protein tyrosine phosphorylation induced by lysophosphatidic acid in rat

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 1, Issue of January 7,$: 645-651, 1994 nnted an U.S.A.

Protein Tyrosine Phosphorylation Induced by Lysophosphatidic Acid in Rat- 1 Fibroblasts EVIDENCE THAT PHOSPHORYLATION OF MAP KINASE IS MEDIATED BY THE Gi-p21" PATHWAY*

(Received for publication, June 7, 1993, and in revised form, August 20, 1993)

Peter L. Hordijk, Ingrid Verlaan, Emile J. van CorvenS, and Wouter H. Moolenaarg From the Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanloan 121, 1066 CX Amsterdam, The Netherlands

Lysophosphatidic acid (LPA) is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. LPA activates its own G protein-coupled receptor(s) leading to stimulation of phospholipase C and inhibition of adenylate cyclase. Furthermore, LPA rapidly activates p21'" through a pertussis toxin-sensitive pathway. In this study, we have examined LPA-induced protein tyrosine phosphorylation in Rat- 1 fibroblasts. LPA action was compared with that of endothelin, which is a stronger activator of phospholipase C than LPA but fails to activate p21'" and to stimulate DNA synthesis in these cells. LPA and, more effec- tively, endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130, 95, and 65-75 kDa. The effect of LPA is dose- and time-dependent, being half-maximal at 3-30 nM and peaking after 2-5 min. Among the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa "focal adhesion kinase" (p12SFAK) but not the 120-kDa p21'" GTPase-activating protein. Furthermore, LPA, like epidermal growth factor, causes tyrosine phosphorylation and activation of the ~ 4 2 1 ~ 4 4 mitogen-activated protein (MAP) kinases, paralleling p21" activation. In contrast, endothelin fails to phosphorylate MAP kinase. Treatment of the cells with pertussis toxin blocks LPA-induced MAP kinase phosphorylation without affecting the other tyrosine phosphorylations. The kinase inhibitor staurosporine (1 PM) blocks LPA-induced, but not epidermal growth factor-induced, activation of p21" and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21'" activation. The results support a model in which LPA-induced phosphorylation of MAP kinase is mediated by p21'", and tyrosine phosphorylation of the other substrates, including ~ 1 2 5 ~ * ~ , is associated with phospholipase C activation.

Lysophosphatidic acid (LPA' 1-acyl-sn-glycerol-3-phosphate) is a simple, water-soluble phospholipid that evokes a

* This work was supported by the Dutch Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Present address: Gene Pharming, 2333 CC Leiden, The Nether- lands.

I To whom correspondence should be addressed. Tel.: 31-20-512- 1971; Fax: 31-20-512-1989. ' The abbreviations used are: LPA, lysophosphatidic acid; PTX,

pertussis toxin; Gi, inhibitory guanine nucleotide-binding protein; EGF, epidermal growth factor; MAP, mitogen-activated protein; TPA, 12-0-tetradecanoylphorbol-13-acetate; pTyr, phosphotyrosine; GAP, p21" GTPase-activating protein.

wide range of biological responses in diverse cell types, appar- ently by activating its cognate G protein-coupled receptor(s) (for review, see Ref. 1). For example, exogenous LPA induces smooth muscle contraction, platelet aggregation, neural shape changes, Dictyostelium chemotaxis and, in fibroblasts, stimulation of DNA synthesis (1-5). LPA is rapidly produced and released by activated platelets and is present in serum at bioactive concentrations, but not in plasma, suggesting a physiological role for LPA in wound healing and inflammation (4, 6, 7). Using a photoreactive LPA analogue, van der Bend et al. (8) recently identified a candidate LPA receptor of an apparent molecular mass of 38-40 kDa in various LPA- responsive cell types, including fibroblasts.

LPA receptor activation triggers various G protein-mediated signal transduction cascades, including stimulation of phospholipases C and D and inhibition of adenylate cyclase (2, 3, 9-11). Furthermore, in common with peptide growth factors, LPA rapidly activates the ~21'"" protooncogene prod- uct in fibroblasts, as measured by the transition from its inactive, GDP-bound state to the active, GTP-bound confor- mation (12). LPA-induced p21'"".GTP accumulation is inhib- ited by PTX, indicating a regulatory role for a heterotrimeric G protein of the Gi subfamily; yet it appears that Gi-mediated p21" activation is independent of known PTX-sensitive effector routes (12). This novel Gi-p21'"" pathway is thought to play a central role in the stimulation of DNA synthesis by LPA (12).

Among the early events following activation of quiescent cells is an increase in tyrosine phosphorylation of cellular proteins. Apart from the growth factor receptor tyrosine kinases, certain G-protein coupled receptors also are capable of stimulating protein tyrosine phosphorylation in their target cells (13-18). The G protein-mediated effector routes by which these receptors stimulate tyrosine phosphorylation are poorly understood, although activation of the phospholipase C-Ca2+- protein kinase C pathway appears to be a common feature. We recently reported that LPA rapidly stimulates p60"'" tyrosine kinase activity in serum-deprived N1E-115 neuroblastoma cells, a response implicated in the LPA-induced reorganization of the actin cytoskeleton and subsequent retraction of developing neurites (4). However, to date, our efforts to detect enhanced p60"'" activity in LPA-treated fibroblasts have been unsuccessful;' in fact, virtually nothing is known about protein tyrosine phosphorylation induced by LPA in fibroblasts.

In this study, we have examined LPA-induced protein tyrosine phosphorylation in quiescent Rat-1 cells. These cells are mitogenically highly responsive not only to LPA but also to EGF (2, 3). Furthermore, we took advantage of the fact that Rat-1 cells express functional endothelin receptors,

* T. Eichholtz and P. L. Hordijk, unpublished observations.

645

646 LPA-induced Tyrosine Phosphorylation and MAP Kinase Activation

which couple to strong stimulation of phospholipid breakdown (3, 10, 11) but fail to mediate activation of p21" and mitogenesis (3, 12). We show here that LPA, like endothelin, rapidly stimulates tyrosine phosphorylation of multiple cellular proteins, among which we have identified the ~ 1 2 5 ' ~ ~ focal adhesion kinase (19). Furthermore, we show that LPA, but not endothelin, stimulates phosphorylation and activation of the ~ 4 2 1 ~ 4 4 MAP kinases; these are serinelthreonine kinases that function within a protein kinase cascade and are a central component of many different signaling pathways (20-25). Using PTX, we were able to dissociate LPA-induced MAP kinase phosphorylation from the other protein tyrosine phosphorylations. Together, our data indicate that LPA-induced MAP kinase phosphorylation is mediated by the Gi- p21" pathway with no apparent role for the phospholipase C-protein kinase C cascade, whereas tyrosine phosphorylation of the other proteins, including ~ 1 2 5 ' * ~ , correlates with phospholipase C activation.

EXPERIMENTAL PROCEDURES

Chemicals and Antibodies-Materials and reagents were obtained from the following sources: 1-oleoyl-LPA, staurosporine, and TPA from Sigma; nitrocellulose from Schleiher & Schuell; acrylamide from Serva; endothelin from Cambridge Research Biochemicals (Cam- bridge, United Kingdom); EGF from Collaborative Research (Bed- ford, MA); PTX from List Biological Laboratories Inc. (Campbell, CA); protein A-Sepharose CL-4B and protein G-Sepharose-4 beads from Pharmacia LKB Biotechnology Inc.; suramin from Bayer; pol- yethyleneimine-cellulose F plates from Merck [32P]orthophosphate, [r-"P]ATP, and the Enhanced ChemiLuminescence system from Amersham Corp.

Anti-phosphotyrosine monoclonal antibody PY20 was from ICN and peroxidase-labeled antibodies from DAKO (Glostrup, Denmark). Normal mouse serum and polyclonal rabbit anti-mouse antibodies were obtained from the Central Laboratory for Blood transfusion (Amsterdam, The Netherlands). Rabbit polyclonal antibodies against p42/p44 MAP kinase and pl20-GAP (produced as fusion proteins in Escherichia coli) and rat monoclonal antibody Y13-259 against p21m were kindly provided by Drs. J. L. Bos and B. Burgering (Utrecht University) and anti-p12fiFAK monoclonal antibody (2A7) by Dr. T. Parsons (University of Virginia at Charlottesville). Polyclonal anti- MAP kinase serum 122 was a generous gift from Dr. c. J. Marshall (Institute of Cancer Research, London).

Cell Culture--Rat-1 cells were grown in Dulbecco's modified Ea- gle's medium supplemented with 7.5% fetal calf serum and were split in a 1: lO ratio at 3-4-day intervals. For experiments, cells in plastic petri dishes or six-well culture plates were grown to 80-90% conflu- ence and then serum-starved for 24 h.

32P Labeling and Immunoprecipitation-Three hours prior to the assay, the medium was replaced by phosphate-free Eagle's modified essential medium (ICN) containing 200 pCi/ml [32P]orthophosphate. Agonists were directly added to the test medium, and, after variable periods of time, the cells were quickly washed with ice-coldphosphate- buffered saline containing 1 mM EDTA, 10 mM sodium fluoride, 0.5 mM sodium vanadate, and 10 mM tetrasodium pyrophosphate. Cells were subsequently scraped in 0.5 ml of ice-cold lysis buffer (10 mM Tris-HC1, pH 7.4,150 mM NaC1, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 10 mM sodium fluoride, 1 mM EDTA, 0.5 mM orthovanadate, 10 mM tetrasodium pyrophosphate, 0.06 trypsin inhibitory units of aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 20 p~ leupeptin). The lysate was centrifuged for 10 min at 14,000 X g and precleared by incubation for 45 min with 50 pl of a 10% solution in lysis buffer of formaline-fixed, heat-killed Staphylococcus aureus Cowan I strain bacteria, coated with rabbit anti-mouse antibodies and normal mouse serum. After centrifugation (10 s, 14,000 X g) and two additional 10-min incubations with S. aureus, the lysates were normalized by trichloroacetic precipitation and counting of aliquots. The lysates were incubated with anti-pTyr antibody (2 h, 4 "C), followed by rabbit anti-mouse-coated S. aureus (45 min, 4 "C). The immunoprecipitates were washed five times with 50 mM Tris-HC1, pH 6.8, 150 mM NaC1, 0.5% Nonidet P-40, 0.5 mM MgCl,, and 0.5 mM sodium orthovanadate. Protein complexes were eluted by boiling in SDS-sample buffer and were analyzed after centrifugation (10 min, 14,000 X g) by SDS-polyacrylamide gel electrophoresis (5-15% gradient). Gels were dried and exposed for 18-120 h a t -70 "C using

Kodak X-AR films with intensifying screens. For immunoprecipitation of PISOGAP, cells grown in IO-cm dishes

were lysed in the buffer used for anti-pTyr immunoprecipitations. Protein content was determined by the method of Lowry et al. (26). The lysates were precleared (30 min, 4 "C) with protein A-Sepharose coated with normal rabbit serum. After incubation with polyclonal anti-GAP serum (2 h, 4 "C) GAP-antibody complexes were captured with protein A-Sepharose (1 h, 4 T ) . Sepharose beads were washed three times with lysis buffer and boiled for 5 min in SDS-sample buffer, and eluted proteins were separated on a 10% polyacrylamide gel and blotted onto nitrocellulose. For immunoprecipitation of ~ 1 2 5 ~ ~ , cells grown in 10-cm dishes were lysed in a buffer containing 50 mM Tris-HC1, pH 7.4, 150 mM NaCl, 2 mM EGTA, 1% Nonidet P-40,0.25% sodium deoxycholate, and the phosphatase and protease inhibitors mentioned above. The lysates were precleared (30 min, 4 "C) with protein A-Sepharose coated with rabbit anti-mouse antibodies and normal mouse serum. Immunoprecipitation was carried out using a n t i - ~ l 2 5 ~ ~ monoclonal antibody 2A7 (2 h, 4 "C) (27) and protein A-Sepharose coated with rabbit anti-mouse antibodies (45 min, 4 "C). The precipitates were washed three times with lysis buffer and analyzed by 10% polyacrylamide gel electrophoresis and blotting.

Zmmunoblotting-For immunoblot analysis of tyrosine-pbos- phorylated proteins or the mobility shift of MAP kinase, cells were stimulated, lysed immediately in SDS-sample buffer, and separated on a 5-15% polyacrylamide gradient or 10% polyacrylamide gel, respectively. Blotting to nitrocellulose was performed at 0.5 A for at least 1 h in 25 mM Tris, 200 mM glycine, and 20% (v/v) methanol. Aspecific binding of antibodies was prevented by incubating the blots in 5% dried milk powder (anti-MAP kinase immunoblots) or 4% bovine serum albumin/l% ovalbumin in TBST (10 mM Tris, pH 8.0, 150 mM NaC1, 0.05% Tween-20; also used for all incubations and washing steps) for 1 h at 37 "C. Next, the blots were incubated (1 h, room temperature) with the anti-pTyr antibody PY20 (1 pg/ml), anti- p42 MAP kinase antiserum (diluted 1:10,000), or the anti-GAP antiserum (diluted 1:lOOO) followed by extensive washing. The blots were subsequently incubated (45 min, room temperature) with peroxidase- labeled rabbit anti-mouse or swine anti-rabbit antibodies (both diluted 1:7500). After washing, immunostained proteins were visualized using the Enhanced ChemiLuminescence detection system according to the instructions of the supplier (Amersham). Immunoblots were reprobed after incubation (30 min, 50 "C) in 67 mM Tris-HCL, pH 6.7, 2% SDS, 100 mM j3-mercaptoethanol. All results shown are representative of at least three independent experiments.

MAP Kinase Activity-MAP kinase activity was analyzed by an immunocomplex kinase assay, essentially as described previously (28). Quiescent cells were stimulated for 5 min, washed with ice-cold phosphate-buffered saline, and lysed in 0.5 ml of lysis buffer (50 mM Tris-HCL, pH 7.5, 1% Triton X-100, 100 mM NaC1, 10 mM sodium fluoride, 5 mM EDTA, 500 p~ sodium orthovanadate, 0.06 trypsin inhibitory units of aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 20 p~ leupeptin). Insoluble material was removed by centrifugation (10 min, 14,000 X g), and protein content was measured (26). The cell lysates were precleared by incubation with protein A-Seph- arose beads coated with normal rabbit serum (1 h, 4 "C), and MAP kinases were immunoprecipitated with protein A-Sepharose beads coated with the anti-MAP kinase antiserum 122 (29) (2 h, 4 "C). The beads were washed four times with lysis buffer, and immunoprecipitated kinase activity toward myelin basic protein was assayed as described (28). The data are expressed as pmol of phosphate incor- porated into the substrate/min/mg of extract protein and are representative of three independent experiments performed in duplicate.

Activation ofp2l""Activation of pHrn was assayed essentially as described previously (12,30). In brief, confluent, serum-deprived cells in six-well tissue culture plates were prelabeled with [3zPjorthoph~~- phate (200 pCi/well) for 4 h. Cells were stimulated and lysed in a 1% Triton X-114 buffer, and p21" was immunoprecipitated using monoclonal antibody Y 13-259 and protein G-Sepharose. Bound nucleotides were separated by thin-layer chromatography using polyethylene- imine-cellulose plates followed by autoradiography.

RESULTS

Protein Tyrosine Phosphorylation Induced by LPA, Endo- thelin, and EGF-Addition of 1-oleoyl-LPA (1 pM) to serum- deprived Rat-1 fibroblasts causes a rapid increase in tyrosine phosphorylation of multiple cellular proteins as detected by immunoprecipitation of 32P-labeled proteins using anti-pTyr antibody PY20 (Fig. 1, left panel). The LPA-induced pattern

LPA-induced Tyrosine Phosphorylation and MAP Kinase Activation

-45- c c

FIG. 1. Stimulation of protein tyrosine phosphorylation in Rat-1 cells. Left panel, Rat-1 cells were labeled with [32P]orthophosphate for 3 h and then stimulated for 5 min with vehicle (C, control), 1-oleoyl-LPA (1 p ~ ) , endothelin (ET; 50 nM), or EGF (50 ng/ml). pTyr-containing proteins were immunoprecipitated from cell lysates using anti-pTyr antibody PY20, and the samples were analyzed by SDS-gel electrophoresis (5-15% gradient gel), as detailed under “Experimental Procedures.” Arrows indicate proteins with increased pTyr content in response to LPA or endothelin. Right panel, cells were stimulated with LPA (1 p ~ ) , endothelin (ET; 50 nM), or EGF (50 ng/ml) for 5 min, and cell lysates were directly analyzed by anti-pTyr immunoblotting using antibody PY20 as described under “Experimental Procedures.” Arrows indicate proteins with increased pTyr content. Stimulation with EGF results in the phosphorylation of a different set of proteins, which includes the autophosphorylated EGF receptor (170-180 kDa; asterisk). Note ap- pearance of additional pTyr-containing proteins of 42/44 kDa in response to LPA and EGF but not endothelin (rightpanel). Molecular mass markers are in kDa. Exposure times: left panel, 120 h; right panel, 10 min.

of protein phosphorylation was compared with that obtained with endothelin or EGF. Endothelin acts through its own G protein-coupled receptor and is a more powerful activator of phospholipases C and D than LPA, without being mitogenic for Rat-1 cells (3, 10, 11). EGF is highly mitogenic without stimulating phospholipid breakdown in these cells (2,3). Fig. 1 (left panel) shows that both LPA and endothelin promote tyrosine phosphorylation of proteins of apparent molecular mass 110-130, 95, and 65-75 kDa. In some experiments, an additional increase in tyrosine phosphorylation of a protein doublet of 210-220 kDa was observed (for example, see Figs. 2A and 3A). We note that, at maximally effective doses, the response to endothelin (50 nM) was consistently of greater magnitude than that evoked by LPA (1 p ~ ) ; it thus appears that there is a positive correlation between the degree of tyrosine phosphorylation and phospholipase activation. The protein phosphorylation pattern induced by EGF is clearly different from that induced by LPA or endothelin (Fig. 1). Here, the major substrates represent the EGF receptor itself (170-180 kDa), together with a set of proteins in the 40-130- kDa range.

LPA-induced protein tyrosine phosphorylation was as- sessed independently of metabolic labeling by anti-pTyr immunoblotting of total cell lysates (Fig. 1, right panel). While the majority of the bands comigrated with those detected in 32P-labeled immunoprecipitates, two additional phosphotyrosyl proteins of 42 and 44 kDa were detected in immunoblots obtained from both LPA- and EGF-treated cells. The p42 and p44 proteins were identified as MAP kinases, as will be demonstrated below. The fact that they were poorly, if at all, precipitated by anti-pTyr antibodies (Fig. 1, left panel) is consistent with previous findings (31). Of note, the ~ 4 2 1 ~ 4 4 proteins were not detectably tyrosine-phosphorylated in response to endothelin (Fig. 1, right panel).

Dose Dependence and Kinetics of LPA-induced Tyrosine

200-

116-

97-

66-

B increase

(9i of maximum)

- 9 8 1 6

647

-log [LPAI (M)

FIG. 2. Dose-response relationship of LPA-induced tyrosine phosphorylation. A , Rat-1 cells were labeled with [32P]orthophosphate for 3 h as in Fig. 1 and then incubated with increasing concentrations of 1-oleoyl-LPA as indicated. Tyrosine-phosphorylated proteins were precipitated using antibody PY20 and analyzed by SDS- gel electrophoresis (5-15% gradient gel) as described under “Experi- mental Procedures.” Molecular mass markers are in kDa; pTyr- containing proteins of 220, 130, 120, 110, 95, and 66-75 kDa are indicated with arrows. Note the presence of a 220-kDa protein band not seen in the experiment of Fig. 1. Exposure time, 120 h. B, the three major pTyr-containing protein bands on the autoradiogram (termed p120, p95, and p66-75) were scanned by laser densitometry, and the relative intensities are presented.

Phosphorylations-To determine the dose-response relationship for LPA-induced tyrosine phosphorylation, 32P-labeled cells were stimulated with varying concentrations of LPA. The pTyr-containing proteins were immunoprecipitated and the intensity of the 110-130-, 95-, and 65-75-kDa bands quantitated by laser densitometry. As shown in Fig. 2, A and B, the threshold dose of LPA is around 1 nM, the half- maximally effective concentration falls within the 3-30 range (depending on the protein band examined), and maximum effects are observed a t -100 nM. This dose-response curve is very similar to that for other LPA-induced early events, such as Ca2+ mobilization, p21’” activation, and neurite retraction (4,9,12); moreover, it overlaps well with the dose dependence determined in competitive LPA receptor binding assays (8). To confirm that the effects of LPA were receptor-mediated, we used suramin, a polyanionic drug that inhibits the binding of LPA to its putative receptor and reversibly blocks the biochemical and biological activities of LPA (3, 4,8, 12). We found that suramin (0.5 mg/ml) completely inhibits LPA- induced tyrosine phosphorylations in Rat-1 cells (not shown).

Kinetic analysis of protein tyrosine phosphorylation in response to LPA (1 p ~ ) shows that LPA action is rapid and transient (Fig. 3, A and B ) ; increased phosphate content in anti-pTyr immunoprecipitates is already detectable within 30 s, and effects are maximal after 2-5 min and then gradually

648 LPA-induced Tyrosine Phosphorylation and MAP Kinase Activation

A Tirne(rnin) 0' 0.5' I' 2' 5' 10' 20'

200-

116- 91 - 66-

B fold increase

"'" I I

A C LPA EGF a-pTyr

200- c-

c

p66-75

0 5 I0 15 20

Time (min)

FIG. 3. Kinetics of LPA-induced tyrosine phosphorylation. A, [32P]orthophosphate-labeled cells were stimulated with LPA (1 p M ) for the indicated periods of time, and pTyr-containing proteins were immunoprecipitated as described under "Experimental Proce- dures." Molecular mass markers are in kDa. Arrows indicate pTyr- containingproteins of 220,130,120,110,95, and 66-75 kDa. Exposure time, 120 h. B, the three major pTyr-containing protein bands on the autoradiogram (p120, p95, and p66-75; cf. Fig. 2) were scanned by laser densitometry, and the relative intensities are presented.

decline over the next 10-20 min. Analysis of the 110-130-kDa Phosphotyrosyl Proteins and

Identification of p125FAK-A major group of phosphotyrosyl proteins in LPA- and endothelin-stimulated Rat-1 cells clus- ters at 110-130 kDa (Fig. 1). Given the stimulatory effect of LPA on p21ra" (12), we examined whether these proteins comprise the 120-kDa GAP (32). In some cell systems, tyrosine phosphorylation of GAP accompanies growth factor- induced p21"" activation (33-35). GAP was immunoprecipitated from LPA- or EGF-stimulated cells and assayed for pTyr content by immunoblotting. As illustrated in Fig. 4A, neither LPA nor EGF induced detectable tyrosine phosphorylation of GAP. EGF did, however, promote a dramatic increase in tyrosine phosphorylation of a 60-65-kDa protein that coprecipitated with GAP and presumably represents the GAP-associated protein p62 (36).

Another candidate phosphotyrosyl protein in the 110-130- kDa range is the focal adhesion kinase, p125FAK, a newly identified tyrosine kinase (19) that becomes rapidly phosphorylated on tyrosine after integrin-mediated cell spreading and adhesion (37-39) and also in neuropeptide-treated Swiss 3T3 cells (40). To test whether ~ 1 2 5 ~ ~ ~ is a constituent of the 110-130-kDa protein band, the kinase was immunoprecipitated from cell lysates using monoclonal antibody 2A7 (27), followed by anti-pTyr immunoblotting. Fig. 4B shows that ~ 1 2 5 ~ ~ ~ is tyrosine-phosphorylated to a relatively high level in unstimulated cells and that both LPA and endothelin induce a small but significant increase in its pTyr content. Again, endothelin was consistently more powerful than LPA in promoting tyrosine phosphorylation of ~ 1 2 5 ~ ~ ~ . There was no significant effect of EGF on ~ 1 2 5 ~ ~ ~ phosphorylation (Fig. 4B and results not shown).

Phosphorylation and Activation of MAP Kinase-The anti- pTyr immunoblotting experiments reveal that LPA and EGF, but not endothelin, induce tyrosine phosphorylation of two

116- 97-

66-

C LPA IiCF a-GAP

B c LPA ET EGF

p125 FAK+ - FIG. 4. Analysis of pl20GAP and ~ 1 2 5 ' ~ ~ phosphorylation.

A, cells were stimulated for 5 min with vehicle (C), LPA (1 pM), or EGF (50 ng/ml). p12OGAP was precipitated using polyclonal anti- GAP antiserum and analyzed by immunoblotting using anti-pTyr antibody; the blots were reprobed using anti-pl2OGAP anti-antiserum to assess proper precipitation of GAP, as indicated in the lower part of A. Markers are in kDa. The Zg arrow indicates heavy chain of precipitating antibody. B, cells were stimulated for 5 min with LPA (1 p ~ ) , endothelin (ET; 50 nM), or EGF (50 ng/ml). ~ 1 2 5 ~ ~ ~ was immunoprecipitated using monoclonal antibody 2A7 and analyzed for pTyr content by anti-pTyr immunoblotting. Equal amounts of ~ 1 2 5 ~ ~ ~ were precipitated, as evidenced by reprobing the blot with monoclonal 2A7 (not shown).

proteins of 42 and 44 kDa (Fig. l), which are likely to represent the ~ 4 2 1 ~ 4 4 MAP kinases. These are activated through phosphorylation on Tyr and Thr residues in response to various external signals including peptide growth factors, phorbol ester, and G protein-coupled receptor agonists (20- 25, 41-43). This activation is readily detectable on anti-MAP kinase immunoblots, where the activated, phosphorylated forms of ~ 4 2 1 ~ 4 4 display a lower electrophoretic mobility (29, 44). Fig. 5 (upper panel) shows that both LPA and EGF induce a rapid shift in mobility of the p42/p44 MAP kinases. In contrast, endothelin fails to induce a detectable mobility shift, a result in line with the anti-pTyr immunoblotting data (Fig. 1, right panel). The protein kinase C-activating phorbol ester TPA (100 ng/ml) induced a small degree of MAP k' mase activation; however, for unknown reasons, TPA-induced MAP kinase phosphorylation in Rat-1 cells was highly variable between different experiments (not shown). It thus appears that although TPA-activated protein kinase C may stimulate MAP kinase phosphorylation to some extent, receptor-mediated phospholipase C-protein kinase C activation is not the route by which LPA acts. This conclusion is supported by further experiments described below.

To confirm that phosphorylation of the MAP kinases is accompanied by increased kinase activity, we performed immunocomplex kinase assays using myelin basic protein as a substrate (28). Stimulation of Rat-1 cells with either LPA or EGF results in enhanced phosphorylation of myelin basic protein in MAP kinase immunoprecipitates (Fig. 5, lower panel), thus showing that MAP kinase phosphorylation parallels increased enzymatic activity.

Kinetic analysis shows that the increase in LPA-induced MAP kinase phosphorylation is detectable after 1 min, reaches a maximum after 5 min, and thereafter declines to prestimulation levels within 10-20 min (Fig. 6, upper panel). In contrast, the EGF-induced mobility shifts are maintained

LPA-induced Tyrosine Phosphorylation and MAP Kinase Activation 649

C LPA ET EGF A C LPA m - + - + "

200- -".I y ? ?

control IJ'A 1 5 1 :

FIG. 5. Phosphorylation and activation of MAP kinase. Up- per panel, Rat-1 cells were stimulated for 5 min with vehicle (C), LPA (1 p ~ ) , endothelin (ET; 50 nM), or EGF (50 ng/ml). Cell lysates were analyzed by SDS-gel electrophoresis followed by immunoblotting with anti-MAP kinase polyclonal antiserum, as described under "Experimental Procedures." The phosphorylated, activated forms of p42/p44 MAP kinases (pp42/pp44) display reduced electrophoretic mobility. Lower panel, immunocomplex kinase assay using myelin basic protein as a substrate. Cells were stimulated for 5 min with vehicle (control), LPA (1 pM), or EGF (50 ng/ml), followed by immunoprecipitation of MAP kinases and an in vitro kinase assay in the presence of myelin basic protein (0.25 mg/ml) as described under "Experimental Procedures." The data represent mean f S.E.

LPA 0.5' I' 2' 5' 1 0 ' 20'

p 4 4 J "4 +pp44

o n "" +Pp42

FIG. 6. Kinetics of LPA- and EGF-induced phosphorylation of MAP kinase. Cells were stimulated with LPA (1 p ~ ; upperpanel) or EGF (50 ng/ml; lower panel) for the indicated periods of time, lysed, and analyzed by immunoblotting for p42/p44 mobility shifts using anti-MAP kinase antiserum (cf. Fig. 5, upper panel).

for up to 30-60 min (Fig. 6, lower panel). We note that the time course from onset to maximum mobility shifts slightly lags behind that observed for ~ 2 1 " ~ activation, which peaks at 2 min after addition of LPA or EGF (12).

Differential Effects of PTX-Treatment of Rat-1 cells with PTX blocks LPA-induced mitogenesis and p21".GTP accumulation but has no effect on LPA-induced phospholipid hydrolysis (2, 11, 12). We examined the effects of PTX on LPA-induced protein tyrosine phosphorylations and MAP kinase activation. Treatment of the cells with 100 ng/ml PTX for 18 h has no effect on LPA-induced tyrosine phosphorylation of the 110-130-, 9 5 , and 65-75-kDa proteins (Fig. 7A); neither did PTX treatment affect tyrosine phosphorylation of ~ 1 2 5 ~ ~ ~ in response to LPA or endothelin (not shown). In contrast, PTX pretreatment completely inhibits tyrosine phosphorylation as well as the mobility shift of p42/p44 MAP kinase (Fig. 7B, upper and lower panels). As expected, the EGF-induced mobility shift of MAP kinase was not affected by PTX (not shown).

Staurosporine Inhibits LPA-induced, but Not EGF-induced, Activation of p21" and MAP Kinase-The alkaloid stauro-

C LPA "

P T X - + - 3 p44+ p42+ - " "

C LPA P I X - + - + "

FIG. 7. Effects of PTX. A , cells were incubated in serum-free medium with (+) or without (-) 100 ng/ml PTX for 18 h. They were then labeled with [32P]orthophosphate for 3 h and stimulated for 5 min with vehicle (C) or LPA (1 p M ) , followed by immunoprecipitation of pTyr-containing proteins using anti-pTyr antibody and analysis by SDS-gel electrophoresis (5-15% gradient gel). The arrows indicate proteins of 130,120,110,95, and 66-75 kDa. Exposure time, 72 h. B, cells were pretreated with PTX as in A , and phosphorylation of p42/ p44 MAP kinase was monitored in immunoblots using anti-pTyr antibody (upper panel) or anti-MAP kinase antiserum (lower panel), as in Figs. 5 and 6. Exposure time, 15 min.

sporine is a potent inhibitor of several serinelthreonine and tyrosine-specific protein kinases (45-47). Addition of staurosporine (1 p ~ ) to Rat-1 cells inhibits LPA-induced MAP kinase phosphorylation (Fig. 8A). This inhibition is selective in that EGF-induced MAP kinase phosphorylation is not affected by the drug (Fig. 8A). We established that staurosporine (1 p ~ ) has no effect on LPA-induced phospholipase C activation, as inferred from Ca2+ mobilization measurements? These results suggest that staurosporine acts upstream of Gi- mediated ~ 2 1 " ~ activation. To test this hypothesis, we monitored LPA- and EGF-induced p2lraS.GTP accumulation in the presence or absence of staurosporine. Fig. 8B shows that, indeed, staurosporine (1 PM) completely inhibits LPA-induced p21"" activation but leaves the response to EG.F unaltered.

These results reveal a previously unrecognized effect of staurosporine and extend our previous studies showing that LPA-induced, but not EGF-induced, p21"" activation is in- hibited by the tyrosine kinase inhibitor genistein (12, 48). However, genistein requires much higher doses than staurosporine for a full inhibitory effect (50 pM uers'sus 1 pM, respectively) (12). Taken together, these pharmacological data hint at the presence of an as yet unidentified protein tyrosine kinase that links Gi to ~ 2 1 " ~ activation.

DISCUSSION

Although LPA has long been known as a critical precursor in the early steps of glycerolipid biosynthesis, its function as a platelet-derived "first messenger" has been recognized only recently (1,6). LPA, at nanomolar doses, binds to, and thereby

K. Jalink, unpublished observations.

650 LPA-induced Tyrosine Phosphorylation and M A P Kinase Actiuation A +staumsporine

C LPAEGF C LPAEGF

B

GTP 1 4 0 b o C LPA EGF C LPA EGF

i slaumrpnne

FIG. 8. Effects of staurosporine on MAP kinase phosphorylation and p21" activation. A , phosphorylation of MAP kinase. Cells were treated for 10 min with either vehicle or staurosporine (1 PM) and then stimulated for 5 min with vehicle (C, control), LPA (1 PM), or EGF (50 ng/ml). Cells were lysed and analyzed for MAP kinase mobility shifts in immunoblots, as in Figs. 5 and 6. Exposure time, 10 min. B, activation of p21". Cells were metabolically labeled with [32P]orthophosphate for 4 h, pretreated for 10 min with either vehicle or 1 PM staurosporine, and then stimulated for 2 min with LPA (1 PM) or EGF (50 ng/ml). The guanine nucleotides bound to p21" were separated by thin-layer chromatography followed by autoradiography as described under "Experimental Procedures." Assays were performed in duplicate as shown. Exposure time, 72 h.

EGF LPA Endothelin J J

4 p r w i n tyrosine phosphorylation

J. MAP-kinase

FIG. 9. Proposed model of early signaling pathways triggered by LPA. In this model, LPA activates its cognate G protein- coupled receptor(s) to trigger three independent effector routes as follows: (i) stimulation of phospholipase C via a PTX-insensitive G protein (putative GJ; (ii) inhibition of adenylate cyclase through PTX-sensitive Gi; and (iii) activation of a Gi-mediated p21" pathway via a putative staurosporine- and genistein-sensitive protein tyrosine kinase. It is not known whether inhibition of adenylate cyclase and p21" activation are mediated by the same Gi protein. Endothelin activates the phospholipase C pathway in Rat-1 cells but does not phosphorylate MAP kinase, whereas EGF does not detectably stimulate phospholipase C activity in these cells. The Gi-p2IrM pathway leads to MAP kinase activation, and the major protein tyrosine phosphorylations are proposed to be linked to phospholipase C activation; it remains possible, however, that enhanced protein tyrosine phosphorylation and phospholipase C activation represent parallel rather than sequential events. For further details, see text. RTK, receptor tyrosine kinase; AC, adenylate cyclase; PLC, phospholipase C.

activates, its cognate G protein-coupled receptor(s) on various target cells (8), but the receptor-mediated signal transduction pathways are not fully understood. Previous studies have shown that LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase (1). Moreover, in fibroblasts, LPA rapidly activates ~21'"" via a novel, PTX- sensitive effector route (12). The latter pathway, rather than phospholipase C activation or adenylate cyclase inhibition, is thought to be critical for LPA-induced mitogenesis (12,49).

In the present study, we have examined LPA-induced protein tyrosine phosphorylation in Rat-1 cells. We have compared the action of LPA with that of endothelin, which activates its own G protein-coupled receptor to trigger phospholipid breakdown but, unlike LPA, does not activate p21'" and is not mitogenic by itself (12,49). Two major novel results are reported here. First, LPA, at 1-100 nM, rapidly stimulates tyrosine phosphorylation of multiple cellular proteins; among these, we have identified the ~ 1 2 5 ~ ~ ~ tyrosine kinase and the ~42144 MAP kinases. Second, by using PTX, we have been able to dissociate LPA-induced MAP kinase phosphorylation from the other tyrosine phosphorylations.

We find that the overall pattern of LPA-induced tyrosine phosphorylations in the 60-220-kDa range is unaltered by PTX treatment and qualitatively similar to that induced by endothelin but different from that induced by EGF. Both LPA and endothelin stimulate phospholipid breakdown in Rat-1 cells, with LPA being a weaker inducer than endothelin (3, 10, 11). Similarly, the overall tyrosine phosphorylation response to LPA is less prominent than that to endothelin. Thus, there is a positive correlation between the degree of receptor-mediated phospholipase C activation and the observed protein tyrosine phosphorylations. A close relationship between these two early events is supported by the fact that other agonists coupling to phospholipase C activation, such as bombesin, vasopressin, angiotensin, and thrombin, also evoke tyrosine phosphorylations in their target cells (14-18). The biochemical steps by which these agonists promote tyrosine phosphorylation remain to be explored, although both protein kinase C activation and a rise in cytosolic-free Ca2+ have been implicated, depending on cell types and agonists (14, 17, 18, 50).

We have identified the ~ 1 2 5 ~ ~ ~ tyrosine kinase as a constituent of the 110-130-kDa phosphotyrosyl proteins induced by LPA and endothelin. The ~ 1 2 5 ~ ~ ~ kinase is predominantly localized in focal adhesions of normal fibroblasts (19) and is rapidly tyrosine-phosphorylated upon cell-extracellular ma- trix interaction or integrin clustering (37-39) and, furthermore, in Swiss 3T3 cells treated with neuropeptides. Although the precise physiological function of ~ 1 2 5 ~ ~ ~ and its mode of regulation by extracellular stimuli are unknown, an appealing possibility is that ~ 1 2 5 ~ ~ ~ participates in the rapid formation of focal adhesions and actin stress fibers as observed in LPA- treated fibroblasts (51).3 In this respect, it is noteworthy that in neuroblastoma cells, LPA-induced changes in the actin cytoskeleton are accompanied by increased p60""" tyrosine kinase activity (4) and that ~ 1 2 5 ~ ~ ~ is a potential substrate for activated p60"" (19, 27, 37). Whether ~60""" activity is similarly enhanced in LPA-treated Rat-1 cells remains to be determined.

A striking difference between the action of LPA and endothelin is that LPA phosphorylates and activates the p42/ p44 MAP kinases, whereas endothelin fails to do so. MAP kinase is activated through an upstream MAP kinase kinase, which is thought to serve as a convergence point of diverse signaling pathways, including the phospholipase C-protein kinase C cascade and the receptor tyrosine kinase-p2lrn"pathway (25). The failure of endothelin to stimulate MAP kinase phosphorylation, despite its potent coupling to the phospholipase C second messenger cascade, is therefore somewhat unexpected and most likely represents a cell-type specific effect; in rat mesangial cells, for example, endothelin does activate MAP kinase via a PTX-insensitive pathway partially dependent on protein kinase C (42). Furthermore, another phospholipase C-activating agonist, bombesin, stimulates MAP kinase activity in 3T3 cells via a pathway that is almost entirely dependent on protein kinase C (43). Taken together, these findings support the view that the relative contributions

LPA-induced Tyrosine Phosphorylation and MAP Kinase Activation 651

of the signaling pathways from receptor to MAP kinase may be very different in different cell types. Whatever the precise biochemical explanation, the failure of endothelin to activate MAP kinase in Rat-1 cells provides a molecular basis for its lack of mitogenicity in these cells.

Several studies have established a cause-effect relationship between activation of p21"" and activation of the MAP kinases (29, 44, 52, 53). Our findings are in line with these reports and indicate that LPA stimulates MAP kinase phosphorylation solely via p21", with no significant contribution of the phospholipase C-protein kinase C pathway. First, agents that block LPA-inducedp21ra8 activation, i.e. PTX and staurosporine, also inhibit MAP kinase phosphorylation, whereas these agents leave the responses to EGF unaltered. Second, comparison between the activation kinetics of p21'" (12) and those of MAP kinase (Fig. 6) reveals that p21" activation parallels but precedes MAP kinase phosphorylation. Third, a phospholipase C-activating agonist that fails to activate p21'"", notably endothelin, is incapable of stimulating MAP kinase phosphorylation. Finally, down-regulation of functional protein kinase C by chronic treatment of the cells with TPA (2) does not attenuate the action of LPA on MAP kinase.'

The present data, along with previous findings, lead us to propose a scheme of the G protein-mediated signal transduction pathways triggered by LPA in fibroblasts (Fig. 9). In this scheme, the signaling routes that LPA shares with endothelin and EGF are also depicted (Fig. 9). Binding of LPA to its G protein-coupled receptor(s) results in activation of at least three independent signaling cascades as follows: (i) stimulation of phospholipase C via a PTX-insensitive G protein (putative G,; 54-56); (ii) PTX-sensitive inhibition of adenylate cyclase; and (iii) activation of p21" via a PTX-sensitive Gi protein (not necessarily the same that couples to inhibition of adenylate cyclase). MAP kinase functions then downstream of p21'", while the major tyrosine phosphorylations (66-75, 95,110-130 kDa; ~ 1 2 5 ~ ~ ~ ) are proposed to be associated with phospholipase C activation. The inhibitory effects observed with staurosporine (Fig. 8) and genistein (12) on LPA-induced p21'"" activation suggest that an intermediate protein tyrosine kinase lies on the route between Gi and p21'". Identification of this putative tyrosine kinase is an obvious challenge for future studies.

While this scheme is the simplest one that is compatible with the available data, it may be too simplistic. In particular, our findings leave open the possibility that phospholipase C activation and increased protein tyrosine phosphorylation actually represent parallel pathways, with phospholipase C and tyrosine kinase (and/or phosphatase) activity being reg- ulated by distinct G protein subunits. A critical test of this hypothesis must await further experiments.

Acknowledgments-We thank Drs. T. Parsons (University of Vir- ginia at Charlottesville), J. L. Bos and B. Burgering (Utrecht Univer- sity), and C. J. Marshall (Institute of Cancer Research, London) for antibodies; K. Jalink for Ca2+ measurements; and D. Schaap for discussions.

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