ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca2+, PKC, and Rho kinase

Steven S. Wu1, Terence Chiu2, and Enrique Rozengurt2

Departments of 1 Pediatrics and 2 Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The G protein-coupled receptor agonists angiotensin II (ANG II) and lysophosphatidic acid (LPA) rapidly induce tyrosine phosphorylation of the cytosolic proline-rich tyrosine kinase 2 (Pyk2) in IEC-18 intestinal epithelial cells. The combined Pyk2 tyrosine phosphorylation induced by phorbol 12,13-dibutyrate, a direct agonist of protein kinase C (PKC), and ionomycin, a Ca2+ ionophore, was equal to that induced by ANG II. Inhibition of either PKC or Ca2+ signaling attenuated the effect of ANG II and LPA, although simultaneous inhibition of both pathways failed to completely abolish Pyk2 tyrosine phosphorylation. Cytochalasin D, which disrupts stress fibers, strongly inhibited the response of Pyk2 to ANG II or LPA. The distinct Rho-associated kinase (ROK) inhibitors HA-1077 and Y-27632, as well as the Rho inhibitor Clostridium botulinum C3 exoenzyme, also significantly attenuated ANG II- and LPA-stimulated Pyk2 tyrosine phosphorylation. Simultaneous inhibition of PKC, Ca2+, and either actin assembly or ROK completely abolished the Pyk2 response. Together, these results show that ANG II and LPA rapidly induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells via separate Ca2+-, PKC-, and Rho-mediated pathways.

IEC-18; paxillin; cytochalasin D; G protein-coupled receptor; Gi; Gq; migration


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTESTINAL EPITHELIAL CELL FUNCTIONS, including growth, motility, differentiation, and transport, are regulated by a combination of environmental factors that include peptide signals, bioactive lipids, adhesion to the extracellular matrix, and cell wounding (2, 7, 13, 33, 47, 55). The nontransformed IEC-18 and IEC-6 intestinal epithelial cell lines, derived from rat small intestinal crypt epithelium (40), have been widely used as a model of epithelial cell proliferation, migration, and differentiation (7, 9, 33, 55). In the "artificial wound" model of epithelial restitution, IEC-6 cell migration is promoted by transforming growth factors-alpha and -beta (TGF-alpha and -beta ), epidermal growth factor (EGF), and lysophosphatidic acid (LPA). Proliferation in culture is also promoted by EGF and TGF-alpha but not by TGF-beta (10, 15, 39, 55). Migration in this model is dependent on cytoskeletal integrity, tyrosine kinase activity, and protein kinase C (PKC) (13, 15, 39). However, many of the mechanisms linking extracellular signals to intestinal epithelial cells responses remain to be elucidated, and the role of focal adhesion-associated kinases in these cells has not been addressed.

Proline-rich tyrosine kinase 2 (Pyk2), also known as calcium-dependent tyrosine kinase (CADTK), related adhesion focal tyrosine kinase (RAFTK), and cell adhesion kinase-beta (CAKbeta ), along with the closely related p125 focal adhesion kinase (p125FAK), constitute a family of nonreceptor protein tyrosine kinases that associate with focal adhesions and are regulated by a variety of extracellular signals (3, 26). Pyk2 lacks Src homology 2 and 3 (SH2 and SH3) domains but contains sequences capable of binding to SH2 and SH3 domains of other proteins, including Src family kinases, the adaptor protein Grb2 (14, 26), and the focal adhesion proteins paxillin (17, 19, 22, 28, 46) and p130CAS (43). In particular, tyrosine-402 (Tyr-402) has been identified as the key site for Pyk2 autophosphorylation and activation (14), with other tyrosine sites contributing to subsequent transphosphorylation and enhanced kinase activity (27, 50). Pyk2 has been found to localize to focal adhesions in response to cell stimulation or extracellular matrix attachment (30). It has also been identified as an element upstream of several pathways leading to transcriptional activation, including the mitogen-activated protein (MAP) kinase cascades [extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK)] (14, 26, 59), the phosphatidylinositol 3-kinase pathway (43), and the nuclear factor-kappa B (NF-kappa B) pathway (49). Additionally, Pyk2 phosphorylates glycogen synthase kinase-3beta (GSK-3beta ), a critical enzyme in the regulation of beta -catenin signaling in intestinal cells (21). Pyk2 therefore has the potential to play a significant role in the transmission and regulation of cellular signals.

Pyk2 is activated by tyrosine phosphorylation in response to various stimuli, including growth factors, neurotransmitters, bioactive lipids, adhesion to the extracellular matrix, and stress signals (16, 17, 22, 26, 30, 59). Organization of the actin cytoskeleton plays an important role in Pyk2 regulation (6, 19, 22). In various cell lines, a number of G protein-coupled receptor (GPCR) agonists induce Pyk2 activation, including angiotensin II (ANG II), bradykinin, cholecystokinin, LPA, and histamine (14, 28, 30, 58, 66). These agonists are known to promote Galpha q-mediated activation of phospholipase C (PLC), generating second messengers that mobilize Ca2+ and activate PKC, which in turn induce Pyk2 tyrosine phosphorylation and activation. However, the relative contributions of Ca2+ mobilization and PKC to Pyk2 activation vary widely among different agonists and cell types (12, 25, 29, 36, 58). The role of these signaling pathways in the regulation of Pyk2 tyrosine phosphorylation has not been studied in normal IEC.

Of these GPCR agonists, ANG II was shown to promote DNA synthesis in IEC (53), and LPA was demonstrated to affect migration and growth in IEC-6 cells (55). Recent work from this laboratory has revealed that in IEC-6 and IEC-18 cells, ANG II and LPA rapidly activate the serine/threonine protein kinase D (PKD), a downstream target of PKC (8). Because ANG II and LPA activate pathways known to be upstream of Pyk2, we examined whether they regulate Pyk2 tyrosine phosphorylation in IEC-6 and IEC-18 cells. Our results show that in these cells, ANG II and LPA induce Tyr-402, Tyr-580, and overall Pyk2 tyrosine phosphorylation in a rapid and concentration-dependent fashion. ANG II- or LPA-induced Pyk2 phosphorylation depends on the integrity of the actin cytoskeleton and is mediated by the distinct actions of PKC, Ca2+, and Rho-associated kinase (ROK). These findings identify a novel role for Rho upstream of Pyk2 signaling and demonstrate that Pyk2 is a point of convergence of PKC-, Ca2+-, and cytoskeleton-dependent signals in IEC.


    MATERIALS AND METHODS
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Cell culture. IEC-18 and IEC-6 cells were purchased from American Type Culture Collection. Stock cultures of these cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in a humidified atmosphere containing 10% CO2 and 90% air at 37°C. For experimental purposes, cells were plated into 100-mm dishes at 4-5 × 105 cells per dish, or into 35-mm dishes at 1 × 105 cells per dish, in DMEM containing 5% FBS and were allowed to grow to confluency (4-6 days) before use.

Lysates and immunoprecipitation. Confluent cultures of IEC-18 or IEC-6 cells were washed twice with serum-free DMEM, equilibrated in the same medium at 37°C for 3 h, and then treated with ANG II, LPA, or other factors as described in the individual experiments. Inhibitors were added to the medium at appropriate times before stimulation. Each confluent 100-mm dish contained ~4 × 106 cells in 5 ml of DMEM final assay volume; each confluent 35-mm dish contained ~5 × 105 cells in 2 ml of DMEM. For immunoprecipitates, stimulation was terminated by aspirating the medium and lysing the cells in 1 ml of ice-cold buffer containing 10 mM Tris, pH 7.6, 1% Triton X-100, 50 mM NaCl, 5 mM EDTA, 0.1 mM sodium orthovanadate, 30 mM disodium pyrophosphate, 50 mM NaF, and 1 mM 4-(2-aminoethyl)-benzonesulfonyl fluoride HCl (lysis buffer). Lysates were clarified by centrifugation at 15,000 rpm for 10 min at 4°C, and the pellets were discarded. Proteins were immunoprecipitated overnight at 4°C with either anti-mouse IgG-agarose linked to monoclonal anti-Tyr(P) (PY-20) or protein A- or protein G-agarose linked to polyclonal anti-Pyk2 antibody. Immunoprecipitates were washed three times with lysis buffer and extracted for 20 min at 95°C in 2× SDS-PAGE sample buffer (200 mM Tris · HCl, pH 6.8, 2 mM EDTA, 6% SDS, 4% 2-mercaptoethanol, 10% glycerol). In the preparation of whole cell lysates, stimulation of experimental dishes was terminated by aspirating the medium, adding 200 µl (small dishes) or 400 µl (large dishes) of 2× SDS-PAGE sample buffer, scraping immediately, and heating for 20 min at 95°C. All samples were then resolved in 8% SDS-PAGE gels.

Western blotting. After SDS-PAGE, proteins were transferred to Immobilon-P membranes (Millipore), which were then blocked for 3 h at room temperature with 5% nonfat dry milk in phosphate-buffered saline (PBS), pH 7.2. Membranes were then incubated overnight at 4°C with anti-Tyr(P) antibody (4G10, 0.5-1 µg/ml), anti-Pyk2 antibody (1 µg/ml), or other antibodies as indicated. The membranes were washed three times with PBS containing 0.1% Tween 20 and then incubated with secondary antibodies [horseradish peroxidase (HRP)-conjugated antibodies to rabbit or mouse, as appropriate; 1:5,000] for 1 h at room temperature. After the membranes were washed an additional four times with PBS-Tween, the immunoreactive bands were visualized by using enhanced chemiluminescence (ECL) detection reagents, and their migration distance was compared with standard high-molecular-weight markers (Bio-Rad). Autoradiograms were scanned by using a GS-710 scanner, and the labeled bands were quantified using the Quantity One program (both also from Bio-Rad). For reprobing, selected membranes were stripped of antibody by incubation at room temperature in 100 mM glycine (pH 2.5) for 10 min and then in 100 mM Tris (pH 8.8) for 10 min. They were then washed and blocked, and antibody incubation was performed as described above.

Measurement of intracellular Ca2+ concentration. Intracellular Ca2+ concentration ([Ca2+]i) was measured with the fluorescent indicator fura 2. Confluent cultures of IEC-18 cells, grown on 9 × 22-mm glass coverslips, were washed twice with Hanks' buffered salt solution (pH 7.2) supplemented with 35 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4, and 0.1% bovine serum albumin (calcium buffer). After being washed, cells were incubated in calcium buffer containing 1 µM fura 2-tetra-acetoxymethyl ester (fura 2-AM) for 30 min at room temperature. Each coverslip was washed twice and incubated for 15 min further in calcium buffer at room temperature and then transferred to a quartz cuvette containing 2 ml of the same buffer at 37°C. After 2 min of equilibration, fluorescence was measured via a Hitachi F-2000 fluorospectrophotometer with dual-excitation wavelengths of 340 nm (lambda 1) and 380 nm (lambda 2) and an emission wavelength of 510 nM while the incubation medium was continually stirred at 37°C. [Ca2+]i was determined from the formula
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB><FR><NU>(R − R<SUB>min</SUB>)</NU><DE>(R<SUB>max</SUB> − R)</DE></FR> × <FR><NU>F<SUB>min</SUB>&lgr;<SUB>2</SUB></NU><DE> F<SUB>max</SUB>&lgr;<SUB>2</SUB></DE></FR>
where R, Rmin, and Rmax are the ratios of the emission at 510 nm after excitation at 340 and 380 nm; Fmax is the fluorescence after the addition of 40 µM digitonin; and Fmin is the fluorescence after the Ca2+ in the solution has been chelated with 10 mM EGTA. The value of the dissociation constant (Kd) used was 224 nM.

Cell migration assay. IEC-18 cells were grown to confluence in 100-mm plastic dishes as previously described, washed twice, and changed to serum-free DMEM for 8 h before wounding. Inhibitors were added before wounding as described in the individual experiments. The cell monolayers were wounded by applying a razor blade to the dish and scraping perpendicularly to the plane of the blade. Four wounds (40 mm long × 6-8 mm wide) were established per dish. Cells were then incubated in serum-free DMEM in the presence or absence of agonists as described in the individual experiments. Experiments were terminated by washing cells twice in PBS, followed by fixing in 10% buffered formalin phosphate at 25°C for 20 min. To quantitate cell migration, cells were observed under phase contrast with a ×10 lens (Plan-Neo, Carl Zeiss) mounted on an upright microscope (Axioskop2, Carl Zeiss). Images were collected with a high-resolution digital camera and software (Spot, Diagnostic Instruments) from 8-10 representative wounded areas per condition initially and at 24 h. Migration was calculated as the number of cells across the cut margin per high-powered field and is presented as means ± SE. Differences between groups were analyzed with the unpaired Student's t-test, with the significance level defined as P < 0.05.

Materials. Anti-Tyr(P) monoclonal antibody (PY20), anti-p125FAK polyclonal rabbit antibody, and anti-Pyk2 polyclonal goat antibody were obtained from Santa Cruz Biotechnology. Anti-Tyr(P) monoclonal antibody (4G10), anti-Pyk2 polyclonal rabbit antibody, and Clostridium botulinum C3 exoenzyme were obtained from Upstate Biotechnology. Phosphospecific polyclonal rabbit antibodies to Pyk2 (pY402 and pY580) were obtained from BioSource International (Camarillo, CA). HRP-linked sheep anti-mouse IgG, HRP-linked donkey anti-rabbit IgG, and ECL reagents were obtained from Amersham. ANG II, LPA, ionomycin, GF-109203X, and agarose-coupled anti-mouse IgG were obtained from Sigma. Protein A-agarose and protein G-agarose were obtained from Roche Diagnostics (Indianapolis, IN). Pertussis toxin (PTX), Ro-318220, and HA-1077 were from Calbiochem. Thapsigargin was obtained from LC Laboratories (Alexis, San Diego, CA). The compound Y-27632 was generously provided by Welfide (Osaka, Japan). An antiserum that specifically recognizes the phosphorylated state of Ser-916 of PKD (pS916) was generously provided by Dr. Doreen Cantrell (Imperial Cancer Research Fund, London, UK). Other items were from standard suppliers or as indicated in the text.


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ABSTRACT
INTRODUCTION
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RESULTS
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ANG II and LPA induce Pyk2 tyrosine phosphorylation in IEC. The IEC-18 and IEC-6 cell lines are known to express a number of GPCRs, including those for the vasoactive octapeptide ANG II and the bioactive lipid LPA (8, 48, 54, 55). To examine the effect of these agonists on Pyk2 tyrosine phosphorylation, which correlates with Pyk2 kinase activation (17, 26), confluent cultures of IEC-18 and IEC-6 cells were stimulated with either 50 nM ANG II or 5 µM LPA and lysed. The extracts were immunoprecipitated with anti-Tyr(P) antibody, separated by SDS-PAGE, and Western blotted with anti-Pyk2 antibody (Fig. 1A). ANG II markedly increased Pyk2 tyrosine phosphorylation 15-fold compared with the basal level in unstimulated IEC-18 cells (mean 6.7% of maximum ± 1.1% SE, n = 16). LPA induced an increase in Pyk2 tyrosine phosphorylation of 9.5-fold compared with baseline (mean 10.5% of maximum ± 1.8% SE, n = 16).


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Fig. 1.   Angiotensin II (ANG II) and lysophosphatidic acid (LPA) induce proline-rich tyrosine kinase 2 (Pyk2) tyrosine phosphorylation in IEC-18 and IEC-6 cells. A: confluent IEC-18 and IEC-6 cells were washed and incubated at 37°C in the absence of agonist (-), with 50 nM ANG II for 1 min, or with 5 µM LPA for 1.5 min. Cells were then lysed, and extracts were immunoprecipitated (IP) with anti-Tyr(P) antibody (PY20), followed by Western blotting (WB) with anti-Pyk2 antibody. B: confluent IEC-18 and IEC-6 cells were washed and incubated as in A with 50 nM ANG II or 5 µM LPA, and cell extracts were immunoprecipitated with anti-Pyk2 antibody, followed by Western blotting with anti-Tyr(P) antibody (4G10). C: the same membranes were stripped of antibody and analyzed further by Western blotting with anti-Pyk2 antibody. D: confluent IEC-18 cells were washed and incubated at 37°C in the absence of agonist (-), with 50 nM ANG II for 1 min, or with 5 µM LPA for 1.5 min. Cells were lysed, and the whole cell lysates were separated by SDS-PAGE, followed by Western blotting with an antibody specific for either Tyr(P)402 or Tyr(P)580 of Pyk2. All autoradiograms shown are representative of at least 3 independent experiments.

To confirm that these findings reflected a change in the phosphorylation state of Pyk2, we stimulated cultures of IEC-18 or IEC-6 cells with ANG II or LPA and immunoprecipitated the cell lysates with anti-Pyk2 antibody, followed by Western blotting with anti-Tyr(P). As demonstrated in Fig. 1B, either agonist once again induced a marked and rapid tyrosine phosphorylation of Pyk2. Stripping these membranes and reprobing with anti-Pyk2 antibody (Fig. 1C) confirmed that equal amounts of total Pyk2 were present in each condition.

Additionally, we found that a broad tyrosine-phosphorylated band in the range of 60-80 kDa was coprecipitated with Pyk2 (Fig. 1B). Paxillin, a multidomain adaptor protein that recruits signaling proteins (including FAK and Src) to focal adhesions, migrates in SDS-PAGE as a heterogeneous band of 68-80 kDa. Paxillin has previously been described as associating with Pyk2 constitutively in liver epithelial cells (28) and HEK-293 kidney cells (17) and inducibly in neutrophils (18). We confirmed the identity of the 68- to 80-kDa band detected in the anti-Pyk2 immunoprecipitates as paxillin by reprobing the membranes with anti-paxillin antibody (data not shown). Equal coimmunoprecipitation of Pyk2 and paxillin was detected in stimulated and unstimulated cells, indicating a constitutive association with the increased tyrosine phosphorylation of Pyk2 and paxillin correlating with one another.

Furthermore, we examined the effect of ANG II and LPA on the phosphorylation state of individual Pyk2 tyrosine residues. Tyr-402, the major site of Pyk2 autophosphorylation upon activation, provides a potential high-affinity binding site for SH2 domains of Src family kinases (14) and is essential for the induction of Pyk2 kinase activity (5). In turn, Src kinases bound at Tyr-402 are able to transphosphorylate Pyk2 at additional sites, including Tyr-580 within the kinase domain, which appears to further enhance Pyk2 kinase activity (27, 50). Parallel cultures of IEC-18 cells were stimulated with ANG II or LPA, and the whole cell lysates were separated by SDS-PAGE, followed by Western blotting with Pyk2 antibodies specific for the phosphorylated Tyr-402 (Fig. 1D, left) or the phosphorylated Tyr-580 sites (Fig. 1D, right). As with tyrosine phosphorylation overall, the phosphorylation of both Tyr-402 and Tyr-580 was rapidly and markedly induced after treatment with ANG II or LPA.

ANG II and LPA induce Pyk2 tyrosine phosphorylation in a time- and dose-dependent manner. The kinetics of Pyk2 tyrosine phosphorylation in response to these two agonists were then studied in detail. ANG II-stimulated Pyk2 phosphorylation in IEC-18 cells peaked rapidly by 1 min, decreased to less than half maximum by 5 min, and approached the baseline level by 20 min after ANG II addition (Fig. 2A). ANG II induced Pyk2 phosphorylation in a dose-dependent fashion, with half-maximal and maximal effects achieved at concentrations of 1 and 30 nM, respectively (Fig. 2B). ANG II induced an equally rapid tyrosine phosphorylation of Pyk2 in IEC-6 cells (Fig. 2C), peaking at 1 min after stimulation and decreasing to half maximum by 5 min.


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Fig. 2.   ANG II induces Pyk2 tyrosine phosphorylation in a time- and dose-dependent manner. A: time course of Pyk2 tyrosine phosphorylation induced by ANG II. Confluent IEC-18 cells were washed and incubated at 37°C with 50 nM ANG II for various times as indicated. Cells were then lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. B: dose response of Pyk2 tyrosine phosphorylation induced by ANG II. Confluent IEC-18 cells were washed and incubated at 37°C for 1 min with various concentrations of ANG II as indicated. Cell lysates were analyzed for Pyk2 phosphorylation as in A. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by ANG II. C: confluent IEC-6 cells were washed and incubated at 37°C with 50 nM ANG II for various times as indicated. Cell extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. Autoradiograms shown are representative of at least 3 independent experiments.

Similarly to ANG II, LPA-induced Pyk2 tyrosine phosphorylation in IEC-18 cells peaked at 1 min, decreased to less than half maximum by 5 min, and approached baseline by 20 min (Fig. 3A). LPA induced Pyk2 phosphorylation in a dose-dependent fashion, with half-maximal and maximal effects occurring at concentrations of 300 nM and 3 µM, respectively (Fig. 3B). The latter concentration is consistent with estimated physiological levels in serum, which range from 2 to 20 µM (34). The time course of LPA-induced Pyk2 tyrosine phosphorylation was similar in IEC-6 cells (Fig. 3C).


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Fig. 3.   LPA induces Pyk2 tyrosine phosphorylation in a time- and dose-dependent manner. A: time course of Pyk2 tyrosine phosphorylation induced by LPA. Confluent IEC-18 cells were washed and incubated at 37°C with 5 µM LPA for various times as indicated. Cells were then lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. B: dose response of Pyk2 tyrosine phosphorylation induced by LPA. Confluent IEC-18 cells were washed and incubated at 37°C for 1.5 min with various concentrations of LPA as indicated. Cell lysates were analyzed for Pyk2 phosphorylation as described in A. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by LPA. C: confluent IEC-6 cells were washed and incubated at 37°C with 5 µM LPA for various times as indicated. Cell extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. Autoradiograms shown are representative of at least 3 independent experiments.

ANG II- or LPA-induced Pyk2 tyrosine phosphorylation is dependent on PKC and Ca2+ mobilization. Many GPCRs, including the receptors for ANG II and LPA, exert their effects by coupling to PLC-mediated breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (31). These second messengers, in turn, mobilize Ca2+ from intracellular stores and activate PKC, respectively. Previous studies have shown Pyk2 activation in response to a variety of stimuli to be dependent on PKC and Ca2+ release (36, 58) or on Ca2+ release alone (12, 29, 37). To study the postreceptor regulation of Pyk2 in IEC-18 cells, we first examined the role of the PLC pathway, focusing on the effects of PKC activation and Ca2+ release.

Cells were treated with phorbol 12,13-dibutyrate (PDBu), a direct PKC agonist; with ionomycin, a Ca2+ ionophore; or with thapsigargin, which specifically inhibits the endoplasmic reticulum Ca2+-ATPase. Ionomycin, at 2.5, 5, and 10 min, induced phosphorylation of Pyk2 to ~50% of the maximal value induced by cell stimulation with ANG II (Fig. 4A). PDBu also partially stimulated Pyk2 but with different kinetics. Its effect increased progressively from 25% of maximum at 2.5 min to 50% of maximum at 10 min. At all three time points tested (2.5, 5, and 10 min), simultaneous addition of ionomycin and PDBu induced increases in Pyk2 phosphorylation equal to the maximal effect induced by ANG II. When thapsigargin was used to elevate [Ca2+]i in place of ionomycin, weaker partial Pyk2 phosphorylation was induced, reaching 25% of maximum at 5 and 10 min, but again the simultaneous use of thapsigargin and PDBu produced an additive effect (Fig. 4B). These findings show that the combination of PKC and Ca2+ can potently induce Pyk2 tyrosine phosphorylation in IEC-18 cells.


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Fig. 4.   Protein kinase C (PKC) and Ca2+ release induce Pyk2 tyrosine phosphorylation in an additive manner. Confluent IEC-18 cells were treated in the presence or absence of 500 nM ionomycin (A), 100 nM thapsigargin (B), or 100 nM phorbol 12,13-dibutyrate (PDBu; A and B) for periods of 2.5, 5, or 10 min as indicated. Parallel cultures of cells were treated with 50 nM ANG II for 1 min. Cells were then lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by ANG II. Autoradiograms shown are representative of at least 3 independent experiments. Cells treated with equivalent amounts of solvent, whether water or DMSO, did not induce any significant change in unstimulated Pyk2 tyrosine phosphorylation (data not shown).

Given the pronounced effect of directly activating PKC and Ca2+ signaling, we next assessed whether these two pathways mediate Pyk2 activation in response to ANG II and LPA. IEC-18 cells were first incubated with inhibitors of PKC or Ca2+ signaling and then stimulated with ANG II or LPA as described above. When cells were pretreated with the selective PKC inhibitors GF-109203X (GF-1) (52, 60) or Ro-318220 (64), ANG II-stimulated Pyk2 phosphorylation was decreased by 50% (Fig. 5A). To inhibit the increase in cytosolic Ca2+ concentration induced by ANG II, the cells were pretreated simultaneously for 30 min with thapsigargin to deplete intracellular Ca2+ stores and with EGTA to chelate extracellular Ca2+. Inhibition of Ca2+ signaling in this manner attenuated ANG II-stimulated Pyk2 tyrosine phosphorylation by ~50%. Pretreatment of cells with both a PKC inhibitor (GF-1 or Ro-318220) and thapsigargin-EGTA decreased Pyk2 tyrosine phosphorylation by 70-80%.


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Fig. 5.   ANG II- or LPA-induced Pyk2 tyrosine phosphorylation is dependent on PKC and Ca2+ release. Confluent IEC-18 cells were treated in either the absence (-) or presence (+) of the following inhibitors: 2 µM GF-109203X (GF-1) for 1 h, 1 µM Ro-318220 for 1 h, or 2 mM EGTA and 50 nM thapsigargin for 30 min (EGTA-Thaps). Cells were then incubated in the absence or presence of either 50 nM ANG II for 1 min (A) or 5 µM LPA for 1.5 min (B). Cells were lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by each agonist. Inhibitors when given alone to cell cultures did not induce any significant change in unstimulated Pyk2 tyrosine phosphorylation (data not shown).

Similar treatments to prevent PKC and Ca2+ signaling were notably less effective in attenuating LPA-induced Pyk2 tyrosine phosphorylation (Fig. 5B). The inhibition of PKC by GF-1 or Ro-318220 diminished LPA-stimulated Pyk2 phosphorylation by 25 and 40%, respectively. In contrast to its effect on ANG II-stimulated Pyk2 tyrosine phosphorylation, pretreatment with thapsigargin-EGTA diminished LPA-stimulated Pyk2 phosphorylation by only 20%. The combination of either GF-1 or Ro-318220 and thapsigargin-EGTA decreased Pyk2 phosphorylation by ~50%. These findings demonstrate that tyrosine phosphorylation of Pyk2 in response to ANG II or LPA is mediated in part by both PKC and Ca2+ signaling in IEC-18 cells but suggest that these signaling pathways are not sufficient to account for the full extent of the ANG II- or LPA-stimulated Pyk2 response.

LPA-induced, but not ANG II-induced, Pyk2 tyrosine phosphorylation is inhibited by PTX. LPA activates several subtypes of heterotrimeric G protein, including Gi, Gq, and G12, which are responsible for transducing LPA signals into a range of different biological responses (34). The PLC-mediated breakdown of PtdIns(4,5)P2 has been shown to occur via both Gi and Gq proteins in different cell types (8, 34). We found that in IEC-18 cells, pretreatment with PTX, which selectively inactivates Gi by ADP-ribosylation, significantly inhibited the response to LPA (Fig. 6A). The maximum inhibitory effect on LPA-stimulated Pyk2 tyrosine phosphorylation (50%) was reached at a concentration of 10 ng/ml. In contrast, ANG II-induced Pyk2 tyrosine phosphorylation was unaffected by preincubation with PTX at 50 ng/ml and only minimally affected at 100 ng/ml (Fig. 6B), consistent with a signaling pathway that involves Gq rather than Gi. In addition, we tested the effects of simultaneously inhibiting Gi, PKC, and Ca2+ release on LPA-induced Pyk2 phosphorylation (Fig. 6C). The inhibitory effect of PTX alone was equivalent to that of Ro-318220 and thapsigargin-EGTA in combination (~50%). When PTX was added to Ro-318220 and thapsigargin-EGTA, the Pyk2 phosphorylation level was not significantly changed. This is consistent with a model in which LPA receptor-induced PLC activation and subsequent PKC activation and Ca2+ mobilization are mediated through Gi upstream of Pyk2 tyrosine phosphorylation.


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Fig. 6.   LPA-induced, but not ANG II-induced, Pyk2 tyrosine phosphorylation is inhibited by pertussis toxin (PTX). A: confluent IEC-18 cells were treated in the presence of various concentrations of PTX as indicated for 3 h and then incubated with 5 µM LPA for 1.5 min. Cells were then lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. B, C: confluent IEC-18 cells were treated in the presence of the indicated concentrations of PTX for 3 h, and in the absence (-) or presence (+) of 1 µM Ro-318220 for 1 h and 2 mM EGTA and 50 nM thapsigargin for 30 min (Ro-EGTA-Th). Cells were then incubated in the absence or presence of either 50 nM ANG II for 1 min (B) or 5 µM LPA for 1.5 min (C), followed by lysis, immunoprecipitation with anti-Tyr(P) antibody (PY20), and Western blotting with anti-Pyk2 antibody. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by each agonist. PTX when given alone to cell cultures did not induce any significant change in unstimulated Pyk2 tyrosine phosphorylation (data not shown).

ANG II- or LPA-induced Pyk2 tyrosine phosphorylation is inhibited by cytochalasin D. Because PKC- and Ca2+-dependent pathways accounted only partially for the GPCR-mediated tyrosine phosphorylation of Pyk2, we examined other potential factors that could play a role in transducing this response. In some cell types, the integrity of the cytoskeleton, and particularly the actin filament network, plays an important role in Pyk2 activation (6, 19, 22). Unlike the closely related kinase p125FAK, Pyk2 exhibits a primarily cytoplasmic distribution pattern (3), although it has been shown to localize to focal adhesions upon stimulation of cells by GPCR agonists or cell adhesion (30). In addition, Pyk2 has been shown to physically associate with a number of focal adhesion proteins (17, 18, 28, 43, 46), including paxillin in IEC-6 and IEC-18 cells, as we have demonstrated (Fig. 1B). We therefore examined the role of the actin cytoskeleton in IEC-18 cells by studying the effect of cytochalasin D, an agent that selectively disrupts actin stress fibers.

Treatment of IEC-18 cells with cytochalasin D significantly inhibited Pyk2 tyrosine phosphorylation in response to either ANG II (Fig. 7A) or LPA (Fig. 7B), attenuating it by 75 and 60%, respectively. Furthermore, when cytochalasin D was combined with the previously described inhibitors of PKC and Ca2+ signaling (Ro-318220 and thapsigargin-EGTA), ANG II-stimulated Pyk2 tyrosine phosphorylation was completely abrogated (Fig. 7A). Similarly, the combination of cytochalasin D and PTX completely blocked the effect of LPA on Pyk2 tyrosine phosphorylation (Fig. 7B). IEC-18 cells pretreated with cytochalasin D exhibited a distinct rounded morphology but remained attached to the culture dish, and immunoprecipitation of cytochalasin D-treated and control cells with anti-Pyk2 antibody demonstrated that equal amounts of total Pyk2 were recovered (data not shown). Therefore, the decreased signal seen reflected reduced phosphorylation of Pyk2 rather than reduced recovery of this protein from cytochalasin D-treated cells.


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Fig. 7.   ANG II- or LPA-induced Pyk2 tyrosine phosphorylation is inhibited by cytochalasin D. A: confluent IEC-18 cells were treated in the absence (-) or presence (+) of 2 µM cytochalasin D (Cyto D) for 2 h, and in the absence or presence of 1 µM Ro-318220 for 1 h and 2 mM EGTA and 50 nM thapsigargin for 30 min (Ro-EGTA-Th). Cells were then incubated with 50 nM ANG II for 1 min and lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. B: confluent IEC-18 cells were treated in the absence (-) or presence (+) of 2 µM cytochalasin D for 2 h and 50 ng/ml PTX for 3 h. Cells were then incubated with 5 µM LPA for 1.5 min and lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by each agonist. Cells treated with cytochalasin D alone did not exhibit any significant change in unstimulated Pyk2 tyrosine phosphorylation (data not shown). C: confluent IEC-18 cells were treated in the presence (+) or absence (-) of 2 µM cytochalasin D for 2 h and then stimulated with 50 nM ANG II (1 min) or 5 µM LPA (1.5 min). Cells were lysed, and the whole cell lysates were separated by SDS-PAGE, followed by Western blotting with antibody [anti-phosphoserine(916)] recognizing the active phosphorylated form of protein kinase D (PKD). Autoradiogram shown is representative of 3 independent experiments.

To further test whether cytochalasin D interfered with other cell functions, we examined its effect on ANG II- and LPA-induced activation of PKD, a cytoplasmic serine-threonine kinase that is activated via a PKC-dependent pathway in IEC-18 and other cell lines (8, 23). Activated PKD undergoes autophosphorylation on serine-916, which can then be quantified by the use of a specific Ser-916(P) antibody (32). The phosphorylation of PKD on Ser-916 induced by either ANG II or LPA was unaffected by pretreatment with cytochalasin D (Fig. 7C).

ANG II- or LPA-induced Pyk2 tyrosine phosphorylation is dependent on Rho and ROK activity. Given the sensitivity of Pyk2 to disruption of the actin cytoskeleton, we next addressed the role of Rho, an important upstream regulator of actin stress fiber assembly, on Pyk2 tyrosine phosphorylation. Rho proteins belong to a family of small GTPases (also including Rac and Cdc42), which regulate the formation and rearrangement of actin stress fibers and focal adhesions in response to growth factors or cell adhesion (42, 45). Therefore, Rho has the potential to regulate Pyk2 via its effects on cytoskeletal organization. On the other hand, Rho could also influence Pyk2 via the PLC pathway, since it promotes resynthesis of PtdIns(4,5)P2, the major substrate for PLC (45). The effect of the Rho pathway on Pyk2 is not well described, although a recent report has implicated G12/13-mediated Rho activation as being dependent on, but not upstream of, Pyk2 (50).

To test the effect of Rho inhibition in IEC-18 cells, we used the Clostridium botulinum C3 exoenzyme, a toxin that catalyzes ADP-ribosylation and thereby inactivation of Rho subfamily proteins (RhoA, RhoB, and RhoC) (45). Because the purified C3 exoenzyme is neither actively internalized by cells nor readily membrane permeable, prolonged incubation is necessary to allow for passive uptake of C3 into the cytoplasm. Cells were preincubated for 24 h with 2.5 µg/ml of C3 exoenzyme or with an equivalent amount of solvent (50% glycerol), and then stimulated with ANG II or LPA (Fig. 8A). The phosphorylation of Pyk2 at Tyr-402 induced by either agonist was reduced by ~40% in C3-treated cells compared with controls treated with or without solvent. This result suggests that Rho plays a role in mediating Pyk2 tyrosine phosphorylation in response to ANG II and LPA.


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Fig. 8.   ANG II- or LPA-induced Pyk2 tyrosine phosphorylation is dependent on Rho/Rho kinase. A: confluent IEC-18 cells were treated for 24 h with 2.5 µg/ml of C3 exoenzyme (C3), an equivalent volume of 50% glycerol solvent (glyc), or left untreated (-). Cells were then incubated with either 50 nM ANG II or 5 µM LPA for 1.5 min and lysed, and the whole cell lysates were Western blotted with antibody to Tyr(P)402 of Pyk2. Subsequent stripping and reprobing with anti-Pyk2 antibody showed a slight but similar decrease in the amount of total Pyk2 in both C3-treated and solvent-treated cell cultures (data not shown). B, C: confluent IEC-18 cells were treated in the absence (-) or presence (+) of the following inhibitors: 10 µM HA-1077 or 10 µM Y-27632 for 1 h, 50 ng/ml PTX for 3 h, or 1 µM Ro-318220 for 1 h with 2 mM EGTA and 50 nM thapsigargin for 30 min (Ro-EGTA-Th). Cells were then incubated with either 50 nM ANG II for 1 min (B) or 5 µM LPA for 1.5 min (C) and lysed, and extracts were immunoprecipitated with anti-Tyr(P) antibody (PY20), followed by Western blotting with anti-Pyk2 antibody. Values are means ± SE of at least 3 independent experiments and are expressed as a percentage of the maximal level of Pyk2 tyrosine phosphorylation induced by LPA. Autoradiograms shown are representative of at least 3 independent experiments. Cells treated with the inhibitors HA-1077 or Y-27632 alone did not exhibit any significant change in unstimulated Pyk2 tyrosine phosphorylation (data not shown).

To further investigate the effect of the Rho pathway on Pyk2, we focused on the ROK family, comprising two closely related kinases (ROK-I and -II) that have been identified as downstream targets of GTP-bound Rho and mediators of stress fiber assembly (1, 45). To disrupt this pathway, we used the compounds 1-(5-isoquinolone sulfonyl)homopiperazine (HA-1077) and Y-27632, two selective, cell-permeable, and structurally distinct ROK inhibitors. HA-1077 was identified as a potent, preferential inhibitor of ROK (11, 38) that has been shown to prevent GPCR-mediated actin stress fiber assembly (51). The structurally unrelated compound Y-27632 was also identified as a selective inhibitor, affecting ROK 200-fold more than PKC (61) and 20- to 30-fold more than other Rho-dependent kinases (24). The Y-27632 compound, like HA-1077, prevented GPCR-mediated stress fiber formation in fibroblasts (51) and recently was shown in T cells to induce disorganization of cortical actin in an identical manner to that of C3 exoenzyme (44).

Pretreatment of IEC-18 cells with either HA-1077 or Y-27632 inhibited both ANG II-stimulated (Fig. 8B) and LPA-stimulated (Fig. 8C) Pyk2 phosphorylation to ~50% of maximum. Again, immunoprecipitation of control, HA-1077-, or Y-27632-treated cells with anti-Pyk2 antibody demonstrated that equal amounts of total Pyk2 were isolated (data not shown). To address the question of whether ROK inhibition is independent of the PKC- and Ca2+-mediated pathways of Pyk2 activation, IEC-18 cells were preincubated with inhibitors of these pathways in addition to HA-1077 or Y-27632. In cells treated with both an ROK inhibitor and Ro-318220/thapsigargin-EGTA, ANG II-stimulated Pyk2 tyrosine phosphorylation was abrogated completely (Fig. 8B). Similarly, LPA-induced phosphorylation of Pyk2 was blocked by the combination of either HA-1077 or Y-27632 with PTX (Fig. 8C). Thus ROK inhibition combined with PKC/Ca2+ inhibition led to complete suppression of Pyk2 tyrosine phosphorylation.

The results presented in Fig. 8, B and C, suggest that Rho/ROK and PKC/Ca2+ participate in distinct signaling pathways to Pyk2. To test this conclusion further, we investigated the effects of ROK on the PKC and Ca2+ signaling pathways independently. To address the question of whether PKC is affected by ROK inhibition, we again examined the PKC-dependent kinase PKD by using the specific Ser-916(P) antibody. Stimulation of PKD phosphorylation on Ser-916 by either ANG II or LPA was unaffected by HA-1077 or Y-27632, whereas it was dramatically reduced by the PKC inhibitor Ro-318220 (Fig. 9A). We then examined the effect of ROK inhibition on the intracellular Ca2+ response to ANG II or LPA. Either ANG II (10 nM) or LPA (1 µM) induced a rapid increase in [Ca2+]i, peaking within 20 s (Fig. 9B). The [Ca2+]i responses were maximal at these concentrations of ANG II and LPA (data not shown). Pretreatment of cells with HA-1077 (10 µM) or Y-27632 (10 µM) had no significant effect on the Ca2+ response induced by either agonist (Fig. 9C), whereas pretreatment with thapsigargin (50 nM)-EGTA (2mM), as in previous experiments, completely abolished the Ca2+ response to both ANG II and LPA (data not shown). Taken together, these results show that the Rho signaling pathway, via ROK, contributes to the tyrosine phosphorylation of Pyk2 in a manner independent from PKC and intracellular Ca2+ release.


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Fig. 9.   Inhibition of Rho-associated kinase (ROK) does not affect PKC or Ca2+ signaling. A: confluent IEC-18 cells were treated in the absence or presence of the following inhibitors for 1 h: 10 µM HA-1077 (HA), 10 µM Y-27632 (Y27), or 1 µM Ro-318220 (Ro). They were then stimulated with 50 nM ANG II (1 min) or 5 µM LPA (1.5 min) and lysed, and the whole cell lysates were separated by SDS-PAGE, followed by Western blotting with antibody [anti-phosphoserine(916)] recognizing the active phosphorylated form of PKD. Autoradiogram shown is representative of 3 independent experiments. B, C: IEC-18 cells were grown to confluence on coverslips, incubated in the absence or presence of 10 µM HA-1077 or 10 µM Y-27632 for 1 h, and then stimulated with 10 nM ANG II or 1 µM LPA as described in MATERIALS AND METHODS. Representative [Ca2+]i tracings for ANG II and LPA alone are shown (B), with the time of agonist addition indicated by arrows. Peak increases in intracellular Ca2+ concentration ([Ca2+]i) from baseline were measured for all conditions and are shown in C. Values are means ± SE of at least 3 independent experiments. Stimulation of cells with 50 nM ANG II or 5 µM LPA did not result in any further increase in the peak [Ca2+]i than the concentrations used (data not shown).

Migration of IEC-18 cells in vitro is promoted by LPA, PKC, and Ca2+ mobilization. As described above, a number of observations suggest a possible role for Pyk2 in regulating the cytoskeleton, including the association of Pyk2 with focal adhesion proteins (28, 43, 46) and the sensitivity of Pyk2 tyrosine phosphorylation to cytochalasin D in IEC-18 and other cell types (6, 19). As a first step toward correlating Pyk2 phosphorylation with cytoskeletal function in IEC, we asked whether the upstream regulators that promote Pyk2 phosphorylation in IEC-18 cells also affect migration of these cells. To test this, we used a model of in vitro "wounding" similar to that previously described (10, 55). In control (unstimulated) dishes following a razor blade wound, there was significant migration of cells across the wound margin at 24 h even in the absence of any agonist (Fig. 10, A and B). Treatment of wounded IEC-18 monolayers with PDBu led to a minimal increase in cell migration, whereas ionomycin induced a significant increase of ~75% over baseline. Incubation with both PDBu and ionomycin stimulated cell migration by >100% above baseline, an extent comparable to that stimulated by LPA (Fig. 10, A, C, and D). These results demonstrate that the combination of PKC and Ca2+ signaling, shown above to promote Pyk2 tyrosine phosphorylation, can also potently stimulate in vitro migration of IEC-18 cells.


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Fig. 10.   Migration of IEC-18 cells in vitro is promoted by LPA, protein kinase C (PKC), and Ca2+ mobilization. A: confluent serum-starved monolayers of IEC-18 cells were wounded as described in MATERIALS AND METHODS. Selected conditions were treated before wounding with 50 ng/ml PTX for 3 h or 500 nM cytochalasin D for 2 h. After being wounded, cells were incubated with or without the following agonists for 24 h: 200 nM PDBu, 50 nM ionomycin, or 20 µM LPA. Values are means ± SE of at least 8 fields and are expressed as the number of cells observed across the wound margin at 24 h. * P < 0.01 vs. control. Photographs representative of selected conditions are shown (B-E). Cells treated with an equivalent amount of solvent (DMSO) did not exhibit any significant difference in migration from control (data not shown).

Pretreatment with PTX before wounding had no effect on unstimulated cell migration at 24 h but significantly blocked LPA-stimulated migration, reducing it to near-baseline level (Fig. 10, A and E). Additionally, pretreatment with cytochalasin D before wounding resulted in near-total inhibition of cell migration, either with (Fig. 10A) or without LPA stimulation (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the gastrointestinal tract, the normal barrier and absorptive functions of epithelial cells depend on multiple processes, such as growth, migration, and differentiation, each of which is regulated by a combination of external factors. The IEC-18 and IEC-6 small intestinal cell lines have provided a useful model for studying these functions in culture (7, 9, 33, 40, 55). Although growth factor-induced proliferation and motility have been described in these cells, the associated intracellular signaling mechanisms are not well characterized.

Previous studies of the nonreceptor tyrosine kinase Pyk2 have demonstrated its association with focal adhesion proteins (17, 28, 43, 46), as well as transcriptional activation cascades (14, 26, 49, 59), suggesting the possibility of a role for Pyk2 in the regulation of cytoskeletal organization, gene expression, or DNA synthesis. In the present study, we have found that stimulation of IEC-18 cells with the GPCR agonists ANG II and LPA rapidly induces Pyk2 tyrosine phosphorylation in a concentration-dependent manner. This rapid change reflects the sum of phosphorylations at multiple sites and correlates here with increased phosphorylation both at Tyr-402, the primary autophosphorylation site, and at Tyr-580, a site of subsequent transphosphorylation by Src family kinases. Additionally, we have identified distinct but convergent pathways upstream of Pyk2, including one dependent on the Rho effector kinase ROK.

The vasoactive octapeptide ANG II is best known for its role in the regulation of fluid balance, vascular tone, and cardiovascular remodeling (20, 31, 57). ANG II acts as a growth factor for multiple cell types including vascular smooth muscle cells (57), cardiac fibroblasts (63), hepatic stellate cells (4), and some IEC (Ref. 53; T. Chiu, unpublished observations). In many of these tissues, ANG II also induces Pyk2 activity (28, 57, 63). The bioactive lipid LPA, which is released during cell injury primarily by platelets and fibroblasts (34), enhances proliferation in fibroblasts, endothelial cells, and smooth muscle cells (34, 35) and stimulates migration of IEC-6 cells in culture (55), as well as inducing Pyk2 activation in cells of neural origin (14). In our laboratory, ANG II and LPA have also recently been described as activators of the novel serine/threonine PKD in IEC-18 and IEC-6 cells (8).

Both ANG II- and LPA-specific GPCRs are known to trigger PKC activation and intracellular Ca2+ release via PLC (34, 62). In IEC-18 cells, simultaneous activation of PKC (by phorbol ester) and elevation of [Ca2+]i (by ionomycin) induced full Pyk2 tyrosine phosphorylation, and conversely, ANG II-stimulated Pyk2 phosphorylation was significantly diminished by either PKC or Ca2+ inhibition. This finding contrasts with some reports of PKC-independent Pyk2 activation in cell types including liver epithelial cells (29), cardiac fibroblasts (37), and HEK-293 kidney cells (12), but it is in accord with other studies showing a requirement for both PKC and Ca2+ in pancreatic acinar cells (58). Of note, both PKC inhibition and Ca2+ depletion have a lesser effect on LPA-stimulated, compared with ANG II-stimulated, Pyk2 phosphorylation, suggesting a greater relative contribution of non-PLC-dependent pathways in the response to LPA.

We also found that ANG II and LPA mediate Pyk2 tyrosine phosphorylation through at least two distinct classes of heterotrimeric G protein, Gi and Gq. As in IEC-6 cells (8) but in contrast to fibroblasts (34), LPA appears to mediate PKC and Ca2+ signaling via PTX-sensitive Gi in IEC-18 cells. In contrast, the failure of PTX to inhibit ANG II-induced Pyk2 phosphorylation is consistent with ANG II receptor coupling to Gq, as described in IEC-6 (8) and other cell types (31, 65). Significantly, inhibition of PKC and Ca2+ signaling (either directly or through Gi) attenuates, but does not completely abrogate, Pyk2 tyrosine phosphorylation.

A number of findings emphasize the importance of cytoskeletal organization to the regulation of Pyk2 in IEC. Tyrosine phosphorylation of Pyk2 was strongly inhibited by cytochalasin D in IEC-18 cells, as seen in other cell types (6, 19, 22) and also as described for the related kinase p125FAK (52). The constitutive association of the adaptor protein paxillin with Pyk2 in IEC-18 and IEC-6 cells and the increased paxillin tyrosine phosphorylation following ANG II or LPA treatment provide further evidence for a link between Pyk2 and the cytoskeleton. Here, as in GN4 liver epithelial cells (28), paxillin may be a direct substrate for Pyk2, although the action of an associated intermediate kinase (such as a member of the Src family) has not been ruled out.

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton and the assembly of focal adhesions (42, 45, 52). Rho proteins were shown to be critical for cell migration after experimental wounding in both normal (47) and polyamine-deficient IEC-6 cells (41). However, the mechanisms responsible for effecting this response are incompletely understood. In the present study, we identify a novel role for Pyk2 downstream of Rho activation. Specifically, inhibition of Rho by C3 exoenzyme, as well as exposure to two structurally unrelated inhibitors of the ROK family, HA-1077 and Y-27632, significantly diminished ANG II- and LPA-stimulated Pyk2 tyrosine phosphorylation. Furthermore, the combination of PTX with either cytochalasin D or ROK inhibitors completely abolished the response to LPA. This suggests that, distinct from the Gi/Gq-mediated, PLC-dependent branch of the Pyk2 phosphorylation pathway, the remaining branch of this GPCR-mediated response involves the sequential activation of Rho and ROK upstream of stress fiber formation. Pyk2, therefore, has the potential to integrate both Rho-dependent and -independent signals after GPCR stimulation. Since the initial submission of this article, it has been reported that in T lymphocytes, Pyk2 activation occurred after inhibition of the Rho/ROK pathway (44), contrasting with our finding that Pyk2 phosphorylation is Rho dependent. These observations warrant further experimental work in other cell types.

Considering the activation of Pyk2 induced by ANG II and LPA, as well as its association with focal adhesion proteins, it becomes possible to hypothesize a role for Pyk2 in mediating the cellular effects of these agonists. IEC migration in vitro, as well as wound healing in vivo, are enhanced by LPA (55). We show here that the direct stimulation of both PKC activation and Ca2+ release can promote cell migration to an equivalent degree as LPA. Our results also suggest that LPA-enhanced migration is mediated via PTX-sensitive Gi and is dependent on actin cytoskeletal integrity. Previous work demonstrated that functional RhoA was necessary for EGF-stimulated migration in IEC (47). Because these upstream elements are all shown here to regulate Pyk2 tyrosine phosphorylation in IEC-18 cells, the findings are compatible with a potential role for Pyk2 in cell migration. Though these preliminary data indicate correlation rather than causality, they provide an important background for further investigation. Results from our laboratory and others have also shown that ANG II promotes proliferation in IEC (Ref. 53; T. Chiu, unpublished observations). Support for a possible role of Pyk2 in the cellular response to injury comes from reports of Pyk2 linkage to MAP kinase (14, 26, 59), phosphatidylinositol 3-kinase (43), and NF-kappa B signaling pathways (49), which in turn affect transcription of genes relevant to proliferation and inflammation. In addition to the role of Pyk2 in growth and migration, key areas for further research include downstream targets of Pyk2 in intestinal cells and the potential for cross talk between Pyk2 and PKD, which have been proposed as responsible for mediating similar functions. In this context, the recent demonstration that Pyk2 stimulates GSK-3beta tyrosine phosphorylation and activation (21) is of considerable interest in view of the role of GSK-3beta in the regulation of beta -catenin signaling in IEC and the importance of this pathway in colon cancer (56).

In conclusion, our results demonstrate that Pyk2 is rapidly tyrosine phosphorylated in intestinal epithelial cells in response to the GPCR agonists ANG II and LPA via pathways that are partially dependent on both PKC and Ca2+ release. LPA, in contrast with ANG II, induces Pyk2 tyrosine phosphorylation through a PTX-sensitive pathway involving Gi. Disruption of the actin cytoskeleton by cytochalasin D strongly attenuates Pyk2 activity, as does inhibition of Rho/ROK, which functions distinctly from PKC and Ca2+ signaling and represents a novel element in the pathway leading from GPCR stimulation to Pyk2. We therefore propose that Pyk2 represents a point of integration for Gi, Gq, and cytoskeletal signaling in intestinal epithelial cells.


    ACKNOWLEDGEMENTS

We thank the Welfide Corporation (Osaka, Japan) for providing the Rho kinase inhibitor Y-27632.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-17294, DK-55003, DK-56930, and P50-CA-090388 (to E. Rozengurt).

S. Wu is a National Institute of Child Health and Human Development Fellow of the Pediatric Scientist Development Program (K12-HD-00850). T. Chiu is a recipient of an American Gastroenterological Association/AstraZeneca Fellowship/Faculty Transition Award.

Address for reprint requests and other correspondence: E. Rozengurt, 900 Veteran Avenue, Warren Hall, Room 11-124, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1786 (E-mail: erozengurt{at}mednet.ucla.edu).

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

First published February 6, 2002;10.1152/ajpcell.00323.2001

Received 17 July 2001; accepted in final form 1 February 2002.


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ABSTRACT
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RESULTS
DISCUSSION
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