©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Guanosine 5`-3-O-(Thio)triphosphate Stimulates Tyrosine Phosphorylation of p125 and Paxillin in Permeabilized Swiss 3T3 Cells
ROLE OF p21(*)

(Received for publication, October 31, 1994; and in revised form, January 20, 1995)

Michael J. Seckl (1) Narito Morii (2) Shuh Narumiya (2) Enrique Rozengurt (1)(§)

From the  (1)Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom and the (2)Faculty of Medicine, Second Department of Pharmacology, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Addition of guanosine 5` - 3 - O - (thio) triphosphate (GTPS) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands. Specifically, GTPS stimulated tyrosine phosphorylation of both focal adhesion kinase (p125) and paxillin. GTPS induced tyrosine phosphorylation was dose-dependent (EC of 2.5 µM) and reached maximum levels after 1.5 min for the M(r) 110,000-130,000 band and 2 min for the M(r) 70,000-80,000 paxillin band. Guanosine 5`-O-(2-thiodiphosphate) inhibited GTPS-induced tyrosine phosphorylation with an IC of 100 µM. Protein kinase C did not mediate GTPS-induced tyrosine phosphorylation. Varying the Ca concentration from 0 to 6 µM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTPS to induce tyrosine phosphorylation. GTPS was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg. Furthermore, 30 µM AlF(4) only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21, markedly reduced the ability of GTPS to stimulate tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands including p125 and paxillin in permeabilized Swiss 3T3 cells. Furthermore, a peptide of p21(p21) inhibited GTPS-induced tyrosine phosphorylation in a dose-dependent manner (IC 1 µM). This peptide also inhibited tyrosine phosphorylation of p125 and paxillin. In contrast, 20 µM p21 peptide failed to inhibit GTPS-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTPS stimulates tyrosine phosphorylation of p125 and paxillin and that a functional p21 is implicated in this process.


INTRODUCTION

Tyrosine phosphorylation has recently been implicated in the intracellular signaling of neuropeptides that act as potent cellular growth factors through receptors with seven transmembrane helices (1, 2, 3, 4, 5, 6, 7, 8) . Bombesin and other mitogenic neuropeptides stimulate tyrosine phosphorylation of multiple proteins in Swiss 3T3 cells, a useful model system for the elucidation of signal transduction pathways leading to cell proliferation(9) . The tyrosine-phosphorylated proteins include broad bands of M(r) 110,000-130,000 and 70,000-80,000(2, 4, 10) . The focal adhesion associated protein p125, (^1)a novel cytosolic tyrosine kinase which lacks Src homology domains 2 and 3(11, 12) , has been identified as a prominent tyrosine-phosphorylated protein migrating within the M(r) 110,000-130,000 band stimulated by neuropeptides in Swiss 3T3 cells(6, 13) . Paxillin, another focal adhesion associated protein (14, 15) has been shown to comprise the M(r) 70,000-80,000 tyrosine-phosphorylated protein band stimulated by neuropeptides in these cells(16) . The rapidity of neuropeptide-stimulated tyrosine phosphorylation is consistent with p125 and paxillin functioning in a neuropeptide-activated tyrosine kinase pathway.

Recent evidence demonstrates that a variety of other agents that modulate cell growth and differentiation including platelet-derived growth factor(17) , the bioactive lipid LPA(18, 19, 20) , sphingosine(21) , tumor promoting phorbol esters(13) , extracellular matrix proteins (22, 23, 24, 25, 26) , and transforming variants of pp60(23, 27) , induce coordinated tyrosine phosphorylation of p125 and paxillin. In all cases, the induction of tyrosine phosphorylation of these proteins required the integrity of the actin cytoskeleton(13, 17, 20, 21, 25) . Furthermore, both tyrosine phosphorylation and the cytoskeletal changes induced by LPA and bombesin require functional p21 protein(20, 28, 29, 30) . These findings support the existence of a tyrosine kinase pathway involving p125 and paxillin, but the components of this signal transduction pathway and their upstream and downstream interactions have not been fully identified.

Cell permeabilization has provided a useful approach to study protein phosphorylation and also to introduce guanine nucleotides to assess the contribution of G proteins in signal transduction in Swiss 3T3 cells (31, 32, 33) . However, tyrosine phosphorylation of p125 and paxillin has not been demonstrated in any permeabilized cell preparation. In the present study, we show that the nonhydrolyzable GTP analogue, GTPS, induced tyrosine phosphorylation of multiple proteins including p125 and paxillin in SLO permeabilized Swiss 3T3 cells. Our results suggest that p21predominantly mediates this process.


EXPERIMENTAL PROCEDURES

Cell Culture

Stock cultures of Swiss 3T3 fibroblasts were maintained in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO(2) and 90% air at 37 °C. For experimental purposes, cells were plated in 33-mm Nunc Petri dishes at 10^5 cells/dish in DMEM containing 10% fetal bovine serum and used after 6-8 days when the cells were confluent and quiescent.

Streptolysin O-Permeabilization of Cells

Previous studies have shown that the pore size induced by SLO depends on both the concentration and length of exposure to the agent(34) . In our own preliminary studies, cultures of Swiss 3T3 cells in 33-mm dishes were labeled for 5 h in 1 ml of DMEM containing [^3H]uridine (1 µCi/ml; 1 µM) to label the UTP pool. The dishes were then washed in DMEM twice prior to incubating with increasing concentrations (0.05-2.4 units/ml) of SLO dissolved in permeabilization solution (see following section) for 10 min at 37 °C. Maximum labeled UTP release occurred at 0.4 units/ml SLO. In further experiments we demonstrated that maximum release of labeled UTP occurred after a 1-min exposure to 0.4 unit/ml SLO (data not shown).

Tyrosine Phosphorylation in Permeabilized Cells

Confluent and quiescent cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM prior to incubation in permeabilization solution at 37 °C. This comprised 120 mM KCl, 30 mM NaCl, 2.5 mM MgCl(2), 1 mM K(2)HPO(4), 10 mM PIPES, 2 mM EGTA, 0.5 mM CaCl(2), KOH (to give pH 7.2), 1 mM ATP, and SLO at 0.4 unit/ml in the presence or absence of various compounds. One min later the cells were stimulated by addition of GTPS at the concentrations indicated in the figure legends. The incubation was terminated after a further 1.5 min unless otherwise specified by cell lysis at 4 °C in 1 ml of a solution containing 10 mM Tris/HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na(3)VO(4), 50 µM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100 (lysis buffer). In experiments where the concentration of free calcium in the permeabilization solution was increased this was achieved by increasing the total amount of CaCl(2) added without altering the EGTA concentration and the free calcium concentration calculated as described previously(35) . In experiments where permeabilized cells were stimulated with 30 µM AlF(4) this was made up freshly to give a final concentration of 30 µM AlCl(3) and 10 mM NaF(36) .

Magnesium Dose Response in Permeabilized Cells

Confluent and quiescent cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM prior to incubation in modified permeabilization solution at 37 °C lacking ATP and MgCl(2) and containing 2 mM EDTA with or without 5 µM GTPS. Increasing volumes of a 1 M solution of MgCl(2) was added to the modified permeabilization solution to give the indicated free magnesium concentration. The free magnesium concentration was calculated using the following equation: [Mg] = [Mg] times {1 + [EDTA]/([Mg] + K) + [GTP]/([Mg] + K)}. The equilibrium constants used were K = 1 µM and K = 60 µM(37) . After 1 min the solution was exchanged for standard permeabilization solution to allow the kinase reaction to occur and the incubation was continued for a further 1.5 min. The cells were then lysed as described above.

Immunoprecipitations

Lysates were centrifuged at 15,000 times g for 20 min and the supernatants were incubated for 4 h at 4 °C with anti-mouse IgG agarose-linked mAbs directed against phosphotyrosine (Py72), p125 (mAb 2A7), or paxillin (mAb 165) as indicated. The immunoprecipitates were washed 3 times with lysis buffer and further analyzed by SDS-PAGE and Western blotting. Cells from parallel cultures treated in an identical fashion were suspended by trypsinization and counted using a Coulter counter to ensure equal numbers of cells per condition.

Western Blotting

Immunoprecipitates were fractionated by SDS-PAGE and the proteins were then transferred to Immobilon membranes. Membranes were blocked using 5% non-fat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 3-5 h in phosphate-buffered saline containing 0.05% Tween 20 and 1 µg/ml of both Py20 and 4G-10 anti-Tyr(P) mAbs. Immunoreactive bands were visualized using I-labeled sheep anti-mouse IgG followed by autoradiography. Autoradiograms were scanned using an LKB Ultrascan XL densitometer, and labeled bands were quantified using the Ultrascan XL internal integrator. The values expressed represent percentages of the maximal increase above control values.

SDS-Polyacrylamide Gel Electrophoresis

Slab gel electrophoresis was performed essentially according to the method of Laemmli(38) . Specifically, the slab gels were 1.5-mm thick with 1.5 cm of a 4% acrylamide stacking gel and 12 cm of 8% acrylamide resolving gel. Samples (100 µl) were electrophoresed at 20 V for 30 min, then run overnight at 50 V and finally at 150 V for 30 min before terminating the run. Radioactivity was detected at -70 °C using Fuji x-ray film with exposure times of 12-72 h.

Down-regulation of PKC

Phorbol ester-sensitive PKCs are down-regulated in Swiss 3T3 cells by prolonged pretreatment with PDBu (39, 40) . In the present study confluent and quiescent cultures were pretreated with 800 nM PDBu for 48 h in conditioned medium which was depleted of growth promoting activity.

Preparation of Recombinant C3 Exoenzyme

The C3 exoenzyme gene (41) was modified by polymerase chain reaction-mediated site-directed mutagenesis to produce a recombinant C3 exoenzyme that lacks the signal peptide and has the dipeptide Met-Ala attached to Ser of the mature exoenzyme. Polymerase chain reaction was performed with the cloned gene as the template and with the synthetic oligonucleotides (5`-ACTGTTCATATGGCTAGCTATGCAGATACTTTCACA-3` and 5`-TTATTGGATCCTATTATTTAAATATCATTGCTGTAA-3`) as primers. The amplified fragment was cleaved with NdeI and BamHI and ligated with a pET-3a vector(42) . After confirming the DNA sequence, the recombinant plasmid, pET-3(R)/C3, was introduced into Escherichia coli BL21 (DE3)pLysE and expressed(41, 42, 43) .

C3 Exoenzyme Pretreatment of Cells

Swiss 3T3 cells were seeded at a density of 1 times 10^5 per 30-mm dish in 2 ml of DMEM supplemented with 10% fetal bovine serum. At 72 h, recombinant C3 exoenzyme at a final concentration of 7.5 µg/ml or diluent was added to the medium. After being cultured for a further 48 h the cells were washed twice with DMEM and then incubated in DMEM:Waymouth's (1:1, v/v) in the presence or absence of C3 exoenzyme at 15 µg/ml for 24 h. This protocol has previously been shown to ADP-ribosylate p21 in intact Swiss 3T3 cells, as shown by the fact that cell homogenates from C3 exoenzyme-treated cells contain markedly reduced levels of native p21 available for [P]ADP-ribosylation by externally added C3 exoenzyme (data not shown)(29, 44) .

Cell-free Kinase Assay

Confluent and quiescent cultures of Swiss 3T3 cells were washed twice in DMEM and then incubated in the presence or absence of 10 nM bombesin for 10 min at 37 °C. The cells were then lysed and the lysates immunoprecipitated with anti-mouse IgG agarose-linked mAbs directed against Py72 for 4 h. The immunoprecipitates were washed three times with lysis buffer and twice with 50 mM HEPES, pH 7.4, 0.1 mM EDTA, 0.01% Brij, 75 mM NaCl (kinase buffer) and resuspended in 20 µl of this buffer. Kinase reactions were initiated by addition of 10 mM MgCl(2) and 100 µM [-P]ATP (20 µCi) and performed in a total volume of 30 µl at 30 °C for 20 min. The immunoprecipitates were then washed three times with lysis buffer and analyzed by SDS-PAGE followed by autoradiography.

Materials

Bombesin and agarose-linked anti-mouse IgG were obtained from Sigma. Anti-Tyr(P) mAb, clone Py72, was obtained from the hybridoma development unit, Imperial Cancer Research Fund, London, United Kingdom. Py20 anti-Tyr(P) mAb and the mAb directed against paxillin (mAb 165) were from ICN, High Wycombe, UK. 4G-10 anti-Tyr(P) mAb and mAb 2A7 directed against p125 were from TCS Biologicals LTD., Buckingham, UK. Anti-p125 mAb for Western blotting was obtained from AFFINITI Research Products Ltd., Nottingham, UK. I-Sheep anti-mouse IgG (50 mCi/mg) and [-P]ATP were from Amersham, UK. SLO was obtained from Welcome Diagnostics, UK. All other reagents used were of the purest grade available.


RESULTS

GTPS Stimulates Tyrosine Phosphorylation of Multiple Substrates Including p125 and Paxillin in Permeabilized Swiss 3T3 Cells

To determine whether GTPS could stimulate tyrosine phosphorylation of proteins in permeabilized Swiss 3T3 cells, quiescent cultures of these cells were permeabilized with 0.4 IU/ml SLO for 1 min and then incubated in the presence or absence of 5 µM GTPS for an additional 1.5 min. Lysates of the permeabilized cells were immunoprecipitated with a specific anti-Tyr(P) mAb, and the immunoprecipitates analyzed by Western blotting using a mixture of Py20/4G-10 anti-Tyr(P) mAbs. Fig. 1A, left, shows that GTPS stimulated tyrosine phosphorylation of a group of bands migrating with an apparent M(r) 110,000-130,000 and 70,000-80,000. Additional experiments showed that the optimal SLO concentration for this purpose was 0.4 IU/ml and that GTPS did not stimulate tyrosine phosphorylation in intact cells (data not shown). In 10 independent experiments, a 1.5-min exposure to 5 µM GTPS stimulated tyrosine phosphorylation of the M(r) 110,000-130,000 band by 4.5 ± 0.2 (S.E.)-fold above basal levels. In contrast, addition of 5 µM ATPS instead of GTPS failed to stimulate tyrosine phosphorylation in permeabilized cells (data not shown).


Figure 1: A, identification of proteins which are tyrosine phosphorylated by GTPS in permeabilized Swiss 3T3 cells. Confluent and quiescent cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM and then incubated in permeabilization solution containing 0.4 IU/ml SLO for 1 min. The incubation was continued for a further 1.5 min in the presence or absence of 5 µM GTPS as described under ``Experimental Procedures.'' The cultures were then lysed and the lysates immunoprecipitated with the anti-Tyr(P) mAb Py72 (left, PY), p125 mAb2A7 (middle, FAK), or anti-paxillin mAb 165 (right, PAX). The immunoprecipitates were analyzed by immunoblotting with a 1:1 mixture of 4G-10 and Py20 anti-Tyr(P) mAbs. Autoradiographs shown are representative of at least two independent experiments. B, time course GTPS-stimulated tyrosine phosphorylation. Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM and permeabilized with 0.4 IU/ml SLO for 1 min prior to addition of 5 µM GTPS. The cultures were then lysed and immunoprecipitates of tyrosine-phosphorylated proteins were analyzed by anti-Tyr(P) immunoblotting. Scanning densitometry of both the M(r) 110,000-130,000 (circles) and 70,000-80,000 (triangles) bands is shown. Each point is representative of three independent experiments ± S.E. and is expressed as a percentage of the maximum response. C, dose response of GTPS-induced tyrosine phosphorylation. Cultures of Swiss 3T3 cells were permeabilized for 1 min and incubated in the presence or absence of increasing concentrations of GTPS for a further 1.5 min before lysis. Scanning densitometry of both the M(r) 110,000-130,000 (circles) and 70,000-80,000 (triangles) bands is shown. Each point is representative of three independent experiments and is expressed as a percentage of the maximum response. For clarity, only the error bars (S.E.) for the M(r) 110,000-130,000 band are shown.



The pattern of tyrosine phosphorylation induced by GTPS in permeabilized Swiss 3T3 cells was identical to that previously shown to be induced by bombesin and other agents (see Introduction for details) in intact cells. These stimuli are known to increase tyrosine phosphorylation of p125, which migrates within the M(r) 110,000-130,000, and paxillin which corresponds to the M(r) 70,000-80,000 band. We therefore examined whether GTPS also stimulates tyrosine phosphorylation of these proteins. Accordingly, lysates of permeabilized Swiss 3T3 cells stimulated with 5 µM GTPS were immunoprecipitated with either anti-p125 or paxillin mAbs and the resulting immunoprecipitates were Western blotted with anti-Tyr(P) mAbs. As shown in Fig. 1A, GTPS markedly stimulated tyrosine phosphorylation of both p125 (5 ± 2-fold) and paxillin (4 ± 1.2-fold) in permeabilized cells.

Tyrosine phosphorylation was a rapid consequence of the addition of GTPS to permeabilized Swiss 3T3 cells. Fig. 1B demonstrates an increase in tyrosine phosphorylation of the M(r) 110,000-130,000 group of bands after 45 s. Maximum tyrosine phosphorylation of this band was reached after 1.5 min of incubation with GTPS. Tyrosine phosphorylation of the M(r) 70,000-80,000 band, corresponding to paxillin, was delayed by 30 s, reaching a maximum 2 min after addition of GTPS (Fig. 1B). GTPS stimulated tyrosine phosphorylation of both the M(r) 110,000-130,000 and 70,000-80,000 bands in a dose-dependent fashion with an identical EC of 2.5 µM. Maximum tyrosine phosphorylation was achieved at 5 µM GTPS (Fig. 1C).

Addition of GDPbetaS inhibited tyrosine phosphorylation of both the M(r) 110,000-130,000 and 70,000-80,000 bands induced by 5 µM GTPS in a dose-dependent manner with an IC of 100 µM (Fig. 2). Increasing the concentration of GTPS to 500 µM almost completely reversed (90%) the inhibitory effect of 250 µM GDPbetaS, a concentration which reduced tyrosine phosphorylation stimulated by 5 µM GTPS by 75% (Fig. 2, inset). These results suggest that a G protein is involved in tyrosine phosphorylation stimulated by GTPS in permeabilized Swiss 3T3 cells.


Figure 2: Effect of GDPbetaS on tyrosine phosphorylation stimulated by GTPS. Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM and permeabilized with 0.4 IU/ml SLO in the presence or absence of increasing concentrations of GDPbetaS. After 1 min 5 µM GTPS was added to the dishes and the incubation continued for a further 1.5 min. The cultures were then lysed and immunoprecipitates of tyrosine-phosphorylated proteins were analyzed by anti-Tyr(P) immunoblotting. Scanning densitometry of both the M(r) 110,000-130,000 (circles) and 70,000-80,000 (triangles) bands is shown. Each point is representative of three independent experiments and is expressed as a percentage of the maximum response. For clarity, only the error bands (S.E.) for the M(r) 110,000-130,000 are shown. Inset, parallel cultures were permeabilized in the presence (lane 2) or absence (lane 3) of 250 µM GDPbetaS for 1 min prior to addition of 500 µM GTPS. Lane 1 shows the effect of 250 µM GDPbetaS alone. An autoradiograph of a representative experiment is shown.



Role of PKC and Ca on GTPS-induced Tyrosine Phosphorylation

GTPS is known to stimulate the activation of heterotrimeric G proteins and consequently PIP(2)-PLC thereby elevating diacylglycerol leading to activation of PKC in Swiss 3T3 cells(31) . It is also known that activated PKC can induce tyrosine phosphorylation of the M(r) 110,000-130,000 and 70,000-80,000 bands including the proteins p125 and paxillin(13) . Consequently, we determined whether PKC could be involved in the downstream signaling of tyrosine phosphorylation stimulated by GTPS. Addition of 200 nM PDBu to permeabilized cells weakly (2-fold) stimulated tyrosine phosphorylation of the M(r) 110,000-130,000 and 70,000-80,000 bands even after 4 min of incubation (Fig. 3A). Indeed, shorter incubations with PDBu produced an even smaller response (data not shown). Thus, tyrosine phosphorylation induced by PDBu was considerably slower and weaker than the effect of GTPS. The staurosporine-related compound, GF109203X, is a selective inhibitor of PKC in Swiss 3T3 cells at concentrations which have no effect on cAMP-dependent kinase or on platelet-derived growth factor, epidermal growth factor, and insulin receptor tyrosine kinases(45) . Fig. 3A (left) shows that a 1-h pretreatment with 3.5 µM GF109203X had no effect on tyrosine phosphorylation stimulated by GTPS in permeabilized Swiss 3T3 cells but abolished the slight effect of PDBu. This suggested that PKC did not mediate GTPS-stimulated tyrosine phosphorylation. In order to further substantiate this result, phorbol ester-sensitive PKCs were down-regulated by prolonged pretreatment with PDBu. As shown in Fig. 3A, tyrosine phosphorylation induced by GTPS was not inhibited by PDBu pretreatment. These results indicated that PKC was not involved in tyrosine phosphorylation induced by GTPS.


Figure 3: Panel A, role of PKC in GTPS-stimulated tyrosine phosphorylation. Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM and incubated in the absence or presence of 3.5 µM GF109203X for 1 h. The cells were then permeabilized with SLO in the absence or presence (GF) of 3.5 µM GF109203X for 1 min and the incubation was continued with (+) or without(-) addition of 5 µM GTPS for a further 1.5 min or 200 nM PDBu for a further 4 min. Parallel dishes were pretreated either in the absence or presence (PDBu) of 800 nM PDBu for 40 h prior to permeabilization and stimulation with (+) or without(-) 5 µM GTPS or 200 nM PDBu. The cultures were then lysed and immunoprecipitates of tyrosine-phosphorylated proteins were analyzed by anti-Tyr(P) immunoblotting. An autoradiograph of a representative experiment is shown. In parallel cultures, both pretreatment with 3.5 µM GF109203X and PDBu completely blocked tyrosine phosphorylation induced by 200 nM PDBu in intact cells (data not shown) in agreement with results published previously(13, 16, 20, 21) . Panel B, effect of Ca concentration on tyrosine phosphorylation in either resting or GTPS-stimulated permeabilized Swiss 3T3 cells. Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM and permeabilized with SLO in the presence or absence of increasing concentrations of Ca as described under ``Experimental Procedures.'' After 1 min, 5 µM GTPS was (solid bars) or was not (open bars) added for a further 1.5 min. The cultures were then lysed and immunoprecipitates of tyrosine-phosphorylated proteins were analyzed by anti-Tyr(P) immunoblotting. The bar graph shown represents scanning densitometry from autoradiographs of tyrosine-phosphorylated proteins migrating with an apparent M(r) 110,000-130,000 and is expressed as a percentage of the maximum response of three independent experiments ± S.E.



The induction of tyrosine phosphorylation in some cell systems has been shown to be dependent on Ca oscillations(5, 46) . Permeabilization provides a convenient procedure to examine directly the effects of increasing concentrations of Ca on tyrosine phosphorylation. Cells were permeabilized for 1 min in permeabilization solution which contained increasing concentrations of Ca and then incubated in the presence or absence of 5 µM GTPS for 1.5 min prior to lysis. Fig. 3B shows that increasing Ca concentrations did not induce any significant tyrosine phosphorylation in the absence of GTPS. This was true even at 6 µM Ca, a concentration known to directly activate PIP(2)-PLC in permeabilized Swiss 3T3 cells(32) . Furthermore, raising the Ca concentration up to 600 nM had no effect on the ability of GTPS to stimulate tyrosine phosphorylation of the M(r) 110,000-130,000 band in permeabilized Swiss 3T3 cells. Interestingly, at 6 µM Ca, some inhibition of GTPS-stimulated tyrosine phosphorylation was seen. Thus, increasing Ca concentrations do not directly stimulate tyrosine phosphorylation or affect GTPS-induced tyrosine phosphorylation in permeabilized Swiss 3T3 cells.

Effect of [Mg] and AlF(4) on Tyrosine Phosphorylation Induced by GTPS

GTPS stimulated activation of both heterotrimeric G proteins and small GTP binding proteins is dependent on Mg concentration. Thus millimolar concentrations of Mg are required for GTPS to bind and activate heterotrimeric G proteins(47) . In marked contrast, nanomolar concentrations of Mg are required for GTPS to bind and activate small GTP binding proteins(37, 48) . The effect of various concentrations of Mg on GTPS-stimulated tyrosine phosphorylation was tested utilizing a two-stage procedure. During the first stage cultures were permeabilized for 1 min in modified permeabilization solution containing increasing concentrations of free Mg in the presence or absence of 5 µM GTPS. Since this solution did not contain ATP, tyrosine kinase activity was blocked. In the second stage, the modified permeabilization solution was exchanged for standard permeabilization solution, which contained both ATP and Mg to allow the kinase reaction to occur. After a further 1.5 min, the cells were lysed and analyzed for protein tyrosine phosphorylation. Table 1shows that even in the absence of exogenously added Mg, GTPS stimulated tyrosine phosphorylation of the M(r) 110,000-130,000 band by 4.6-fold. Further increasing the Mg concentration did not significantly change the stimulation of tyrosine phosphorylation by GTPS, although both the basal and GTPS-stimulated tyrosine phosphorylation increased. Interestingly, 30 µM AlF(4) which has been shown to activate heterotrimeric but not small GTP binding proteins(49) , was only weakly able to stimulate tyrosine phosphorylation in permeabilized Swiss 3T3 cells (1.8 ± 0.3 fold, Table 1). These results suggested that tyrosine phosphorylation stimulated by GTPS was largely mediated by a small GTP binding protein(s).



Involvement of p21 in GTPS-stimulated Tyrosine Phosphorylation

p21, a member of the Ras small G protein superfamily, has been implicated in mitogen-induced actin stress fibers and focal adhesions(28, 50) , the distinct sites in the plasma membrane where p125 and paxillin are localized (11, 12, 14, 15) . The Clostridium botulinum exoenzyme, C3 ADP-ribosyltransferase, has been shown to ADP-ribosylate the Asn of p21 and thereby prevents its interaction with downstream targets(41, 51, 52) . To assess the role of p21 in GTPS-stimulated tyrosine phosphorylation, cultures of Swiss 3T3 cells were treated with C3 exoenzyme using a protocol that resulted in ADP-ribosylation of p21 (see ``Experimental Procedures''). Pretreatment with C3 exoenzyme markedly inhibited tyrosine phosphorylation of both the M(r) 110,000-130,000 (60% reduction) and 70,000-80,000 (67% reduction) group of bands stimulated by GTPS (Fig. 4, upper panel). Furthermore, C3 pretreatment inhibited GTPS-stimulated tyrosine phosphorylation of p125 and paxillin by 70% (Fig. 4, middle and lower panels, respectively). These results suggest that p21 predominantly mediates the effect of GTPS and consequently, that p21 lies upstream of tyrosine phosphorylation.


Figure 4: Role of p21 as a mediator of GTPS-stimulated tyrosine phosphorylation: effect of C3 exoenzyme pretreatment. Swiss 3T3 cells in 33-mm dishes were incubated in DMEM supplemented with 10% fetal bovine serum in the presence (+) or absence(-) of C3 exoenzyme for 48 h at 7.5 µg/ml as described under ``Experimental Procedures.'' The cells were then washed twice with DMEM and treated for a further 24 h with 15 µg/ml C3 exoenzyme in DMEM:Waymouth's (1:1, v/v) prior to permeabilization with SLO for 1 min and then incubation for another 1.5 min in the presence (+) or absence(-) of 5 µM GTPS as indicated. The cultures were then lysed and the lysates immunoprecipitated with anti-Tyr(P) mAb Py72 (upper), anti-p125 mAb2A7 (middle), or anti-paxillin mAb165 (lower). The immunoprecipitates were analyzed by immunoblotting with anti-Tyr(P) mAbs. An autoradiograph of a representative experiment is shown.



To obtain further evidence that p21 was involved in tyrosine phosphorylation stimulated by GTPS, we synthesized a peptide corresponding to the effector domain of p21 (p21). This approach was based on previous work demonstrating that amino acid residues within p21 are necessary for actin reorganization (53) and that an effector domain peptide of p21 (p21) blocks the interaction of p21 with p74(54) . Cells were permeabilized in the presence or absence of 20 µM p21 peptide for 1 min and then exposed to 5 µM GTPS for a further 1.5 min. Fig. 5A (top) shows that tyrosine phosphorylation of both the M(r) 110,000-130,000 and 70,000-80,000 bands induced by GTPS was completely inhibited by p21. In contrast, addition of p21 at 20 µM did not affect GTPS-stimulated tyrosine phosphorylation. Importantly, Fig. 5, top, also demonstrates that 20 µM p21 could specifically inhibit p125 (middle panel) and paxillin (lower panel) tyrosine phosphorylation induced by GTPS. However, addition of an identical concentration of p21 failed to inhibit p125 and paxillin tyrosine phosphorylation stimulated by GTPS. Both p21 and p21 at concentrations up to 20 µM did not affect tyrosine phosphorylation in the absence of GTPS. The inhibitory effect of p21 was dose dependent with an IC of 1 µM (Fig. 5, bottom). These results provide additional evidence that p21 predominantly mediates the stimulatory effect of GTPS on tyrosine phosphorylation.


Figure 5: Effect of p21 and p21effector peptides on GTPS-stimulated tyrosine phosphorylation. Upper panel, identification of tyrosine-phosphorylated proteins which are inhibited by the p21 effector peptide. Cultures of Swiss 3T3 cells in 33-mm dishes were washed twice in DMEM and permeabilized with SLO in the presence or absence of 20 µM of either the p21 (Rho) or the p21 (Ras) effector peptides for 1 min prior to incubation for a further 1.5 min in the presence or absence of 5 µM GTPS. The cells were then lysed and the lysates immunoprecipitated with anti-Tyr(P) mAb Py72 (upper), anti-p125 mAb2A7 (middle), or anti-paxillin mAb165 (lower). The immunoprecipitates were immunoblotted with anti-Tyr(P) mAbs. Autoradiographs shown are representative of at least two independent experiments. Lower panel, dose response of rhop21effector peptide on GTPS-stimulated tyrosine phosphorylation. Identical cultures were permeabilized in the presence of increasing concentrations of the p21 effector peptide (closed symbols) or 20 µM p21 effector peptide (open symbols) for 1 min prior to incubation for another 1.5 min in the presence or absence of 5 µM GTPS. The cells were then lysed and immunoprecipitates of tyrosine-phosphorylated proteins were analyzed by anti-Tyr(P) immunoblotting. Scanning densitometry of both the M(r) 110,000-130,000 (circles) and 70,000-80,000 (triangles) bands is shown. Each point is representative of two independent experiments and is expressed as a percentage of the maximum response.



We verified that the effector peptides were not inhibiting bombesin-stimulated tyrosine phosphorylation in a cell-free kinase assay. Confluent and quiescent cultures of Swiss 3T3 cells were incubated in the presence or absence of 10 nM bombesin for 10 min, a concentration and time known to induce maximum tyrosine phosphorylation(10) . The cells were then lysed and the lysates immunoprecipitated with anti-mouse IgG agarose-linked mAbs directed against Py72 for 4 h. A cell-free in vitro kinase assay was performed in the presence or absence of either 20 µM p21 or p21 peptides. Neither peptide affected basal or bombesin-stimulated phosphorylation of proteins in this kinase assay (data not shown).


DISCUSSION

We demonstrate for the first time that GTPS can rapidly stimulate tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands in permeabilized cells. The pattern of GTPS-induced tyrosine phosphorylation is similar to that recently reported for neuropeptides (2, 6, 13, 16) and LPA (20) in intact cells. Indeed, GTPS specifically induces tyrosine phosphorylation of the focal adhesion associated proteins, p125, and paxillin. Interestingly, tyrosine phosphorylation of the M(r) 70,000-80,000 paxillin band lagged behind the M(r) 110,000-130,000 band by 30 s which would be consistent with previous in vitro evidence suggesting that paxillin is a substrate of p125(55) .

GTPS-stimulated tyrosine phosphorylation could be inhibited by GDPbetaS in a dose-dependent fashion. This indicates that GTPS-stimulated tyrosine phosphorylation is mediated by a G protein. We have previously shown that GTPS can activate PIP(2)-PLC in permeabilized Swiss 3T3 cells(31, 32, 33) . PIP(2)-PLC catalyzes the hydrolysis of inositol phospholipids into diacylglycerol and inositol 1,4,5-trisphosphate which activate PKC and mobilize Ca, respectively(56) . Activation of PKC by phorbol esters or membrane permeable diacylglycerols has been shown to induce tyrosine phosphorylation of p125 and paxillin in intact Swiss 3T3 cells(13, 16) . It was therefore possible that GTPS stimulates tyrosine phosphorylation via heterotrimeric G protein mediated activation of PKC. In this respect, integrin-induced stimulation of p125 tyrosine phosphorylation appears to be mediated by PKC(26) . In contrast, our results show that GTPS stimulates tyrosine phosphorylation of both the M(r) 110,000-130,000 band and 70,000-80,000 paxillin band through a PKC-independent pathway as judged by the fact that neither down-regulation nor inhibition of PKC prevented GTPS-mediated tyrosine phosphorylation.

In liver epithelial cells, angiotensin II increases tyrosine phosphorylation of cellular components, including a M(r) 125,000 band, apparently through a Ca-dependent pathway(5) . It has also been reported that the induction of Ca oscillations are required for GPIIb-IIIa-induced tyrosine phosphorylation of a M(r) 125,000 protein in cells expressing this integrin(46) . It is likely but unproven that these M(r) 125,000 bands are related to p125. In view of these findings we tested whether Ca could stimulate tyrosine phosphorylation in permeabilized Swiss 3T3 cells. Our results show that increasing the Ca concentration in the absence of GTPS, to mimic Ca mobilization from internal stores, failed to increase tyrosine phosphorylation over background levels. Furthermore, the presence or absence of Ca had little effect on GTPS-induced tyrosine phosphorylation. These results strongly suggest that tyrosine phosphorylation occurs via a Ca independent pathway in permeabilized Swiss 3T3 cells. This is in agreement with our previous reports showing that neuropeptides, LPA, and sphingosine induce tyrosine phosphorylation via a PKC- and Ca-independent pathway in intact Swiss 3T3 cells(6, 13, 16, 17, 20, 21) .

GTPS has been shown to bind and irreversibly activate both heterotrimeric and small GTP binding proteins. It was therefore important to assess the contribution of these two classes of G proteins in mediating GTPS-induced tyrosine phosphorylation. The fact that GTPS could induce tyrosine phosphorylation almost to the same degree at nanomolar and millimolar concentrations of Mg suggests that a small GTP binding protein predominantly mediates GTPS-induced tyrosine phosphorylation(37, 47, 48) . This data is supported by the fact that AlF(4), a direct activator of heterotrimeric G proteins(49) , only weakly stimulated tyrosine phosphorylation in permeabilized Swiss 3T3 cells.

Tyrosine phosphorylation of p125 and paxillin stimulated by bombesin, LPA, and sphingosine is closely related to changes in the organization of actin microfilaments induced by these ligands in Swiss 3T3 cells. Disruption of the cytoskeleton with cytochalsin D inhibits the ability of these agents to stimulate tyrosine phosphorylation(13, 16, 20, 21) . Furthermore, both tyrosine phosphorylation and the cytoskeletal changes induced by LPA and bombesin require functional p21 protein(20, 28, 29) . Consequently, it was of interest to determine whether p21 mediated GTPS-induced tyrosine phosphorylation of p125 and paxillin in permeabilized cells.

The C. botulinum C3 exoenzyme has been shown to ADP-ribosylate the Asn of p21, a residue within the effector domain of this small G protein and thereby prevents the interaction of p21 with its downstream targets(41, 51, 52) . Pretreatment with C3 exoenzyme markedly inhibits GTPS-induced tyrosine phosphorylation of multiple substrates including p125 and paxillin. These results suggest that p21 predominantly mediates the effect of GTPS and consequently, that p21 lies upstream of tyrosine phosphorylation. Interestingly, we have recently shown that pretreatment with C3 exoenzyme also inhibits bombesin and endothelin-induced tyrosine phosphorylation in intact cells(30) .

In a separate approach to test whether p21 mediates GTPS-induced tyrosine phosphorylation we used a synthetic peptide corresponding to the effector domain of p21 (p21). This region of p21 and more recently p21 has been identified as necessary for actin reorganization(53, 57) . Furthermore, an effector domain peptide of p21 (p21) can block the ability of p21 to interact with p74in vitro(54) . We reasoned that a p21 peptide could interfere with the interaction between this small G protein and its effector(s). The peptide p21 inhibited tyrosine phosphorylation of both the M(r) 110,000-130,000 and 70,000-80,000 bands induced by GTPS in a dose-dependent fashion. In contrast, p21 at identical concentrations did not affect GTPS-stimulated tyrosine phosphorylation in permeabilized Swiss 3T3 cells. Importantly, p21 but not p21 could specifically inhibit p125 and paxillin tyrosine phosphorylation induced by GTPS. These results provide additional evidence that p21 predominantly mediates GTPS-induced tyrosine phosphorylation. The mechanism(s) linking p21 activation with tyrosine phosphorylation of p125 and paxillin warrants further investigation.

In conclusion, using permeabilized Swiss 3T3 cells we demonstrate for the first time that GTPS can induce tyrosine phosphorylation of multiple substrates including p125 and paxillin. As these effects can occur at nanomolar Mg concentrations and are blocked by either C3 exoenzyme or p21 peptide, we suggest that the small GTP binding protein p21 predominantly mediates GTPS-induced tyrosine phosphorylation.


FOOTNOTES

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

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: FAK, focal adhesion kinase; anti-Tyr(P), anti-phosphotyrosine; C3 toxin, Clostridium botulinum C3 exoenzyme; DMEM, Dulbecco's modified Eagle's medium; GDPbetaS, guanosine 5`-3-O-(thio)diphosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate; LPA, lysophosphatidic acid; mAb, monoclonal antibody; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; PIP(2)-PLC, phosphatidylinositol 4,5-bisphosphate-specific phospholipase C; SLO, streptolysin O; Py72, phosphotyrosine; PIPES, 1,4-piperazineethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. S. Rankin for her help with the C3 exoenzyme assay.


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