THE JOURNL OF IOLOGIL HEMISTRY 0 1994 by The merian Soiety for iohemistry and Moleular iology In. Vol. 269 No. 1 Issue of January 7$: 645-651 1994 nnted an U.S.. Protein Tyrosine Phosphorylation Indued by Lysophosphatidi id in Rat- 1 Fibroblasts EVIDENE THT PHOSPHORYLTION OF MP KINSE IS MEDITED Y THE Gi-p21 PTHWY* (Reeived for publiation June 7 1993 and in revised form ugust 20 1993) Peter L. Hordijk Ingrid Verlaan Emile J. van orvens and Wouter H. Moolenaarg From the Division of ellular iohemistry The Netherlands aner Institute Plesmanloan 121 1066 X msterdam The Netherlands Lysophosphatidi aid () is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. ativates its own G protein-oupled reeptor(s) leading to stimulation of phospholipase and inhibition of adenylate ylase. Furthermore rapidly ativates p21' through a pertussis toxin-sensitive pathway. In this study we have examined -indued protein tyrosine phosphorylation in Rat- 1 fibroblasts. ation was ompared with that of endothelin whih is a stronger ativator of phospholipase than but fails to ativate p21' and to stimulate DN synthesis in these ells. and more effetively endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130 95 and 65-75 kda. The effet of is dose- and time-dependent being half-maximal at 3-30 nm and peaking after 2-5 min. mong the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa foal adhesion kinase (p12sfk) but not the 120-kDa p21' GTPase-ativat- (2 3 9-11). Furthermore in ommon with peptide growth fators rapidly ativates the ~21' protoonogene proding protein. Furthermore like epidermal growth ut in fibroblasts as measured by the transition from its fator auses tyrosine phosphorylation and ativation inative GDP-bound state to the ative GTP-bound onforof the ~421~44 mitogen-ativated protein (MP) mation (12). -indued p21'.gtp aumulation is inhibkinases paralleling p21 ativation. In ontrast en- ited by PTX indiating a regulatory role for a heterotrimeri dothelin fails to phosphorylate MP kinase. Treatment G protein of the Gi subfamily; yet it appears that Gi-mediated of the ells with pertussis toxin bloks -indued p21 ativation is independent of known PTX-sensitive ef- MP kinase phosphorylation without affeting the fetor routes (12). This novel Gi-p21' pathway is thought to other tyrosine phosphorylations. The kinase inhibitor play a entral role in the stimulation of DN synthesis by staurosporine (1 PM) bloks -indued but not epi- (12). dermal growth fator-indued ativation of p21 and mong the early events following ativation of quiesent * This work was supported by the Duth aner Soiety. The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked aduertisement in aordane with 18 U.S.. Setion 1734 solely to indiate this fat. 4 Present address: Gene Pharming 2333 Leiden The Netherlands. I To whom orrespondene should be addressed. Tel.: 31-20-512-1971; Fax: 31-20-512-1989. ' The abbreviations used are: lysophosphatidi aid; PTX pertussis toxin; Gi inhibitory guanine nuleotide-binding protein; EGF epidermal growth fator; MP mitogen-ativated protein; TP 12-0-tetradeanoylphorbol-13-aetate; ptyr phosphotyrosine; GP p21 GTPase-ativating protein. wide range of biologial responses in diverse ell types apparently by ativating its ognate G protein-oupled reeptor(s) (for review see Ref. 1). For example exogenous indues smooth musle ontration platelet aggregation neural shape hanges Dityostelium hemotaxis and in fibroblasts stimulation of DN synthesis (1-5). is rapidly produed and released by ativated platelets and is present in serum at bioative onentrations but not in plasma suggesting a physiologial role for in wound healing and inflammation (4 6 7). Using a photoreative analogue van der end et al. (8) reently identified a andidate reeptor of an apparent moleular mass of 38-40 kda in various responsive ell types inluding fibroblasts. reeptor ativation triggers various G protein-mediated signal transdution asades inluding stimulation of phospholipases and D and inhibition of adenylate ylase MP kinase onsistent with an intermediate protein ells is an inrease in tyrosine phosphorylation of ellular kinase linking the reeptor to p21' ativation. proteins. part from the growth fator reeptor tyrosine The results support a model in whih -indued phosphorylation of MP kinase is mediated by p21' kinases ertain G-protein oupled reeptors also are apable and tyrosine phosphorylation of the other substrates of stimulating protein tyrosine phosphorylation in their target inluding ~ 125~*~ is assoiated with phospholipase ells (13-18). The G protein-mediated effetor routes by whih ativation. these reeptors stimulate tyrosine phosphorylation are poorly understood although ativation of the phospholipase -a2+protein kinase pathway appears to be a ommon feature. We reently reported that rapidly stimulates p60' ty- Lysophosphatidi aid (' 1-ayl-sn-glyerol-3-phos- rosine kinase ativity in serum-deprived N1E-115 neurophate) is a simple water-soluble phospholipid that evokes a 645 blastoma ells a response impliated in the -indued reorganization of the atin ytoskeleton and subsequent retration of developing neurites (4). However to date our efforts to detet enhaned p60' ativity in -treated fibroblasts have been unsuessful;' in fat virtually nothing is known about protein tyrosine phosphorylation indued by in fibroblasts. In this study we have examined -indued protein tyrosine phosphorylation in quiesent Rat-1 ells. These ells are mitogenially highly responsive not only to but also to EGF (2 3). Furthermore we took advantage of the fat that Rat-1 ells express funtional endothelin reeptors * T. Eihholtz and P. L. Hordijk unpublished observations.
646 -indued Tyrosine Phosphorylation and MP Kinase tivation whih ouple to strong stimulation of phospholipid breakdown Kodak X-R films with intensifying sreens. (3 10 11) but fail to mediate ativation of p21 and mito- For immunopreipitation of PISOGP ells grown in IO-m dishes genesis (3 12). Weshow here that like endothelin were lysed in the buffer used for anti-ptyr immunopreipitations. Protein ontent was determined by the method of Lowry et al. (26). rapidly stimulates tyrosine phosphorylation of multiple el- The lysates were preleared (30 min 4 ) with protein -Sepharose lular proteins among whih we have identified the ~ 125'~~ oated with normal rabbit serum. fter inubation with polylonal foal adhesion kinase (19). Furthermore we show that but not endothelin stimulates phosphorylation and ativation of the ~421~44 MP kinases; these are serinelthreonine kinases that funtion within a protein kinase asade and are a entral omponent of many different signaling pathways (20-25). Using PTX we were able to dissoiate -indued MP kinase phosphorylation from the other protein tyrosine phosphorylations. Together our data indiate that -indued MP kinase phosphorylation is mediated by the Gip21 pathway with no apparent role for the phospholipase -protein kinase asade whereas tyrosine phosphorylation of the other proteins inluding ~125'*~ orrelates with phospholipase ativation. EXPERIMENTL PROEDURES hemials and ntibodies-materials and reagents were obtained from the following soures: 1-oleoyl- staurosporine and TP from Sigma; nitroellulose from Shleiher & Shuell; arylamide from Serva; endothelin from ambridge Researh iohemials (ambridge United Kingdom); EGF from ollaborative Researh (edford M); PTX from List iologial Laboratories In. (ampbell ); protein -Sepharose L-4 and protein G-Sepharose-4 beads from Pharmaia LK iotehnology In.; suramin from ayer; polyethyleneimine-ellulose F plates from Merk [32P]orthophosphate [r-p]tp and the Enhaned hemiluminesene system from mersham orp. nti-phosphotyrosine monolonal antibody PY20 was from IN and peroxidase-labeled antibodies from DKO (Glostrup Denmark). Normal mouse serum and polylonal rabbit anti-mouse antibodies were obtained from the entral Laboratory for lood transfusion (msterdam The Netherlands). Rabbit polylonal antibodies against p42/p44 MP kinase and pl20-gp (produed as fusion proteins in Esherihia oli) and rat monolonal antibody Y13-259 against p21m were kindly provided by Drs. J. L. os and. urgering (Utreht University) and anti-p12fifk monolonal antibody (27) by Dr. T. Parsons (University of Virginia at harlottesville). Polylonal anti- MP kinase serum 122 was a generous gift from Dr.. J. Marshall (Institute of aner Researh London). ell ulture--rat-1 ells were grown in Dulbeo's modified Eagle's medium supplemented with 7.5% fetal alf serum and were split in a 1:lO ratio at 3-4-day intervals. For experiments ells in plasti petri dishes or six-well ulture plates were grown to 80-90% onfluene and then serum-starved for 24 h. 32P Labeling and Immunopreipitation-Three hours prior to the assay the medium was replaed by phosphate-free Eagle's modified essential medium (IN) ontaining 200 pi/ml [32P]orthophosphate. gonists were diretly added to the test medium and after variable periods of time the ells were quikly washed with ie-oldphosphatebuffered saline ontaining 1 mm EDT 10 mm sodium fluoride 0.5 mm sodium vanadate and 10 mm tetrasodium pyrophosphate. ells were subsequently sraped in 0.5 ml of ie-old lysis buffer (10 mm Tris-H1 ph 7.4150 mm Na1 1% sodium deoxyholate 0.1% SDS 1% Nonidet P-40 10 mm sodium fluoride 1 mm EDT 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 entrifuged for 10 min at 14000 X g and preleared by inubation for 45 min with 50 pl of a 10% solution in lysis buffer of formaline-fixed heat-killed Staphyloous aureus owan I strain bateria oated with rabbit anti-mouse antibodies and normal mouse serum. fter entrifugation (10 s 14000 X g) and two additional 10-min inubations with S. aureus the lysates were normalized by trihloroaeti preipitation and ounting of aliquots. The lysates were inubated with anti-ptyr antibody (2 h 4 ) followed by rabbit anti-mouse-oated S. aureus (45 min 4 ). The immunopreipitates were washed five times with 50 mm Tris-H1 ph 6.8 150 mm Na1 0.5% Nonidet P-40 0.5 mm Mgl and 0.5 mm sodium orthovanadate. Protein omplexes were eluted by boiling in SDS-sample buffer and were analyzed after entrifugation (10 min 14000 X g) by SDS-polyarylamide gel eletrophoresis (5-15% gradient). Gels were dried and exposed for 18-120 h at -70 using anti-gp serum (2 h 4 ) GP-antibody omplexes were aptured with protein -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% polyarylamide gel and blotted onto nitroellulose. For immunopreipitation of ~ 1 2 5 ells ~ ~ grown in 10-m dishes were lysed in a buffer ontaining 50 mm Tris-H1 ph 7.4 150 mm Nal 2 mm EGT 1% Nonidet P-400.25% sodium deoxyholate and the phosphatase and protease inhibitors mentioned above. The lysates were preleared (30 min 4 ) with protein -Sepharose oated with rabbit anti-mouse antibodies and normal mouse serum. Immunopreipitation was arried out using anti-~l25~~ monolonal antibody 27 (2 h 4 ) (27) and protein -Sepharose oated with rabbit anti-mouse antibodies (45 min 4 ). The preipitates were washed three times with lysis buffer and analyzed by 10% polyarylamide gel eletrophoresis and blotting. Zmmunoblotting-For immunoblot analysis of tyrosine-pbosphorylated proteins or the mobility shift of MP kinase ells were stimulated lysed immediately in SDS-sample buffer and separated on a 5-15% polyarylamide gradient or 10% polyarylamide gel respetively. lotting to nitroellulose was performed at 0.5 for at least 1 h in 25 mm Tris 200 mm glyine and 20% (v/v) methanol. speifi binding of antibodies was prevented by inubating the blots in 5% dried milk powder (anti-mp kinase immunoblots) or 4% bovine serum albumin/l% ovalbumin in TST (10 mm Tris ph 8.0 150 mm Na1 0.05% Tween-20; also used for all inubations and washing steps) for 1 h at 37. Next the blots were inubated (1 h room temperature) with the anti-ptyr antibody PY20 (1 pg/ml) antip42 MP kinase antiserum (diluted 1:10000) or the anti-gp antiserum (diluted 1:lOOO) followed by extensive washing. The blots were subsequently inubated (45 min room temperature) with peroxidaselabeled rabbit anti-mouse or swine anti-rabbit antibodies (both diluted 1:7500). fter washing immunostained proteins were visualized using the Enhaned hemiluminesene detetion system aording to the instrutions of the supplier (mersham). Immunoblots were reprobed after inubation (30 min 50 ) in 67 mm Tris-HL ph 6.72% SDS 100 mm j3-meraptoethanol. ll results shown are representative of at least three independent experiments. MP Kinase tivity-mp kinase ativity was analyzed by an immunoomplex kinase assay essentially as desribed previously (28). Quiesent ells were stimulated for 5 min washed with ie-old phosphate-buffered saline and lysed in 0.5 ml of lysis buffer (50 mm Tris-HL ph 7.5 1% Triton X-100 100 mm Na1 10 mm sodium fluoride 5 mm EDT 500 p~ sodium orthovanadate 0.06 trypsin inhibitory units of aprotinin 1 mm phenylmethylsulfonyl fluoride and 20 p~ leupeptin). Insoluble material was removed by entrifugation (10 min 14000 X g) and protein ontent was measured (26). The ell lysates were preleared by inubation with protein -Sepharose beads oated with normal rabbit serum (1 h 4 ) and MP kinases were immunopreipitated with protein -Sepharose beads oated with the anti-mp kinase antiserum 122 (29) (2 h 4 ). The beads were washed four times with lysis buffer and immunopreipitated kinase ativity toward myelin basi protein was assayed as desribed (28). The data are expressed as pmol of phosphate inorporated into the substrate/min/mg of extrat protein and are representative of three independent experiments performed in dupliate. tivation ofp2ltivation of phrn was assayed essentially as desribed previously (1230). In brief onfluent serum-deprived ells in six-well tissue ulture plates were prelabeled with [3zPjorthoph~~phate (200 pi/well) for 4 h. ells were stimulated and lysed in a 1% Triton X-114 buffer and p21 was immunopreipitated using monolonal antibody Y 13-259 and protein G-Sepharose. ound nuleotides were separated by thin-layer hromatography using polyethyleneimine-ellulose plates followed by autoradiography. RESULTS Protein Tyrosine Phosphorylation Indued by Endothelin and EGF-ddition of 1-oleoyl- (1 pm) to serumdeprived Rat-1 fibroblasts auses a rapid inrease in tyrosine phosphorylation of multiple ellular proteins as deteted by immunopreipitation of 32P-labeled proteins using anti-ptyr antibody PY20 (Fig. 1 left panel). The -indued pattern
-indued TyrosinePhosphorylation and MP Kinase tivation 647 200-116- 9766-45- FIG. 1. Stimulation of protein tyrosine phosphorylation in Rat-1 ells. Left panel Rat-1 ells were labeled with [32P]orthophosphate for 3 h and then stimulated for 5 min with vehile ( ontrol) 1-oleoyl- (1 p ~ )endothelin (ET; 50 nm) or EGF (50 ng/ml). ptyr-ontaining proteins were immunopreipitated from ell lysates using anti-ptyr antibody PY20 and the samples were analyzed by SDS-gel eletrophoresis (5-15% gradient gel) as detailed under Experimental Proedures. rrows indiate proteins with inreased ptyr ontent in response to or endothelin. Right (ET; 50 panel ells were stimulated with (1 p ~ ) endothelin nm) or EGF (50 ng/ml) for 5 min and ell lysates were diretly analyzed by anti-ptyr immunoblotting using antibody PY20 as desribed under Experimental Proedures. rrows indiate proteins with inreased ptyr ontent. Stimulation with EGF results in the phosphorylation of a different set of proteins whih inludes the autophosphorylated EGF reeptor (170-180 kda; asterisk). Note appearane of additional ptyr-ontaining proteins of 42/44kDa in response to and EGF but not endothelin (rightpanel).moleular mass markers are in kda. Exposure times: left panel 120 h; right panel 10 min. inrease (9iof maximum) - 9 8 1 6 -log [I (M) FIG.2. Dose-responserelationship of -induedtyrosine phosphorylation. Rat-1 ells were labeled with [32P]orthophosphate for 3 h as in Fig. 1 and then inubated with inreasing onentrations of 1-oleoyl-as indiated. Tyrosine-phosphorylated proteins were preipitated using antibody PY20 and analyzed by SDSgel eletrophoresis (5-15% gradient gel) as desribed under Experimental Proedures. Moleular mass markers are in kda; ptyrontaining proteins of220 13012011095 and 66-75 kda are indiated with arrows. Note the presene of a 220-kDa protein band not seen in the experiment of Fig. 1. Exposure time 120 h. the three major ptyr-ontaining protein bands on the autoradiogram (termed p120 p95 and p66-75) were sanned by laser densitometry and therelative intensities are presented. of protein phosphorylation was ompared with that obtained with endothelin or EGF. Endothelin ats through its own G protein-oupled reeptor and is a more powerful ativator of phospholipases and D than without being mitogeni for Rat-1 ells (3 10 11).EGF is highly mitogeni without stimulating phospholipid breakdown in these ells (23). Fig. 1 (left panel) shows that both and endothelin promote tyrosine phosphorylation of proteins of apparent moleular mass 110-130 95 and 65-75 kda. In some experiments an additional inrease in tyrosine phosphorylation of a protein doublet of 210-220 kda was observed (for example see Figs. Phosphorylations-To determine the dose-response relation2 and 3). We note that at maximally effetive doses the ship for -indued tyrosine phosphorylation 32P-labeled response to endothelin (50 nm) was onsistently of greater ells were stimulated with varying onentrations of. magnitude than that evoked by (1p ~ )it; thus appears The ptyr-ontaining proteins were immunopreipitated and that there is a positive orrelation between the degree of the intensity of the 110-130-95- and 65-75-kDa bands tyrosine phosphorylation and phospholipase ativation. The quantitated by laser densitometry. s shown in Fig. 2 and protein phosphorylation pattern indued by EGF is learly the threshold dose of is around 1 nm the halfdifferent from that indued by or endothelin (Fig. 1). maximally effetive onentration falls within the 3-30 range Here the major substrates represent the EGF reeptor itself (depending on the protein band examined) and maximum (170-180 kda) together with a set of proteins in the 40-130- effets are observed a t -100 nm. This dose-response urve is kda range. very similar to thatfor other -indued early events suh -indued proteintyrosine phosphorylation was as- as a2+mobilization p21 ativation and neurite retration sessed independently of metaboli labeling by anti-ptyr im- (4912); moreover it overlaps well with the dose dependene munoblotting of total ell lysates (Fig. 1 right panel). While determined in ompetitive reeptor binding assays (8). the majority of the bands omigrated with those deteted in To onfirm that the effets of were reeptor-mediated 32P-labeledimmunopreipitates two additional phosphotyro- we used suramin a polyanioni drug that inhibits the binding syl proteins of 42 and 44 kda were deteted in immunoblots of to its putative reeptor and reversibly bloks the obtained from both - and EGF-treatedells. The p42 and biohemial and biologial ativities of (3 48 12). We p44 proteins were identified as MP kinases as willbe found that suramin (0.5 mg/ml) ompletely inhibits demonstrated below. The fat that theywere poorly if at all indued tyrosine phosphorylations in Rat-1 ells (not shown). preipitated by anti-ptyr antibodies (Fig. 1 left panel) is Kineti analysis of proteintyrosine phosphorylation in onsistent with previous findings (31). Of note the ~ 4 2 1 ~ 4 4response to (1p ~ shows ) that ation is rapid and proteins were not detetably tyrosine-phosphorylated in re- transient (Fig. 3 and ) ; inreased phosphate ontent in sponse to endothelin (Fig. 1 right panel). anti-ptyr immunopreipitates is already detetable within 30 Dose Dependene and Kinetis of -indued Tyrosine s and effets are maximal after 2-5 min and then gradually
-indued Tyrosine Phosphorylation and MP Kinase tivation 648 Tirne(rnin) 0' 0.5' I' 2' 5' 10' 20' - 200- a-ptyr EGF 20011697-11691 - 66-66- a-gp IiF fold inrease ' I I p66-75 p125 FK+ ET EGF FIG.4. nalysis of pl20gp and ~ 1 2 5 phosphorylation. ' ~ ~ ells were stimulated for 5 min with vehile () (1 p M ) or 0 5 I0 15 20 EGF (50 ng/ml). p12ogp was preipitated using polylonal antigp antiserum and analyzed by immunoblotting using anti-ptyr Time (min) FIG. 3. Kinetis of -indued tyrosine phosphorylation. antibody; the blots were reprobed using anti-pl2ogp anti-antiserum [32P]orthophosphate-labeledells were stimulated with (1 to assess proper preipitation of GP as indiated in the lower part of. Markers are in kda. The Zg arrow indiates heavy hain of p M ) for the indiated periods of time and ptyr-ontaining proteins were immunopreipitated as desribed under Experimental Proe- preipitating antibody. ells were stimulated for 5 min with (ET; 50 nm) or EGF (50 ng/ml). ~ 1 2 was 5 ~ ~ ~ dures. Moleular mass markers are in kda. rrows indiate ptyr- (1 p ~ )endothelin immunopreipitated using monolonal antibody 27 and analyzed ontainingproteins of 22013012011095 and 66-75 kda. Exposure time 120 h. the threemajor ptyr-ontaining protein bands on the for ptyr ontent by anti-ptyr immunoblotting. Equal amounts of 5 ~preipitated ~ ~ as evidened by reprobing the blot with autoradiogram (p120 p95 and p66-75; f. Fig. 2) were sanned by ~ 1 2 were monolonal 27 (not shown). laser densitometry and the relative intensities are presented. deline over the next 10-20 min. nalysis of the 110-130-kDa Phosphotyrosyl Proteins and Identifiation of p125fk- major group of phosphotyrosyl proteins in - and endothelin-stimulated Rat-1 ells lusters at 110-130 kda (Fig. 1).Given the stimulatory effet of on p21ra (12) we examined whethertheseproteins omprise the 120-kDa GP (32). In some ell systems tyrosine phosphorylation of GP aompanies growth fatorindued p21 ativation (33-35). GP was immunopreipitated from - or EGF-stimulated ells and assayed for ptyr ontent by immunoblotting. s illustrated in Fig. 4 neither nor EGF indued detetabletyrosinephosphorylation of GP. EGF did however promote a dramati inrease in tyrosine phosphorylation of a 60-65-kDa protein that opreipitated with GP and presumably represents the GP-assoiated protein p62 (36). nother andidate phosphotyrosyl protein in the 110-130kDa range is the foal adhesion kinase p125fka newly identified tyrosine kinase (19) that beomes rapidly phosphorylated on tyrosine after integrin-mediated ell spreading and adhesion (37-39) and also in neuropeptide-treated Swiss 3T3 ells (40). To testwhether ~ 1 2 is 5a onstituent ~ ~ ~ of the 110-130-kDa protein band the kinase was immunopreipitated from ell lysates using monolonal antibody 27 (27) followedby anti-ptyr immunoblotting. Fig. 4 shows that ~ 1 2 is 5tyrosine-phosphorylated ~ ~ ~ to a relatively high level inunstimulated ells and that both and endothelin indue a small but signifiant inrease in its ptyr ontent. gain endothelin was onsistently more powerful than in promoting tyrosine phosphorylation of ~ 1 2 5There ~ ~ was ~. no signifiant effet of EGF on ~ 1 2 phosphorylation 5 ~ ~ ~ (Fig. 4 and results not shown). Phosphorylation and tivation of MP Kinase-The antiptyr immunoblotting experiments reveal that and EGF but not endothelin indue tyrosine phosphorylation of two proteins of42 and 44 kda (Fig. l) whih are likely to represent the ~ 4 2 1 ~ 4MP 4 kinases. Theseare ativated through phosphorylation on Tyr and Thr residues in response to various external signals inluding peptide growth fators phorbol ester and G protein-oupled reeptor agonists (2025 41-43). This ativation is readily detetable on anti-mp kinase immunoblots where the ativated phosphorylated forms of ~ 4 2 1 ~ display 44 a lower eletrophoreti mobility (29 44).Fig. 5 (upper panel) shows that both and EGF indue a rapid shift in mobility of the p42/p44 MP kinases. In ontrast endothelin fails to indue a detetable mobility shift a result in line with the anti-ptyrimmunoblotting data (Fig. 1 right panel). Theprotein kinase -ativating phorbol ester TP (100 ng/ml) indued a small degree of MP k'mase ativation; however for unknown reasons TP-indued MP kinase phosphorylation in Rat-1 ells was highly variable between different experiments (not shown). It thus appears that althoughtp-ativatedprotein kinase may stimulate MP kinase phosphorylation to some extent reeptor-mediated phospholipase -protein kinase ativation is not theroute by whih ats. This onlusion is supported by further experiments desribed below. T o onfirm that phosphorylation of the MP kinases is aompanied by inreased kinase ativity we performed immunoomplex kinase assays using myelin basi protein as a substrate (28). Stimulation of Rat-1 ells with either or EGF results in enhaned phosphorylation of myelin basi proteinin MP kinase immunopreipitates (Fig.5 lower panel) thus showing that MP kinase phosphorylation parallels inreased enzymati ativity. Kineti analysis shows that the inrease in -indued MP kinase phosphorylation is detetable after 1 min reahes a maximum after 5 min and thereafter delines to prestimulation levels within 10-20 min (Fig. 6 upper panel). In ontrast theegf-indued mobility shifts are maintained
-indued TyrosinePhosphorylation and MP Kinase tivation ET EGF - + m - + 200-649. I y?? ontrol IJ' FIG. 5. Phosphorylation and ativation of MP kinase. Upper panel Rat-1 ells were stimulated for 5 min with vehile () (1p ~ )endothelin (ET; 50 nm) or EGF (50 ng/ml). ell lysates were analyzed by SDS-gel eletrophoresis followed by immunoblotting with anti-mp kinase polylonal antiserum as desribed under Experimental Proedures. The phosphorylated ativated forms of p42/p44 MP kinases (pp42/pp44) display redued eletrophoreti mobility. Lower panel immunoomplex kinase assay using myelin basi protein as a substrate. ells were stimulated for 5 min with vehile (ontrol) (1 pm) or EGF (50 ng/ml) followedby immunopreipitation of MP kinases and anin vitro kinase assay in the presene of myelin basi protein (0.25 mg/ml) as desribed under Experimental Proedures. The data represent mean f S.E. 0.5' I' 2' 5' 151: 10' 20' -3 P T X - + p 4 4 + p42+ PIX-+-+ FIG. 7. Effets of PTX. ells were inubated in serum-free medium with (+) or without (-) 100 ng/ml PTX for 18 h. They were then labeled with [32P]orthophosphatefor 3 h and stimulated for 5 min with vehile ()or (1p M ) followed byimmunopreipitation of ptyr-ontaining proteins using anti-ptyr antibody and analysis by SDS-gel eletrophoresis (5-15% gradient gel). The arrows indiate proteins of 13012011095 and66-75 kda. Exposure time 72 h. ells were pretreated with PTX asin and phosphorylation of p42/ p44 MP kinase was monitored in immunoblots using anti-ptyr antibody (upper panel) or anti-mp kinase antiserum (lowerpanel) as in Figs. 5 and 6. Exposure time 15 min. sporine is a potent inhibitor of several serinelthreonine and tyrosine-speifi protein kinases (45-47). ddition of staurosporine (1 p ~ to)rat-1 ells inhibits -indued MP p 4 4 J +pp44 kinase phosphorylation (Fig. 8). This inhibition is seletive +Pp42 in that EGF-indued MP kinase phosphorylation is not o n affeted by the drug (Fig. 8). We established that staurosfig.6. Kinetis of - and EGF-indued phosphorylation porine (1p ~ has ) no effet on -indued phospholipase of MP kinase. ells werestimulated with (1p ~upperpanel) ; ativation as inferred from a2+mobilization measurements? or EGF (50 ng/ml; lower panel) for the indiated periods of time lysed and analyzed by immunoblotting for p42/p44 mobility shifts These results suggest that staurosporine ats upstream of Gimediated ~ 2 1 ativation. ~ To test thishypothesis we moniusing anti-mp kinase antiserum (f. Fig. 5 upper panel). tored - and EGF-indued p2lras.gtpaumulation in for up to 30-60 min (Fig. 6 lower panel). We note that the the presene or absene of staurosporine. Fig. 8 shows that time ourse from onset to maximum mobility shifts slightly indeed staurosporine (1 PM) ompletely inhibits -inlags behind that observed for ~ 2 1 ativation ~ whih peaks dued p21 ativation but leaves the response to EG.F unaltered. at 2 min after addition of or EGF(12). These results reveal a previously unreognized effet of Differential Effets of PTX-Treatment of Rat-1 ells with staurosporine and extend our previous studies showing that PTX bloks -indued mitogenesis and p21.gtp au-indued but not EGF-indued p21 ativation is inmulation but hasno effet on -indued phospholipid hibited by the tyrosine kinase inhibitor genistein (12 48). hydrolysis (2 11 12). We examined the effets of PTX on -indued proteintyrosinephosphorylations and MP However genistein requires muh higher doses than stauroskinase ativation. Treatment of the ells with 100 ng/ml PTX porine for a full inhibitory effet (50 pm uers'sus 1pM respefor 18 h has no effet on -indued tyrosine phosphoryla- tively) (12). Taken together these pharmaologial data hint tion of the 110-130- 9 5 and 65-75-kDa proteins (Fig. 7 ) ; at the presene of an as yet unidentified protein tyrosine ~ neither did PTX treatment affet tyrosine phosphorylation kinase that links Gi to ~ 2 1 ativation. of ~ 1 2 in5response ~ ~ to ~ or endothelin (not shown). In DISUSSION ontrast PTXpretreatment ompletely inhibits tyrosine phosphorylation as well as themobility shift of p42/p44 MP lthough has long been known as a ritial preursor kinase (Fig. 7 upper and lower panels). s expeted the in the early steps of glyerolipid biosynthesis its funtion as EGF-indued mobility shift of MP kinase was not affeted a platelet-derived first messenger has been reognized only by PTX (notshown). reently (16). at nanomolar doses binds to and thereby Staurosporine Inhibits -induedbut Not EGF-indued tivation ofp21 and MP Kinase-The alkaloid staurok. Jalink unpublished observations. 4
-indued Tyrosine Phosphorylation and M P Kinase tiuation 650 In the present studywe have examined -indued protein tyrosine phosphorylation in Rat-1 ells. We have omparedtheation of withthat of endothelin whih ativates its own G protein-oupled reeptor totrigger phospholipid breakdown but unlike does not ativatep21' and is not mitogeni by itself(1249). Twomajor novel results are reportedhere. First at 1-100 nm rapidly stimulates tyrosine phosphorylationof multiple ellular proteins; among these we have identified the ~ 1 2 tyrosine 5 ~ kinase ~ ~ and the ~42144 MP kinases. Seond by using PTX we have been able todissoiate -indued MP kinase phosphorylation from the other tyrosinephosphorylations. We find that the overall pattern of -indued tyrosine GTP 1 4 0 b o phosphorylations in the 60-220-kDa range is unaltered by PTX treatment and qualitatively similar to thatindued by EGF EGF islaumrpnne endothelin but different from that indued by EGF. oth phospholipid breakdown in FIG. 8. Effets of staurosporine onmp kinase phosphoryl- and endothelin stimulate ation and p21 ativation. phosphorylation of MP kinase. Rat-1 ells with beinga weaker induer than endothelin ells were treated for 10 min with either vehile or staurosporine (1 (3 10 11). Similarly the overall tyrosine phosphorylation PM) and then stimulated for 5 min with vehile ( ontrol) (1 response to is less prominent than that to endothelin. PM) or EGF (50 ng/ml). ells were lysed and analyzed for MP Thus there is a positive orrelation between the degree of kinase mobility shifts in immunoblots as in Figs. 5 and 6. Exposure time 10 min. ativation of p21. ells were metabolially labeled reeptor-mediated phospholipase ativation and the observed protein tyrosinephosphorylations. lose relationship with [32P]orthophosphate for 4 h pretreated for 10 min with either by the fat that vehile or 1 PM staurosporine and then stimulated for 2 min with between these two early events is supported (1 PM) or EGF (50 ng/ml). The guanine nuleotides bound to other agonists oupling to phospholipase ativation suh p21 were separated by thin-layer hromatography followed by au- as bombesinvasopressinangiotensin and thrombin also toradiography as desribed under ExperimentalProedures. ssays evoke tyrosine phosphorylations in their targetells (14-18). were performed in dupliate as shown. Exposure time 72 h. The biohemial steps by whih these agonists promote tyrosine phosphorylation remain tobe explored although both EGF Endothelin protein kinase ativation and a rise in ytosoli-free a2+ J J have been impliated depending on ell types and agonists (14 17 18 50). We have identified the ~ 1 2 tyrosine 5 ~ ~kinase ~ as a onstituent of the 110-130-kDa phosphotyrosyl proteins indued by and endothelin. The ~ 1 2 kinase 5 ~is predominantly ~ ~ loalized in foal adhesions of normal fibroblasts (19) and is rapidly tyrosine-phosphorylated upon ell-extraellular ma4 p r w i n tyrosine trix interation or integrin lustering (37-39) and furtherphosphorylation more in Swiss 3T3 ells treated with neuropeptides. lthough the preise physiologial funtion of ~ 1 2 and 5 its ~ mode ~ ~of MP-kinase regulation by extraellular stimuli are unknown appealing an possibility is that~ 1 2 partiipates 5 ~ ~ ~in therapid formation FIG.9. Proposed model of early signaling pathways trigfibers as observed in gered by. In this model ativates its ognateg protein- of foal adhesions and atin stress treated fibroblasts (51).3In thisrespet it isnoteworthy that oupled reeptor(s) to trigger three independent effetor routes as follows: (i) stimulation of phospholipase via a PTX-insensitive G in neuroblastoma ells-indued hanges in the atin protein(putative GJ; (ii)inhibition of adenylate ylase through ytoskeleton are aompanied by inreased p60 tyrosine PTX-sensitive Gi; and (iii) ativation of a Gi-mediatedp21 pathway kinase ativity (4) and that ~ 1 2 is 5a potential ~ ~ ~ substrate via a putative staurosporine- andgenistein-sensitive protein tyrosine kinase. It is not known whether inhibition of adenylate ylase and for ativated p60 (19 27 37). Whether ~60 ativity is similarly enhaned in -treated Rat-1 ells remains to be p21 ativation are mediated by the same Gi protein. Endothelin ativates the phospholipase pathway in Rat-1 ells but does not determined. phosphorylate MP kinase whereas EGF does not detetably stim striking differene between the ation of and enulate phospholipase ativity in these ells. The Gi-p2IrMpathway dothelin is that phosphorylates and ativates the p42/ leads tompkinaseativationandthe major proteintyrosine p44 MP kinases whereas endothelin fails to do so. MP phosphorylations are proposed to be linked tophospholipase atikinase kinase vation; it remains possible however that enhaned protein tyrosine kinase is ativated through an upstream MP phosphorylation and phospholipase ativation represent parallel whih is thought to serve as a onvergene point of diverse the phospholipase-protein rather than sequential events. For further details see text. RTK signalingpathwaysinluding reeptor tyrosine kinase; adenylate ylase; PL phospholipase kinase asadeand thereeptor tyrosine kinase-p2lrnpath. way (25). The failure of endothelin to stimulate MPkinase phosphorylation despite its potentoupling to the phosphoativates its ognate G protein-oupled reeptor(s) onvarious lipaseseondmessengerasade is therefore somewhat signal transdution unexpeted and most likely represents aell-type speifi target ells (8)but the reeptor-mediated endothelin does pathwaysarenot fully understood.previousstudies have effet; in rat mesangial ells for example shown that stimulates GTP-dependent phosphoinositide ativate MP kinasevia a PTX-insensitive pathwaypartially hydrolysis and inhibits adenylate ylase (1). Moreover in dependent on protein kinase (42). Furthermore another fibroblasts rapidly ativates ~21' via a novel PTX- phospholipase-ativatingagonist bombesin stimulates sensitive effetor route (12). The latter pathway rather than MP kinase ativity in 3T3 ells via a pathway thatis almost phospholipase ativation or adenylateylase inhibition is entirely dependent on proteinkinase (43). Taken together thought tobe ritial for -indued mitogenesis (1249). these findings support theview that therelative ontributions +staumsporine EGF J. EGF
-indued Tyrosine Phosphorylation of the signaling pathways from reeptor to MP kinase may be very different in different ell types. Whatever the preise biohemial explanation the failure of endothelin to ativate MP kinase in Rat-1 ells provides a moleular basis for its lak of mitogeniity in these ells. Several studies have established a ause-effet relationship between ativation ofp21 and ativation of the MP kinases (29 44 52 53). Our findings are in line with these reports and indiate that stimulates MP kinase phosphorylation solely via p21 with no signifiant ontribution of the phospholipase -protein kinase pathway. First agents that blok -induedp21ra8 ativation i.e. PTX and staurosporine also inhibit MP kinase phosphorylation whereas these agents leave the responses to EGF unaltered. Seond omparison between the ativation kinetis of p21' (12) and those of MP kinase (Fig. 6) reveals that p21 ativation parallels but preedes MP kinase phosphorylation. Third a phospholipase -ativating agonist that fails to ativate p21' notably endothelin is inapable of stimulating MP kinase phosphorylation. Finally down-regulation of funtional protein kinase by hroni treatment of the ells with TP (2) does not attenuate the ation of on MP kinase.' The present data along with previous findings lead us to propose a sheme of the G protein-mediated signal transdution pathways triggered by in fibroblasts (Fig. 9). In this sheme the signaling routes that shares with endothelin and EGF are also depited (Fig. 9). inding of to its G protein-oupled reeptor(s) results in ativation of at least three independent signaling asades as follows: (i) stimulation of phospholipase via a PTX-insensitive G protein (putative G; 54-56); (ii) PTX-sensitive inhibition of adenyl- ate ylase; and (iii) ativation of p21 via a PTX-sensitive Gi protein (not neessarily the same that ouples to inhibition of adenylate ylase). MP kinase funtions then downstream of p21' while the major tyrosine phosphorylations (66-75 95110-130 kda; ~ 1 2 5 are ~ proposed ~ ~ ) to be assoiated with phospholipase ativation. The inhibitory effets observed with staurosporine (Fig. 8) and genistein (12) on -indued p21' ativation suggest that an intermediate protein tyrosine kinase lies on the route between Gi and p21'. Identifiation of this putative tyrosine kinase is an obvious hallenge for future studies. While this sheme is the simplest one that is ompatible with the available data it may be too simplisti. In partiular our findings leave open the possibility that phospholipase ativation and inreased protein tyrosine phosphorylation atually represent parallel pathways with phospholipase and tyrosine kinase (and/or phosphatase) ativity being regulated by distint G protein subunits. ritial test of this hypothesis must await further experiments. knowledgments-we thank Drs. T. Parsons (University of Virginia at harlottesville) J. L. os and. urgering (Utreht University) and. J. Marshall (Institute of aner Researh London) for antibodies; K. Jalink for a2+ measurements; and D. Shaap for disussions. REFERENES 1. Moolenaar W. H. Jalink K. and van orven E. J. (1992) Reu. Physiol. iohem. Pharmaeol. 11948-65 2. van orven E. J. Groenink. Jalink K. Eihholtz T. and Moolenaar W. H. (1989) ell 6945-54 3. van orven E. J. van Rijswijk Jalink K. van der end R. L. van ' P. L. Hordijk unpublished observations. and MP Kinase tivation 651 litterswijk W. J. and Moolenaar W. H. (1992) iohem. J. 281163-169 4. Jalink K. Eihholtz T. Postma F. R. van orven E. J. and Moolenaar W. H. (1993) ell Growth & Dzffer. 4247-255 5. Jalink. K.. Moolenaar. W. H.. and van Duvn. -.. (1993) Pro. Natl. ad. Si. 'U S.. 90 1867-1861' 6. Eihholtz T. Jalink K. Fahrenfort I. and Moolenaar W. H. (1993) iokm. J. 291677-680 7. Tigyi G. and Miledi R. (1992) J. id. hem. 26721360-21367 8. van der end R. L. runner J. Jalink K. van orven E. J. Moolenaar W. H. and van litterswijk W. J. (1992) EMOJ. 112495-2501 9. Jall~k K.o~n.o~en E. J. and Moolenaar W. H. (1990) J. wl. hem. zoo LLLJL-UZJY 10. Plevin R. MaNulty E. E. Palmer S. and Wakelam M. J. 0. (1991) iohem. J. 280609-615 11. van der end R. L. de Widt J. van orven E. J. Moolenaar W. H. and van litterswijk W. J. (1992b) iohem. J. 286235-240 12. van orven E. J. Hordijk P. L. Medema R. H. os J. L. and Moolenaar W. H. (1993) Pro. Natl. ad. Si. U. S.. 90 1259~1261 13. Golden. and rugge J. S. (1989) Pro. Natl. ad. Set. U. S.. 86901-9n.5 14. Hukle W. R. Prokop.. Dy R.. Herman. and Earp S. (1990) Mol. ell. iol. 106290-6298 15. Leeb-Lundberg L. M. F. and Song X.-H. (1991) J. iol. hem. 286 7746-7749 16. Zahary I. Gil J. Lehmann W. Sinneth-Smith J. and Rozengurt E. (1991) Pro. Natl. ad. Si. U. S.. 884577-4581 17. Fore T. Kyriakis J. M. vruh J. and onventre J. V. (1991) J. iol. hem. 2666650-6656 18. Offermans S. ombien E. and Shultz G. (1993) iohem. J. 290 27-32 19. Shler M. D. orgman. obb. S. Vines R. R. Reynolds.. and Parsons T. J. (1992) Pro.Natl. qd. Sei. U. S.. 89 5192-5196 20. Sturgill W. and Wu J. (1991) mhm. wphys. ta 1092350-357 21. obb M. H. oulton T. G. and Robbins D. J. (1991) eu Regul. 2 965- R7R 22. Th;'x& G. (1992) eu 68 1-4 23. rews.m. lessandri and Erikson R. L. (1992) ell Growth & Differ. 3135-142 24. Nishida E. and Gotoh Y. (1993) Trends iohem. Sei. 18128-131 25. Lange-arter. Pleiman. M. Gardner. M. lumer K. J. and Johnson G. L. (1993) Siene 260 315-319 26. Lowy 0. H. Rosebrough N. J. Farr. L. and Randall R. J. (1951) J. wl. hem. 193265-275 27. Kanner S.. Reynolds.. Vines R. R. and Parsons J. Th. (1990) Pro. Natl. ad. Si. U. S.. 873328-3332 28. Kahan... Seuwen. K.. Melohe.. S.. and Pouvssermr. J. (1992).. J. iol. km. 267 13369-13375 29. Leevers S. J. and Marshall. J. (1992) EMO J. 11569-574 30. urgering. M. T. Medema R. H. Maassen J.. van de Wetering M. L. van der Eb. J. Mormik F. and os J. L. (1991) EMO J. 10 1103-1109 31. Kazlaukas. and oo er J (1988) J. ell. iol. 106 1395-1402 32. Mormik F. (1989) 8eU'665-8 33. Ellis. Moran M. Mormik F. and Pawson T. (1990) Nature 343 377-381 34. Torti M. Marti K.. ltshuler D. Yamamoto K. and Lapetina E. G. (1992) J. iol. hem. 26782934298 35. Molloy. J. Fleming T. P. ottaro D. uadro and aronson S.. (1992) Mol. ell. iol. 123903-3909 36. Wong G. Muller O. lark R. onroy L. Moran M. F. Polakis P. Mormik F. (1992) ell 69551-558 37. Guan J-L. and Shalloway D. (1992) Nature 368690492 38. Kornberg L. Ea H. S. Parsons J. T. Shaller M. and Juliano R.L. (1992) 2. '. lal. F hem. 267 23439-23442 39. umdge K. Turner E. and Romer L. H. (1992) J. eu iol. 119893-903 40. Zahary I. Sinneth-Smith J. and Rozengurt E. (1992) J. iol. hem. 26719031-19034 41. I'llemain G. Pouyssegur J. and Weber M. J. (1991) ell Regul. 2 675-684 42. Wang Y. Simonson M. S. Pouyssegur J. and Dunn J. (1992) iohem. J. 287589-594 43. Pay L: DFkerJ. and Saltiel. R. (1993) iohem. J. 289283-287 44. de ne8 Smlts M. M urgering. M. T. Leevers S. J. Marshall.. J. and os J. L. (1992) Nature 367602-604 45. Ruegg U. T. and urgess G. M. (1989) Trends PhurmoL Si. 10 218-220 46. Fallon R. J. (1990) iohem. iophys. Res. ommun. 170 1191-1196 47. Serist J. P. Sehgal. I. Powis G. and braham R. T. (1990) J. iol. hem. 26620394-20400 48. kiyama Y. and Ogawara H. (1991) Methods Enzyml. 201362-370 49. Moolenaar W. H. (1991) ell Growth & Differ. 2359-364 50. Vostal J. G. Jakson W. L. and Shulman N. R. (1991) J. iol. hem. 26616911-16916 51. Ridle J and Hall. (1992) ell 70389-399 52. Wood: K: W. Sarneki. Roberts T. M. and lenis J. (1992) ell 68 1041 -- ---1- nsn -- - 53. Thomas S. M. DeMaro M. Drangelo G. Halegoua S. and rugge J. (1992) ell 68 1031-1040 54. Taylor S. J. hae H. Z. Rhee S. G. and Exton J. (1991) Nature 360 51 - fi-51 - R 55. Lee. H. Park D. Wu D. Rhee S. G. and Simon M. I. (1992) J. iol. hem. 26716044-16047 56. Sternweis P.. and Smrka. V. (1992) Trends iohem. Si. 17502-506