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proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca, Argentina
(Requests for offprints should be addressed to A R de Boland; Email: aboland{at}criba.edu.ar)
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Abstract |
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s and Gß, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gß subunits by preincubation of cells with a Gß antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gß
–c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2. |
Introduction |
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(Gentili et al. 2001a) and the mitogen-activated protein kinases, ERK1 and ERK2 (Gentili & de Boland 2000) which leads to an increase in DNA synthesis (Gentili et al. 2001b). Initial studies on the mechanisms underlying PTH activation of the enterocyte tyrosine phosphorylation revealed that the cytosolic tyrosine kinase c-Src plays a central role in these processes, demonstrating that pharmacological inhibition of c-Src abolishes PTH-dependent PLC phosphorylation, the ERK cascade activation (Gentili & de Boland 2000, Gentili et al. 2001a) and ERK-induced enterocyte proliferation (Gentili et al. 2001b). The c-Src tyrosine kinase has been shown to regulate a diverse number of cellular effects including stimulating and inhibiting cell growth (Roche et al. 1995, Broome & Hunter 1996), regulating cell adhesion (Parsons & Parsons 1997, Cary et al. 2002), and regulating apoptosis (Carragher et al. 2001).
The heterotrimeric guanine nucleotide binding (G) proteins control diverse biological processes by conveying signals from cell-surface receptors to intracellular effectors. They are a family of proteins that transduce an extracellular signal to an intracellular response via a seven helical transmembrane receptor (G protein-coupled receptor, GPCR). Upon activation, the receptor facilitates the exchange of GDP for GTP in the G subunit. G is then thought to dissociate from the Gß heterodimer, allowing both complexes to individually activate a number of effectors (Neer 1995, Hamm 1998). Although function was originally ascribed to the GTP-bound -subunit, it is now well established that the ß-dimer plays active roles in the signaling process through upstream recognition of receptors and downstream regulation of effectors (Clapham & Neer 1997). Molecular cloning has identified at least 5 ß- and 12 -subunit genes in the mouse and human genomes. Structurally, -subunits are the most diverse, with four subgroups that show less than 50% identity to each other (Balcueva et al. 2000). Moreover, -subunits exhibit very different temporal (Morishita et al. 1999, Schuller et al. 2001) and spatial (Betty et al. 1998) patterns of expression. These characteristics suggest that -subunits have heterogeneous functions. Free Gß interacts with a large assortment of effector proteins, including phospholipases (Rhee & Bae 1997), adenylyl cyclases (Sunahara et al. 1996), ion channels (Schneider et al. 1997), and G protein-coupled receptor kinases (Pitcher et al. 1992). There are, however, G protein-coupled receptor responses, such as MAP kinase activation (Koch et al. 1994, Luttrell et al. 1996, 1997), receptor internalization (Liu et al. 1997, Lin et al. 1998), and organelle transport (Stow & Heimann 1998, Jamora et al. 1999) that are mediated through the Gß subunit but which have not definitively been linked to known Gß effectors. Although activation of G protein–coupled receptors can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present study, we identified c-Src tyrosine kinase as a direct effector of G proteins and characterized the G proteins subunits involved in the mechanism by which PTH regulates ERKs via c-Src tyrosine kinase.
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Materials and Methods |
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Synthetic rat PTH(1–34), immobilon P (polyvinylidene difluoride, PVDF) membranes, enolase and protein A-Sepharose were from Sigma Chemical Co. (St Louis, MO, USA). Rabbit polyclonal anti-phosphotyrosine antibody, rabbit anti-phospho c-Src Y527 and Y416 antibodies were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-PLC, anti-P-ERK, anti-ERK and anti-c-Src antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). G protein antibodies were generously provided by Dr Maria Julia Marinissen (NIH, MD, USA). Secondary antibody goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG and the Super Signal CL-HRP substrate system for enhanced chemiluminiscence (ECL) were obtained from Amersham Corp. (Arlington Heights, IL, USA). [-32P]ATP (3000 Ci/mmol) was from New England Nuclear (Chicago, IL, USA). All other reagents were of analytical grade.
Animals
Three-month-old male Wistar rats were fed with standard rat food (1.2% Ca, 1.0% phosphorus), with water available ad libitum, and were maintained on a 12 h light-12 h darkness cycle. Animals were killed by cervical dislocation. Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals (1996 7th edn) Washington, DC: National Academy Press, aka National Research Council Guide).
Isolation of duodenal cells
Duodenal cells were isolated as described previously (Massheimer et al. 1994). The method employed yields preparations containing only highly absorptive epithelial cells that are devoid of cells from the upper villus or crypt (Weiser 1973). The duodenum was excised, washed and trimmed of adhering tissue. The intestine was slit lengthwise, cut into small segments (2 cm length) and placed into solution A: 96 mM NaCl, 1.5 mM KCl, 8 mM KH2PO4, 5.6 mM Na2HPO4, 27 mM Na citrate, pH 7.3, for 10 min at 37 °C. The solution was discarded and replaced with solution B (isolation medium): 154 mM NaCl, 10 mM NaH2PO4, 1.5 mM EDTA, 0.5 mM dithio-threitol (DTT), 5.6 mM glucose, pH 7.3, for 15 min at 37 °C with vigorous shaking. The cells were sedimented by centrifugation at 155 x g for 10 min, washed twice with 154 mM NaCl, 10 mM NaH2PO4, 5.6 mM glucose, pH 7.4 and resuspended in the incubation medium (solution D): 154 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM MgCl2, 10 mM NaMOPS pH 7.4, 5.6 mM glucose, 0.5% BSA, 1 mM CaCl2, 2.5 mM glutamine. All the above steps were performed under a 95% O2 : 5% CO2 atmosphere using oxygenated solutions. The enterocytes were used between 20 and 60 min after their isolation. Cell viability was assessed by trypan blue exclusion in dispersed cell preparations; 85–90% of the cells were viable for at least 150 min.
In vitro treatments
Isolated duodenal cells were pre-equilibrated in solution D for 15 min and then exposed for short intervals (15 s–10 min) to PTH (10–8 M). After treatment, enterocytes were lysed in 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 25 mM NaF, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 0.25% sodium deoxycholate and 1% NP40. Insoluble material was pelleted in a microcentrifuge at 14 000 r.p.m. for 10 min. The protein content of the clear lysates was determined according to the method of Bradford (1976).
Immunoprecipitation
Lysate aliquots (500–700 µg protein) were incubated overnight at 4 °C with anti-phosphotyrosine antibody, followed by precipitation of the complexes with protein A conjugated with Sepharose. The immune complexes were washed three times with cold immunoprecipitation buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF, 0.2 mM sodium orthovanadate, 1% Triton X-100 and 1% NP40), two times with PBS and then subjected to Western blot analysis.
Co-immunoprecipitation
Co-immunoprecipitation assays were performed under native conditions in order to preserve protein–protein associations. After hormone treatment, cells were lysed (15 min at 4 °C) in 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 3 mM KCl, 0.5 mM EDTA, 0.2 mM Na3VO4 (OV), 1 mM NaF, 1 mM PMSF, 6 µg/ml leupeptin, 8 µg/ml aprotinin, and 1% Tween-20. Lysates were clarified by centrifugation (14 000 x g, 10 min) and immunoprecipitation of the supernatants was performed with anti-Gs, anti-Gß or anti-c-Src antibodies, the precipitated immunocomplexes were washed and processed as described above. To confirm co-inmmunoprecipitation of both proteins, immunoprecipitation and immunoblotting were performed with the same antibodies used in reverse order.
SDS-PAGE and immunoblotting
Immunoprecipitated proteins (or lysate proteins) dissolved in Laemmli sample buffer were separated on SDS-polyacrylamide (10%) gels (Laemmli 1970) and electrotransferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 2 h at room temperature in TBST (50 mM Tris–HCl, pH 7.4, 200 mM NaCl, 1% Tween 20 containing 1% dry milk). Anti-phospho c-Src (Tyr527), anti-phospho c-Src (Tyr416), anti-c-Src, anti- phospho ERKs (p42 and p44 isoforms), anti-Gs, or anti-Gß antibodies were allowed to react with the membrane overnight at 4 °C. Next, the membranes were washed three times in TBST, incubated with a 1:10 000 dilution of peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature and washed three additional times with TBST. The membranes were then visualized using an enhanced chemiluminescent technique, according to the manufacturer’s instructions. Images were obtained with a model GS-700 Imaging Densitometer from Bio-Rad (Hercules, CA, USA) by scanning at 600 dpi and printing at the same resolution. Bands were quantified using the Molecular Analyst program (Bio-Rad).
Measurement of c-Src kinase activity
Cell lysates (700 µg protein) were prepared followed by immunoprecipitation of c-Src as described above. After three washes with immunoprecipitation buffer and two washes with kinase buffer (50 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 1 mM DTT, 0.1 mM sodium orthovanadate), the immune complexes were incubated at 30 °C for 10 min in kinase buffer (30 µl/sample) containing enolase as an exogenous substrate for Src (2.5 µg/assay), 50 µM ATP and [-32P]ATP (2 µCi/assay). To terminate the reaction, the phosphorylated product was separated from free isotope on ion-exchange phosphocellulose filters (Whatman P-81). Papers were immersed immediatly onto ice-cold 75 mM H3PO4, washed (1 x 5 min, 3 x 20 min) and counted in a scintillation counter.
Measurement of adenylyl cyclase activity
Adenylyl cyclase activity was determined by measuring the cAMP generated after hormonal treatment of microsomal membranes (Farndale et al. 1994). Microsomal membranes were isolated by centrifugation at 100 000 x g (60 min). Microsomal protein (75 µg) was incubated in 500 µM ATP, 10 mM MgCl2,10 mM phosphocreatine, 50 U/ml creatine kinase, 100 µM isobutylxanthine, 1 mM DTT, 10 mM Tris–HCl, pH 7.4, for 3 min at 30 °C. Treatment was stopped by the addition of perchloric acid (6%) followed by centrifugation. Cyclic AMP was measured in the supernatant by a protein binding assay (Tovey et al. 1974) using a commercial kit.
Statistical evaluation
Statistical significance of the data was evaluated using Student’s t-test (Snedecor & Cochran 1967) and probability values below 0.05 (P < 0.05) were considered significant. Results are expressed as means ± standard deviation (S.D.) from the indicated set of experiments.
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Results |
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-32P]ATP and enolase, acting as exogenous c-Src substrate, were added. As shown in Fig. 1
, PTH caused a time-dependent increase in Src kinase activity in rat enterocytes. Phosphorylation of the c-Src substrate is biphasic, with an early phase peaking at 30 s (+120%) and a second phase gradually increasing up to 5 min of treatment with the hormone (+220%). No time-dependent changes in c-Src activity were observed under basal conditions (data not shown). We then investigated PTH–induced changes in tyrosine phosphorylation of c-Src. Enterocytes were treated with PTH (10–8 M, 30 s-5 min) and cell lysates were immunoprecipitated with anti-P tyrosine antibody, followed by Western blotting with a specific anti-cSrc antibody. As shown in Fig. 2, PTH increased the level of tyrosine phosphorylation of c-Src with a kinetics roughly similar to that found for the increase in kinase activity. Then we monitored the phosphorylation state of Tyr527 and Tyr416 of c-Src. To that end, enterocytes were exposed to 10–8 M PTH (15 s-10 min), followed by Western blot analysis of cell lysates with anti-c-Src-phospho Tyr527 and anti-c-Src-phospho Tyr416. Total c-Src was measured in the same immunoblot by stripping the membrane and reincubating with anti-c-Src antibody. As shown in Fig. 3A, PTH transiently induces the dephosphorylation of Tyr527 at 15 s (– twofold), and increases the phosphorylation on Tyr416 of c-Src, with maximal effects at 5 min (+ threefold) (Fig. 3B).
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, BAPTA-AM fully suppressed PTH-dependent c-Src Tyr416 phosphorylation. In the presence of EGTA, the phosphorylation of this residue is also abolished, except at 1 min, the latter suggesting that the PTH-stimulated release of calcium from intracellular stores is responsible for the early effect of PTH on c-Src activation. Chelation of extracellular or intracellular Ca2+ also inhibited hormone-dependent rapid dephosphorylation of c-Src Tyr527 (Fig. 4B). These results point to a role for Ca2+ as an upstream activator of c-Src in rat enterocytes exposed to PTH.
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s and Gß subunits in PTH-stimulated enterocytes. As shown in Fig. 5, association of both subunits to c-Src was clearly detectable under basal conditions and stimulation with PTH significantly increased the formation of the complex. The hormone induces the greatest association of the kinase with Gs and Gß at 30 and 15 s respectively. Similar results were obtained when the antibodies were used in reverse order (not shown). These results suggest that G proteins may be required for PTH-induced c-Src activation. It is likely that cSrc association with G protein subunits changes the enzyme conformation leading to increased accessibility of the active site to substrates. It has been reported that direct G protein regulation of c-Src does not involve the dephosphorylation of Tyr527 (Ma et al. 2000). Therefore, in an attempt to further evaluate whether PTH-induced G protein-Src association stimulates the PTH-mediated increase in c-Src Tyr416 autophosphorylation, enterocyte homogenates were preincubated on ice for 10 min in the presence of anti-Gß or anti-Gs antibodies, followed by exposure to 10–8 M PTH(1–34) for 5 min. Proteins were resolved by electrophoresis, electroblotted into PVDF membranes and incubated with anti-c-Src-phospho Tyr416. In order to evaluate the equivalence of c-Src kinase content among the different experimental conditions, blotted membranes were re-probed with anti-Src antibody. When enterocyte homogenates were preincu-bated with anti-Gß antibody followed by a brief exposure to PTH, the effect of the hormone on c-Src Tyr416 phosphorylation was abolished (Fig. 6). Incubation with anti-Gs did not alter the PTH-dependent increase in c-Src Tyr416 phosphorylation. In rat enterocytes, we have previously reported that PTH activates the MAP kinases, ERK1 and ERK2, by a mechanism dependent on c-Src, the adenylyl cyclase pathway and Ca2+ (Gentili & de Boland 2000, Gentili et al. 2001b). To examine whether Gß and Gs subunits could mediate PTH-dependent activation of ERKs, we determined whether the hormone effect is sensitive to antibody blockade of these G protein subunits. To that end, enterocyte homogenates were preincubated in the presence or absence of anti-Gß or anti-Gs antibodies followed by a brief exposure to 10–8 M PTH(1–34). Then, cell lysates were probed with an anti-phospho ERKs antibody, which recognizes both the 42 and 44 kDa species. As shown in Fig. 7, and in agreement with our previous results (Gentili & de Boland 2000, Gentili et al. 2001b), PTH induces a marked increase in the phosphorylation of both isoforms. Hormone-dependent ERK1 and ERK2 tyrosine phos-phorylation was sensitive to antibody treatment of Gß subunits, suggesting that c-Src via the dimer Gß activates the mitogen-activated protein kinases in cells stimulated with PTH. Antibody blockade of Gs had no effect on PTH-dependent increase in ERK1 and ERK2 phos-phorylation. The efficacy of the antibody against Gs was tested by evaluating its effects on adenylyl cyclase activity. As shown in Table 1, incubation of microsomal membranes with PTH (10–8 M) induced a 200% increase in adenylyl cyclase activity, an effect that was not seen in membranes preincubated with anti-Gs.
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Discussion |
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In the present work, we demonstrate, for the first time, that PTH rapidly increases, in a biphasic manner, the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells. The biphasic nature of c-Src activity has also been observed in rat colonocytes stimulated with the steroid hormone 1,25(OH)2-vitamin D3 (Khare et al. 1997). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), which rapidly undergoes re-phosphorylation most likely catalyzed by Csk as discussed above. This is followed by a second event of activation related to the phosphorylation at Tyr416. In line with these observations, there is evidence demonstrating that PTH activates c-Src in osteoblastic cells through changes in tyrosine phosphorylation (Izbicka et al. 1994). Our results show that removal of external Ca2+ and chelation of intracellular Ca2+ suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca2+ is an upstream activator of c-Src in enterocytes stimulated with PTH. Extracellular Ca2+ has been shown to be required for the activation of the Src kinase pathways in pancreatic acinar cells (Tsunoda et al. 1996). Furthermore, Src family kinases have been implicated in the control of receptor-operated Ca2+ influx in various cell types (Niklinska et al. 1992, Lee et al. 1993, Bonaccorsi et al. 1995, Fleming et al. 1995). Of interest, in colonic smooth muscle cells, the biphasic activation of ceramide-induced Src kinase activity was shown to be dependent on extracellular Ca2+ in the second, sustained phase of activation (Ibitayo et al. 1998).
c-Src family tyrosine kinases emerged to play a role in heterotrimeric G protein signaling. There is evidence suggesting the importance of c-Src in GPCR-signal transduction pathways, but the biochemical mechanisms used by G proteins to activate c-Src remains largely elusive. Recent studies demonstrated that Gs and Gi directly stimulate the kinase activity of the downregulated c-Src and found that activated G proteins interact with the kinase domain of c-Src (Ma et al. 2000). Our results indicate that the G protein subunits, Gs and Gß, are associated with c-Src in basal conditions and this association increases two- to threefold in rat intestinal cells treated with PTH. Blocking of Gß subunits by preincubation of cells with a Gß antibody abolished hormone-dependent c-Src Tyr416 phosphorylation, indicating that c-Src is a down effector of Gß complexes. In line with this observation, it has been reported that Gß subunits, when overexpressed in COS-7 cells, increased (by ~ twofold) c-Src Tyr416-autophosphorylation (Luttrell et al. 1996). Moreover, in other G protein signaling systems, Gß plays significant roles in downstream effector activation (Clapham & Neer 1997).
There is increasing evidence suggesting that Src tyrosine kinases have a pivotal role in the regulation of various cellular processes (Erpel & Courtneidge 1995). Members of this family are thought to be involved in signal transduction mechanisms linked to the mitogen-activated protein kinases (ERK1 and ERK2) cascade underlying the regulation of cell proliferation and differentiation by agonists of receptor tyrosine kinases or heterotrimeric G protein-coupled receptors (Marshall 1995). While the tyrosine kinase growth factor receptors transmit signals to ERKs in a well defined multistep process, the stimulation of ERK activity by G protein-coupled receptors may be mediated by different classes of G proteins, including Gs, Gi, Gq/11, ß complexes, or Gi/o (Liebmann 2001, Marinissen & Gutkind 2001). We found that blockade of Gß subunits by preincubation of cells with a Gß antibody abolished PTH-dependent ERK1/ERK2 activation in rat intestinal cells. In agreement with these observations, it was reported (Luttrell et al. 1996) that Gß subunit-mediated formation of Shc–c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors. Nishida et al.(2000) reported that inhibition of the ß subunit attenuates hydrogen peroxide (H2O2)-induced ERK activation via c-Src in rat neonatal cardiomyocytes. c-Src can also activate Ras through the transactivation of the epidermal growth factor receptor and related receptors following stimulation of GPCRs coupled to either Gi or Gq (Della Rocca et al. 1997, Andreev et al. 2001, Shah & Catt 2002).
In summary, the results of this work show that PTH activates c-Src in intestinal cells through conformational changes via G proteins and a calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gß–c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2 (Fig. 8).
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Acknowledgements |
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Received 11 October 2005
Accepted 26 October 2005
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