August 2001 First published online 14 September 2001
Abstract Escherichia coli heat stable enterotoxin (STa) binds to isolated rat intestinal epithelial cells and triggers a cascade reaction including increase of intracellular calcium levels ([Ca2 ]i ) and membrane bound protein kinase C (PKC) activity. In response to STa, the cytosolic PKC activity falls from 110 to 35 nmol with increase of membrane bound PKC activity from 15 to 78 nmol. Furthermore, the increase of PKC activity induced by STa treatment was always preceded by an increase in [Ca2 ]i . Cytosolic [Ca2 ]i was significantly higher (161 nM) in STa treated cells as compared to untreated cells (51.3 nM). In addition, immunoblot performed on extracts of STa treated rat enterocytes with a monoclonal antibody against PKC K showed a prominent band of PKC K. Translocation of PKC K could be blocked by dantrolene, a drug which inhibits the mobilisation of [Ca2 ]i from the intracellular store. Our results, therefore, provide evidence for the role of [Ca2 ]i in STa treated cells for the translocation of PKC K from cytosol to membrane. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Protein kinase C; Rat enterocyte ; Heat stable enterotoxin
1. Introduction Heat stable enterotoxin (STa) produced by Escherichia coli binds to its receptor guanylyl cyclase type C (GC-C) leading to the activation of the enzyme GC-C resulting in a rise of cGMP in intestinal epithelial cells [1]. Besides cGMP, another signal transduction pathway has been implicated in the induction of intestinal secretion by STa enterotoxin. Previous reports from our laboratory [2^4] and others [5,6] suggest that STa action involves a chain reaction associated with a rise of cytosolic Ca2 and stimulation of diacylglycerol (DG) and phospholipid dependent membrane bound protein kinase C (PKC) activity. PKC is present mainly in the cytosol of resting cells. Activation of PKC by Ca2 and DG or phorbol ester appears to involve the redistribution of enzyme from cytosol to a membrane associated site during stimulation [7,8]. A rise in cytosolic Ca2 alone can bring about redistribution but activation of PKC requires a second interaction with
* Corresponding author : Tel. : +91 (33) 3506619; Fax: +91 (33) 350-6790. E-mail address : [email protected] (P.C. Sen).
phosphatidylserine (PS) and DG. There is abundant evidence that PKC activation in the intestine triggers a secretion of ions and £uid [9]. The PKC activation in response to STa and phosphorylation of GC-C is well established [10] ; however, the physiological relevance of these interactions has not yet been explained. In the present communication, we demonstrate a striking association between inositol trisphosphate (IP3 ) induced elevation of cytosolic Ca2 and the physical translocation of PKC and provide evidence that PKC K is the major translocatable PKC which may have a potential role in the activation of particulate GC-C in STa treated rat enterocytes. 2. Materials and methods 2.1. Reagents Puri¢ed STa and PS, diolein, histone IIIs, dithiothreitol (DTT), phenylmethylsulfonyl £uoride (PMSF), leupeptin and protein kinase C K antibody were obtained from Sigma Chemicals (St. Louis, MO, USA). [Q-32 P]ATP (speci¢c activity 4000 cpm mol31 ) was purchased from Bhabha
0378-1097 / 01 / $20.00 ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 7 3 - 1
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Atomic Research Centre, Hyderabad, India. All other chemicals were of analytical grade and obtained from SRL, India. 2.2. Preparation of rat epithelial cells Rat intestinal epithelial cells were prepared from jejunum as described earlier [11]. The prepared cells were suspended in balanced salt solution (BSS) containing 135 mM NaCl, 4.5 mM KCl, 5.6 mM glucose, 0.5 mM MgCl2 , 10 mM HEPES and 1 mM CaCl2 , pH 7.4 plus 10 Wg ml31 leupeptin. Cell numbers and viability were determined with a haemocytometer and trypan blue exclusion, respectively. 2.3. Preparation of cytosolic and of membrane bound PKC and assay of enzyme activity Epithelial cells (4U106 cells ml31 ) were treated with 6 ng STa and incubated at 37³C for 45 s, 1 min and 2 min. The reaction was stopped by addition of ice cold BSS and the membrane was prepared as described earlier [3]. After centrifugation at 100 000Ug for 60 min, the supernatant was separated from the pellet and used for measuring cytosolic PKC, while the pellet was resuspended in 20 mM Tris^HCl (pH 7.5) and homogenised in the presence of 1% Triton X-100 to solubilise the membrane bound proteins and recentrifuged at 100 000Ug. Supernatant collected after centrifugation was used as the source of membrane PKC. The enzyme was assayed with histone IIIs and pseudosubstrates (PKC substrate based on the classical PKC pseudosubstrate sequence [Ser25 ] PKC fragment 19^31 (RFARKGSLRQKNV) and another substrate containing Thr as phosphate acceptor (VRKRTLRRL) were used for the assay) [12] in the presence of Ca2 , PS and diolein as described previously [3]. 2.4. Detection of PKC by immunoblotting [13] Epithelial cells treated with STa (6 ng) were harvested and suspended in 20 mM Tris bu¡er (pH 7.5), containing
0.25 M sucrose, 10 mM EGTA, 1 mM EDTA, 1 mM PMSF, 10 Wg leupeptin ml31 and 2 mM DTT, homogenised and centrifuged as before at 4³C. The fractions (50 Wg protein) were mixed with one-fourth volume of 5ULaemmli sodium dodecyl sulfate sample bu¡er [14], boiled and subjected to 10% SDS^PAGE, transferred to nitrocellulose membranes and the paper was blocked with 3% bovine albumin overnight. The nitrocellulose blot was incubated with a 1:5000 diluted rabbit monoclonal antisera against PKC K for 2 h at room temperature. Rabbit anti-mouse antibody conjugated to alkaline phosphatase at a dilution of 1:3000 in 2% bovine albumin was used to recognise the band on nitrocellulose membrane. Immunoreactive bands were visualised with NBT-BCIP reagent as the chromogenic substrate. 2.5. Measurement of intracellular [Ca2+] The £uorescent dye fura-2 AM was used as a probe for trapping intracellular free calcium ([Ca2 ]i ). The method was followed as described earlier [4]. In short, the epithelial cells were loaded with the fura-2 AM. Fluorescence was measured at 37³C with excitation at 340 nm and emission at 495 nm. 3. Results and discussion In our earlier studies [3], we reported that membrane bound PKC activity was increased ¢ve-fold in STa treated rat enterocytes compared to normal. STa had no direct role on PKC activity. We also studied the subcellular distribution of PKC activity in both treated and untreated cells [14]. It was observed that at time zero (control), more than 90% of the total PKC activity was found in the cytosolic fraction and decreased after 45 s of STa treatment. Membrane bound activity of PKC was increased and reached a maximum after 1 min of STa incubation. We have further extended the study by measuring cytosolic and membrane bound PKC and [Ca2 ]i under di¡erent conditions.
Table 1 Correlation between accumulation of [Ca2 ]i and translocation of PKC from cytosol to membrane in rat enterocytes under di¡erent condition Condition
No addition STa Dantrolene Dantrolene+STa EGTA EGTA+STa
Concentration
^ 3 ng 6 ng 50 WM 50 WM+6 ng 5 mM 5 mM+6 ng
[Ca2 ]i (nM) 51.3 þ 2.2 105.0 þ 5.8 165.0 þ 14.0 49.7 þ 4.0 65.0 þ 3.5 34.0 þ 4.0 109.0 þ 7.0
PKC activity (nmol (mg protein)31 min31 ) Membrane
Cytosol
15.0 þ 3.2 50.6 þ 8.2 98.4 þ 9.4 18.8 þ 1.2 29.0 þ 2.8 12.0 þ 2.8 51.0 þ 6.5
110 þ 8.5 78 þ 5.2 35 þ 2.6 102 þ 7.6 85 þ 3.7 105 þ 7.0 80 þ 3.5
Cells (4U106 ml31 ) were preincubated with STa for 1 min or with either dantrolene or EGTA for 15 min and 10 min respectively at 37³C, followed by treatment with 6 ng STa for further 1 min. The [Ca2 ]i and PKC activity were measured as described in Section 2. Data represent the mean þ S.E.M. (n = 3).
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In the untreated cells, PKC activity was higher in the cytosolic fraction than in membrane fraction (Table 1). In response to STa, the cytosolic PKC activity fell from 110 to 35 nmol with a simultaneous increase of membrane bound PKC activity from 15 to 98 nmol. Furthermore, the decrease of cytosolic PKC was preceded by an increase in [Ca2 ]i . [Ca2 ]i was signi¢cantly higher (165.0 þ 14.0 nM) in STa treated cells as compared to untreated cells (51.3 þ 2.2 nM) which was also reported earlier [4]. The increase of [Ca2 ]i in the enterocytes appears to be linked to a decrease of cytosolic PKC and concomitant increase of membrane bound PKC activity (Table 1). In order to investigate the functional role of [Ca2 ]i in signalling, the translocation of PKC from cytosol to membrane, the en-
zyme activity was measured in both control and STa treated cells in the presence and absence of dantrolene, a drug which inhibits the mobilisation of [Ca2 ]i from some IP3 sensitive intracellular store, and EGTA (5 mM) which chelates the extracellular Ca2 (1 mM) present in the cell suspension bu¡er resulting in no in£ux of Ca2 . It was found that EGTA reduced both the basal and the STa treated rise in [Ca2 ]i from 51 þ 3 to 34 þ 4 nM and from 165 þ 14 to 109 þ 7 nM, respectively. It was reported earlier that the sharp rise of [Ca2 ]i within 1 min after treatment of STa was followed by rapid decay in the presence of dantrolene whereas in the presence of EGTA the sustained rise of [Ca2 ]i was abolished [4]. A signi¢cant inhibition of both [Ca2 ]i (165 vs 65 nM) and PKC (98 vs 29 nmol) activity in membrane was also noted in STa treated cells on addition of dantrolene, although no direct e¡ect on basal [Ca2 ]i and the activity of PKC was observed. The e¡ect of dantrolene on translocation of K PKC was more pronounced than that of EGTA. The increased activity of PKC in STa treated cells as compared to that in control re£ects the role of released endogenous Ca2 which might act as an activator of PKC. Dose related e¡ects of STa on [Ca2 ]i and PKC are also shown in Table 1. The [Ca2 ]i was increased signi¢cantly from a basal concentration of 51 þ 3 nM to 105 þ 3 nM and 165 þ 14 nM within 1 min of treatment with 3 and 6 ng of STa, respectively. The [Ca2 ]i increased as early as 45 s and reached the maximal level within 1 min of STa incubation (6 ng) followed by a sustained phase up to 5 min at least (Fig. 1). At this period the cytosolic PKC activity decreased rapidly (within 1 min) after STa treatment and remained low after that (Fig. 4, top).
Fig. 2. E¡ect of Ca2 on membrane bound PKC activity in rat enterocytes. Cells (4U106 ml31 ) were treated with 6 ng STa for 1 min and the enzyme was assayed in the presence of di¡erent concentrations of Ca2 as mentioned above. Hanes^Woolf plot. Data represent the mean of three separate experiments.
Fig. 3. E¡ect of diolein on membrane bound PKC activity in rat enterocytes. Cells (4U106 ml31 ) were treated with 6 ng STa for 1 min and the enzyme was assayed in the presence of di¡erent concentrations of diolein as mentioned above. Hanes^Woolf plot. Data represent the mean of three separate experiments.
Fig. 1. Time course of STa (6 ng) treatment on intracellular calcium in rat enterocytes. Cells (4U106 ml31 ) were loaded with 5 WM fura-2 AM for 40 min followed by treatment with STa. The arrow indicates the time of addition of STa. The result shown here is a representative of three separate experiments.
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The studies were further elaborated with di¡erent concentrations of Ca2 (Fig. 2) and diolein (Fig. 3) in the presence of a known amount of PS. In Figs. 2 and 3, the negative intercept on the horizontal axis of each Hanes^Woolf plot gives rise to the Ka value for Ca2 and diolein respectively. In control and STa treated cells, the Ka for Ca2 averaged 0.34 mM and 0.10 mM, respectively, while the corresponding value for diolein was 25 WM and 6.25 WM. The particulate protein kinase, which we measured, was not a¡ected either in control or in STa treated cells by addition of cGMP (2 WM), cAMP (1 WM) and calmodulin (60 Wg ml31 ) to the assay system. The change appeared to be speci¢c for PKC activity as kinetic studies showed the enzyme was Ca2 , PS and diolein dependent. Ka values for Ca2 and diolein were much lower in STa treated cells. The results further con¢rm our previous studies [3] showing a considerable enzyme activity in the absence of PS and diolein suggesting that [Ca2 ]i and DG remained high in the STa treated cells. Fig. 4 (bottom) shows the translocation of PKC into epithelial cell membranes with PKC K monoclonal antibody. The 82-kDa PKC K, the predominant isotype (lane 1) in control cells (zero time), followed by decreased PKC K immunological cross reactivity in the cytosol (lanes 2^4) was noted. In contrast, at zero time (lane 5), the PKC K level was found to be minimal in the membrane and increased to a maximum at 1 min (lane 7). These ¢ndings were corroborated by the PKC activity at di¡erent times of STa treatment (Fig. 4, top). The decreased activity
Fig. 4. Top: Time course of STa treatment on PKC activity in rat enterocytes. Bottom: Immunoblot showing the translocation of PKC K from cytosol to membrane following STa treatment at di¡erent times. Cytosolic and membrane fractions from control and treated enterocytes were subjected to 10% SDS^PAGE. After the run was over, proteins were transferred to nitrocellulose membrane and immunoblotting was done as described in Section 2. The data represent one of the three experiments. Lane 1, control cytosol; lanes 2^4, at 45 s, 1 min and 2 min respectively after STa (6 ng) treatment ; lanes 5^8, the corresponding membrane fractions.
Table 2 E¡ect of di¡erent substrates on membrane bound PKC activity in rat enterocytes Substrate
Histone IIIs 25 Ser pseudosubstratea Threonine pseudosubstrateb
PKC activity (nmol (mg protein)31 min31 ) Normal
STa treated
15.0 þ 3.2 25.0 þ 2.0 20.0 þ 2.5
78.4 þ 9.4 212.6 þ 5.8 106.3 þ 3.5
Cells (4U106 ml31 ) were treated with 6 ng STa for 1 min and PKC activity was assayed with di¡erent substrates as described in Section 2. PKC substrates based on the classical PKC pseudosubstrate sequence [Ser25 ]a PKC fragment 19^31 (RFARKGSLRQKNV) and another substrate containing Thrb as phosphate acceptor (VRKRTLRRL) were used for the assay.
shown in this ¢gure (2 min, top and bottom) is probably due to downregulated cleavage of the enzyme. PKC catalyses the phosphorylation of seryl and threonyl residues in many proteins [7] including the GC-C receptor for STa [16]. Histone HI and HIIIs are routinely used to assay this enzyme activity. However, phosphorylation of HIII histone is very non-speci¢c. To overcome this, we have used some pseudosubstrates speci¢c for PKC and it was observed that synthetic peptides were phosphorylated by PKC in the presence of Ca2 , DG and PS in both control and STa treated cells (Table 2). It has already been shown that inhibition of PKC with speci¢c inhibitors [15] reduces the activity of STa induced GC-C suggesting that the translocatable PKC K plays an important role in the activation of GC-C. It was reported previously that PKC phosphorylates the Ser 1029 residue of the STa receptor GC-C in vitro and in vivo causing activation of the enzyme GC-C [17]. Considerable rises in PKC activity (approximately three-fold) with the 25 Ser pseudosubstrate and one-fold with the Thr pseudosubstrate compared to histone were obtained (Table 2). The time course of phosphorylation of the 25 Ser pseudosubstrate by STa treated membrane proceeded linearly and the Km is considerably low (7 WM) (results not shown). The involvement of PKC K in STa mediated action was further con¢rmed in this study as the Km values for PKC LI and LII were much higher than that for PKC K and this pseudosubstrate is known to be a poor substrate for the N, O and P subspecies [12]. PKC translocation to the plasma membrane has been shown to be related to the rise of Ca2 . STa induced translocation of PKC K to the plasma membrane has been implicated in the mobilisation of Ca2 and DG which is the hydrolysed product of phosphatidylinositol bisphosphate by phospholipase C [2,4]. The activation of PKC activity by STa required its translocation from the cytoplasm to the plasma membrane where it is activated through a complex mechanism. Though the question as to precisely where the events occur at the plasma membrane remains unresolved, it is clear that the translocation of PKC is crucial.
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Acknowledgements The work was supported by the Indian Council of Medical Research in the form of awarding an Emeritus Scientistship to Dr. Uma Ganguly [No. 74/6/96-Pers.(EMS)]. We are thankful to Dr. Ratna Ray, USA for supplying protein kinase C K antibody. References [1] Sears, C.L. and Kaper, B.J. (1996) Enteric bacterial toxins: Mechanisms of action and linkage to intestinal secretion. Microbiol. Rev. 60, 167^215. [2] Banik, N.D. and Ganguly, U. (1989) Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells: E¡ect of E. coli heat-stable toxin. FEBS Lett. 250, 201^204. [3] Ghosh Chaudhury, A., Sen, P.C. and Ganguly, U. (1993) Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli. FEMS Microbiol. Lett. 110, 185^190. [4] Ghosh Chaudhury, A. and Ganguly, U. (1995) Evidence for stimulation of the inositol triphosphate- Ca2 signalling system in rat enterocytes by heat-stable enterotoxin of E. coli. Biochim. Biophys. Acta 1267, 131^133. [5] Knoop, F.C., Martig, R.J. and Boetem, W.J. (1990) The e¡ect of Escherichia coli heat-stable enterotoxin on protein kinase activity. Toxicon 28, 493^500. [6] Weikel, C.S., Spann, C.L., Chambers, C.P., Crane, J.K., Linden, J. and Hewlett, E.L. (1990) Phorbol esters enhance the cyclic GMP response of T84 cells to the heat-stable enterotoxin of E. coli (STa). Infect. Immun. 58, 1402^1407.
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[7] Kikkawa, U., Kishimoto, A. and Nishizuka, Y. (1989) The protein kinase C family : Heterogeneity and its implications. Annu. Rev. Biochem. 58, 31^44. [8] Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607^ 614. [9] Fondacaro, J.D. and Henderson, L.S. (1985) Evidence for protein kinase C as a regulator of intestinal electrolyte transport. Am. J. Physiol. 249, C356^C361. [10] Crane, J.K., Weliner, M.S., Bolen, E.J., Sando, J.J., Linden, J., Gnerrant, R.L. and Sears, C.L. (1992) Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of E. coli (STa) and protein kinase C. Infect. Immun. 60, 5004^5012. [11] Banik, N. and Ganguly, U. (1988) Stimulation of phosphoinositides breakdown by the heat-stable E. coli enterotoxin in rat intestinal epithelial cells. FEBS Lett. 236, 489^492. [12] Kemp, B.F. and Pearson, B.R. (1991) Design and use of peptide substrates for protein kinases. Methods Enzymol. 200, 121^130. [13] Towbin, J., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350^ 4354. [14] Laemmili, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680^685. [15] Ganguly, U. and Ghosh Chaudhury, A. (1996) in: Cytokines, Cholera and the Gut (Keersch, G.T. and Kawakami, M., Eds.), pp. 281^ 288. IOS Press. [16] Danko, S., Kim, D.H., Sreter, F.A. and Ikemoto, N. (1985) Inhibitors of Ca2 -release from the isolated sarcoplasmic reticulum. II. The e¡ect of dantrolene on Ca2 -release induced by ca¡eine, Ca2 and depolarization. Biochim. Biophys. Acta 816, 18^24. [17] Crane, J.K. and Sharks, K.L. (1996) Phosphorylation and activation of the intestinal guanylyl cyclase receptor for E. coli heat-stable toxin by protein kinase. Mol. Cell. Biochem. 165, 111^120.
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