Departments of 1 Pediatrics and 2 Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095
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ABSTRACT |
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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
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INTRODUCTION |
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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- and -
(TGF-
and -
), epidermal growth factor (EGF),
and lysophosphatidic acid (LPA). Proliferation in culture is also
promoted by EGF and TGF-
but not by TGF-
(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- (CAK
), 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-
B (NF-
B) pathway (49). Additionally,
Pyk2 phosphorylates glycogen synthase kinase-3
(GSK-3
), a
critical enzyme in the regulation of
-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 Gq-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.
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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
(1) and 380 nm (
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
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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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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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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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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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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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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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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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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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DISCUSSION |
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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-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-3
tyrosine phosphorylation and activation
(21) is of considerable interest in view of the role of
GSK-3
in the regulation of
-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.
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ACKNOWLEDGEMENTS |
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We thank the Welfide Corporation (Osaka, Japan) for providing the Rho kinase inhibitor Y-27632.
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amano, M,
Chihara K,
Kimura K,
Fukata Y,
Nakamura N,
Matsuura Y,
and
Kaibuchi K.
Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase.
Science
275:
1308-1311,
1997
2.
André, F,
Rigot V,
Remacle-Bonnet M,
Luis J,
Pommier G,
and
Marvaldi J.
Protein kinases C-gamma and -delta are involved in insulin-like growth factor I-induced migration of colonic epithelial cells.
Gastroenterology
116:
64-77,
1999[ISI][Medline].
3.
Avraham, H,
Park SY,
Schinkmann K,
and
Avraham S.
RAFTK/Pyk2-mediated cellular signaling.
Cell Signal
12:
123-133,
2000[ISI][Medline].
4.
Bataller, R,
Gines P,
Nicolas JM,
Gorbig MN,
Garcia-Ramallo E,
Gasull X,
Bosch J,
Arroyo V,
and
Rodes J.
Angiotensin II induces contraction and proliferation of human hepatic stellate cells.
Gastroenterology
118:
1149-1156,
2000[ISI][Medline].
5.
Blaukat, A,
Ivankovic-Dikic I,
Grönroos E,
Dolfi F,
Tokiwa G,
Vuori K,
and
Dikic I.
Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades.
J Biol Chem
274:
14893-14901,
1999
6.
Brinson, AE,
Harding T,
Diliberto PA,
He Y,
Li X,
Hunter D,
Herman B,
Earp HS,
and
Graves LM.
Regulation of a calcium-dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. Dependence on calcium and the actin cytoskeleton.
J Biol Chem
273:
1711-1718,
1998
7.
Carroll, KM,
Wong TT,
Drabik DL,
and
Chang EB.
Differentiation of rat small intestinal epithelial cells by extracellular matrix.
Am J Physiol Gastrointest Liver Physiol
254:
G355-G360,
1988
8.
Chiu, T,
and
Rozengurt E.
PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC.
Am J Physiol Cell Physiol
280:
C929-C942,
2001
9.
Chiu, T,
Wu SS,
Santiskulvong C,
Tangkijvanich P,
Yee HF,
and
Rozengurt E.
Vasopressin-mediated mitogenic signaling in intestinal epithelial cells.
Am J Physiol Cell Physiol
282:
C434-C450,
2002
10.
Ciacci, C,
Lind SE,
and
Podolsky DK.
Transforming growth factor beta regulation of migration in wounded rat intestinal epithelial monolayers.
Gastroenterology
105:
93-101,
1993[ISI][Medline].
11.
Davies, SP,
Reddy H,
Caivano M,
and
Cohen P.
Specificity and mechanism of action of some commonly used protein kinase inhibitors.
Biochem J
351:
95-105,
2000[ISI][Medline].
12.
Della Rocca, GJ,
van Biesen T,
Daaka Y,
Luttrell DK,
Luttrell LM,
and
Lefkowitz RJ.
Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase.
J Biol Chem
272:
19125-19132,
1997
13.
Dieckgraefe, BK,
Weems DM,
Santoro SA,
and
Alpers DH.
ERK and p38 MAP kinase pathways are mediators of intestinal epithelial wound-induced signal transduction.
Biochem Biophys Res Commun
233:
389-394,
1997[ISI][Medline].
14.
Dikic, I,
Tokiwa G,
Lev S,
Courtneidge SA,
and
Schlessinger J.
A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.
Nature
383:
547-550,
1996[ISI][Medline].
15.
Dionne, S,
D'Agata ID,
Ruemmele FM,
Levy E,
St-Louis J,
Srivastava AK,
Levesque D,
and
Seidman EG.
Tyrosine kinase and MAPK inhibition of TNF-- and EGF-stimulated IEC-6 cell growth.
Biochem Biophys Res Commun
242:
146-150,
1998[ISI][Medline].
16.
Eguchi, S,
Iwasaki H,
Inagami T,
Numaguchi K,
Yamakawa T,
Motley ED,
Owada KM,
Marumo F,
and
Hirata Y.
Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells.
Hypertension
33:
201-206,
1999
17.
Felsch, JS,
Cachero TG,
and
Peralta EG.
Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor.
Proc Natl Acad Sci USA
95:
5051-5056,
1998
18.
Fuortes, M,
Melchior M,
Han H,
Lyon GJ,
and
Nathan C.
Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF.
J Clin Invest
104:
327-335,
1999
19.
Ganju, RK,
Hatch WC,
Avraham H,
Ona MA,
Druker B,
Avraham S,
and
Groopman JE.
RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.
J Exp Med
185:
1055-1063,
1997
20.
Goodfriend, TL,
Elliott ME,
and
Catt KJ.
Angiotensin receptors and their antagonists.
N Engl J Med
334:
1649-1654,
1996
21.
Hartigan, JA,
Xiong WC,
and
Johnson GVW
Glycogen synthase kinase 3 beta is tyrosine phosphorylated by PYK2.
Biochem Biophys Res Commun
284:
485-489,
2001[ISI][Medline].
22.
Hiregowdara, D,
Avraham H,
Fu Y,
London R,
and
Avraham S.
Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin.
J Biol Chem
272:
10804-10810,
1997
23.
Iglesias, T,
Waldron RT,
and
Rozengurt E.
Identification of in vivo phosphorylation sites required for protein kinase D activation.
J Biol Chem
273:
27662-27667,
1998
24.
Ishizaki, T,
Uehata M,
Tamechika I,
Keel J,
Nonomura K,
Maekawa M,
and
Narumiya S.
Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases.
Mol Pharmacol
57:
976-983,
2000
25.
Keely, SJ,
Calandrella SO,
and
Barrett KE.
Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells is mediated by intracellular Ca2+, PYK-2, and p60src.
J Biol Chem
275:
12619-12625,
2000
26.
Lev, S,
Moreno H,
Martinez R,
Canoll P,
Peles E,
Musacchio JM,
Plowman GD,
Rudy B,
and
Schlessinger J.
Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions.
Nature
376:
737-745,
1995[ISI][Medline].
27.
Li, X,
Dy RC,
Cance WG,
Graves LM,
and
Earp HS.
Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase.
J Biol Chem
274:
8917-8924,
1999
28.
Li, X,
and
Earp HS.
Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells.
J Biol Chem
272:
14341-14348,
1997
29.
Li, X,
Lee JW,
Graves LM,
and
Earp HS.
Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.
EMBO J
17:
2574-2583,
1998
30.
Litvak, V,
Tian D,
Shaul YD,
and
Lev S.
Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades.
J Biol Chem
275:
32736-32746,
2000
31.
Matsusaka, T,
and
Ichikawa I.
Biological functions of angiotensin and its receptors.
Annu Rev Physiol
59:
395-412,
1997[ISI][Medline].
32.
Matthews, SA,
Rozengurt E,
and
Cantrell D.
Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/protein kinase Cµ.
J Biol Chem
274:
26543-26549,
1999
33.
McCormack, SA,
Viar MJ,
and
Johnson LR.
Migration of IEC-6 cells: a model for mucosal healing.
Am J Physiol Gastrointest Liver Physiol
263:
G426-G435,
1992
34.
Moolenaar, WH.
Lysophosphatidic acid, a multifunctional phospholipid messenger.
J Biol Chem
270:
12949-12952,
1995
35.
Moolenaar, WH,
Kranenburg O,
Postma FR,
and
Zondag GC.
Lysophosphatidic acid: G-protein signaling and cellular responses.
Curr Opin Cell Biol
9:
168-173,
1997[ISI][Medline].
36.
Murasawa, S,
Matsubara H,
Mori Y,
Masaki H,
Tsutsumi Y,
Shibasaki Y,
Kitabayashi I,
Tanaka Y,
Fujiyama S,
Koyama Y,
Fujiyama A,
Iba S,
and
Iwasaka T.
Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase.
J Biol Chem
275:
26856-26863,
2000
37.
Murasawa, S,
Mori Y,
Nozawa Y,
Masaki H,
Maruyama K,
Tsutsumi Y,
Moriguchi Y,
Shibasaki Y,
Tanaka Y,
Iwasaka T,
Inada M,
and
Matsubara H.
Role of calcium-sensitive tyrosine kinase Pyk2/CAK/RAFTK in angiotensin II-induced Ras/ERK signaling.
Hypertension
32:
668-675,
1998
38.
Nagumo, H,
Sasaki Y,
Ono Y,
Okamoto H,
Seto M,
and
Takuwa Y.
Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells.
Am J Physiol Cell Physiol
278:
C57-C65,
2000
39.
Polk, DB.
Epidermal growth factor receptor-stimulated intestinal epithelial cell migration requires phospholipase C activity.
Gastroenterology
114:
493-502,
1998[ISI][Medline].
40.
Quaroni, A,
Wands J,
Trelstad RL,
and
Isselbacher KJ.
Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.
J Cell Biol
80:
248-265,
1979[Abstract].
41.
Rao, JN,
Li L,
Golovina VA,
Platoshyn O,
Strauch ED,
Yuan JXJ,
and
Wang JY.
Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration.
Am J Physiol Cell Physiol
280:
C993-C1007,
2001
42.
Ridley, AJ,
and
Hall A.
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70:
389-399,
1992[ISI][Medline].
43.
Rocic, P,
Govindarajan G,
Sabri A,
and
Lucchesi PA.
A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by ANG II in vascular smooth muscle.
Am J Physiol Cell Physiol
280:
C90-C99,
2001
44.
Rodriguez-Fernandez JL, Sanchez-Martin L, Rey M, Vicente-Manzanares M,
Narumiya S, Teixido J, Sanchez-Madrid F, and Cabanas C. Rho and
ROCK modulate the tyrosine kinase PYK2 in T cells through regulation of
the activity of the integrin LFA-1. J Biol Chem, 2001.
45.
Sah, VP,
Seasholtz TM,
Sagi SA,
and
Brown JH.
The role of Rho in G protein-coupled receptor signal transduction.
Annu Rev Pharmacol Toxicol
40:
459-489,
2000[ISI][Medline].
46.
Salgia, R,
Avraham S,
Pisick E,
Li JL,
Raja S,
Greenfield EA,
Sattler M,
Avraham H,
and
Griffin JD.
The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells.
J Biol Chem
271:
31222-31226,
1996
47.
Santos, MF,
McCormack SA,
Guo Z,
Okolicany J,
Zheng Y,
Johnson LR,
and
Tigyi G.
Rho proteins play a critical role in cell migration during the early phase of mucosal restitution.
J Clin Invest
100:
216-225,
1997
48.
Sechi, LA,
Valentin JP,
Griffin CA,
and
Schambelan M.
Autoradiographic characterization of angiotensin II receptor subtypes in rat intestine.
Am J Physiol Gastrointest Liver Physiol
265:
G21-G27,
1993
49.
Shi, CS,
and
Kehrl JH.
PYK2 links Gq and G13
signaling to NF-
B activation.
J Biol Chem
276:
31845-31850,
2001
50.
Shi, CS,
Sinnarajah S,
Cho H,
Kozasa T,
and
Kehrl JH.
G13-mediated PYK2 activation.
J Biol Chem
275:
24470-24476,
2000
51.
Sinnett-Smith, J,
Lunn JA,
Leopoldt D,
and
Rozengurt E.
Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130cas.
Exp Cell Res
266:
292-302,
2001[ISI][Medline].
52.
Sinnett-Smith, J,
Zachary I,
Valverde AM,
and
Rozengurt E.
Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation. Role of protein kinase C, Ca2+ mobilization, and the actin cytoskeleton.
J Biol Chem
268:
14261-14268,
1993
53.
Smith, RD,
Corps AN,
Hadfield KM,
Vaughan TJ,
and
Brown KD.
Activation of AT-1 angiotensin receptors induces DNA synthesis in a rat intestinal epithelial (RIE-1) cell line.
Biochem J
302:
791-800,
1994[ISI][Medline].
54.
Smith, RD.
Identification of atypical (non-AT1, non-AT2) angiotensin binding sites with high affinity for angiotensin I on IEC-18 rat intestinal epithelial cells.
FEBS Lett
373:
199-202,
1995[ISI][Medline].
55.
Sturm, A,
Sudermann T,
Schulte KM,
Goebell H,
and
Dignass AU.
Modulation of intestinal epithelial wound healing in vitro and in vivo by lysophosphatidic acid.
Gastroenterology
117:
368-377,
1999[ISI][Medline].
56.
Taipale, J,
and
Beachy PA.
The Hedgehog and Wnt signaling pathways in cancer.
Nature
411:
349-354,
2001[ISI][Medline].
57.
Tang, H,
Zhao ZJ,
Landon EJ,
and
Inagami T.
Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2.
J Biol Chem
275:
8389-8396,
2000
58.
Tapia, JA,
Ferris HA,
Jensen RT,
and
García LJ.
Cholecystokinin activates PYK2/CAK by a phospholipase C-dependent mechanism and its association with the mitogen-activated protein kinase signaling pathway in pancreatic acinar cells.
J Biol Chem
274:
31261-31271,
1999
59.
Tokiwa, G,
Dikic I,
Lev S,
and
Schlessinger J.
Activation of Pyk2 by stress signals and coupling with JNK signaling pathway.
Science
273:
792-794,
1996[Abstract].
60.
Toullec, D,
Pianetti P,
Coste H,
Bellevergue P,
Grand-Perret T,
Ajakane M,
Baudet V,
Boissin P,
Boursier E,
and
Loriolle F.
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J Biol Chem
266:
15771-15781,
1991
61.
Uehata, M,
Ishizaki T,
Satoh H,
Ono T,
Kawahara T,
Morishita T,
Tamakawa H,
Yamagami K,
Inui J,
Maekawa M,
and
Narumiya S.
Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension.
Nature
389:
990-994,
1997[ISI][Medline].
62.
Ushio-Fukai, M,
Griendling KK,
Akers M,
Lyons PR,
and
Alexander RW.
Temporal dispersion of activation of phospholipase C-1 and -
isoforms by angiotensin II in vascular smooth muscle cells. Role of
q/11,
12, and
G protein subunits.
J Biol Chem
273:
19772-19777,
1998
63.
Wang, D,
Yu X,
and
Brecher P.
Nitric oxide inhibits angiotensin II-induced activation of the calcium-sensitive tyrosine kinase proline-rich tyrosine kinase 2 without affecting epidermal growth factor receptor transactivation.
J Biol Chem
274:
24342-24348,
1999
64.
Yeo, EJ,
and
Exton JH.
Stimulation of phospholipase D by epidermal growth factor requires protein kinase C activation in Swiss 3T3 cells.
J Biol Chem
270:
3980-3988,
1995
65.
Yuan, J,
Slice L,
Walsh JH,
and
Rozengurt E.
Activation of protein kinase D by signaling through the alpha subunit of the heterotrimeric G protein Gq.
J Biol Chem
275:
2157-2164,
2000
66.
Zwick, E,
Wallasch C,
Daub H,
and
Ullrich A.
Distinct calcium-dependent pathways of epidermal growth factor receptor transactivation and PYK2 tyrosine phosphorylation in PC12 cells.
J Biol Chem
274:
20989-20996,
1999