Arachidonic acid induces ERK activation via Src SH2 domain association with the epidermal growth factor receptor

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1 & 6 Interntionl Society of Nephrology originl rticle Archidonic cid induces ERK ctivtion vi Src SH2 domin ssocition with the epiderml growth fctor receptor LD Alexnder 1, Y Ding 2, S Algrsmy 2, X-L Cui 2 nd JG Dougls 2 1 Deprtment of Nturl Sciences, The University of Michign-Derorn, Derorn, Michign, USA nd 2 Deprtment of Medicine, Division of Nephrology nd Hypertension, Cse Western Reserve University School of Medicine nd University Hospitls of Clevelnd, Clevelnd, Ohio, USA Within the kidney, ngiotensin II type 2 (AT 2 ) receptor medites phospholipse A 2 (PLA 2 ) ctivtion, rchidonic cid relese, epiderml growth fctor (EGF) receptor trnsctivtion, nd mitogen-ctivted protein kinse ctivtion. Archidonic cid mimics this trnsctivtion y n undetermined mechnism. The role of c-src in mediting ngiotensin II nd rchidonic cid signling ws determined y employing immunocomplex kinse ssy, Western lotting nlysis, nd protein immunolotting on co-precipitted EGF receptor (EGFR) proteins nd grose conjugtes of glutthione S-trnsferse fusion proteins contining the c-src homology 2 (SH2) nd SH3 domins. Angiotensin II induced extrcellulr signl-regulted kinse (ERK) ctivtion in primry cultures of rit proximl tuule cells vi the ctivtion of c-src nd ssocition of the EGFR with the c-src SH2 domin, effects tht were mimicked y rchidonic cid nd its inctive nlogue eicostetrynoic cid. Inhiition of PLA 2 y mepcrine nd methyl rchidonyl fluorophosphte, AT 2 receptor y PD123319, Src fmily kinses y, 1-(tert-utyl)-3-(4-chlorophenyl)-4- minopyrzolo[3,4-d] pyrimidine (PP2) nd c-src y overexpression of dominnt-negtive mutnt of c-src rogted these effects. However, inhiitors of rchidonic cid metolic pthwys did not lock these effects. The present work provides new nd novel prdigm for trnsctivtion of kinse receptor linked to ftty cid, which my pply to ctivtion of vriety of phospholipses nd ccompnying rchidonic cid relese. Kidney Interntionl (6) 69, doi:1.138/sj.ki.5363; pulished online 5 April 6 KEYWORDS: rchidonic cid; ngiotensin II; c-src; EGF receptor trnsctivtion; mitogen ctivted-protein (MAP) kinse Correspondence: LD Alexnder, Deprtment of Nturl Sciences, The University of Michign-Derorn, 491 Evergreen Rod, Derorn, Michign 48128, USA. E-mil: ldlex@umd.umich.edu Received 21 Novemer 3; revised 16 August 5; ccepted 15 Septemer 5; pulished online 5 April 6 Moleculr iologicl oservtions vlidte the existence of two mjor ngiotensin II receptor sutypes (type 1 (AT 1 ) nd type 2 (AT 2 )). These receptor sutypes re phrmcologiclly distinct nd shre only pproximtely 3% sequence homology. While the AT 1 receptor hs een considered to suserve mny of the iologicl functions linked to ngiotensin II, emerging evidence indictes tht the dult AT 2 receptor my lso medite significnt iologicl responses s well. Renl proximl tuule epithelil cells represent n unusul site of ngiotensin II ction, s oth ngiotensin II AT 1 (solterl) nd AT 2 (picl) receptors re present. This rit AT 2 receptor is novel with respect to phrmcologicl chrcteristics, in tht phrmcologiclly it is insensitive to losrtn nd cndesrtn, ut sensitive to PD123319, 1 nd its unique signling is linked to rchidonic cid relese upon ctivtion. 2 4 In contrst to the AT 2 receptor expressed in PC12 cells, which inhiits extrcellulr signl-regulted kinse (ERK) ctivtion, memer of the mitogen-ctivted protein (MAP) kinses, the rit AT 2 receptor ctivtes ERK through Shc/Gr2/Sos nd p21rs. 5,6 Src proteins consist of t lest 14 relted lterntively spliced gene products, which re expressed in tissue-specific mnner. 7 A growing ody of dt indictes tht the Src fmily of protein tyrosine kinses functionlly intercts with vriety of non-receptor nd receptor protein tyrosine kinses, prticulrly the epiderml growth fctor (EGF) receptor (EGFR) From these studies, it is generlly greed tht the ssocition of Src (through Src homology 2 (SH2) domin) with the EGFR results in the mutul stimultion of Src ctlytic ctivity, nd enhnced phosphoryltion of the receptors downstrem trgets. In most cell types, the tyrosine phosphoryltion of the EGFR, in response to ngiotensin II, ws oserved to e medited y the ngiotensin II type 1 (AT 1 ) receptor. 8,9,12 However, in rit proximl tuule cells, the effects of ngiotensin II on EGFR phosphoryltion pper to e medited y AT 2 receptor, 6,13 nd rchidonic cid ppers to mimic nd medite ngiotensin II-induced signl trnsduction. Although mechnisms linking the AT 1 receptor to the tyrosine kinse pthwy exist, unresolved questions Kidney Interntionl (6) 69,

2 o r i g i n l r t i c l e LD Alexnder et l.: Archidonic cid induces c-src ctivtion persist s to the mechnism(s) y which the AT 2 receptor nd rchidonic cid, ftty cid, re linked to the tyrosine kinse pthwy. In the present study, we exmined whether ngiotensin II stimultes c-src nd the role of c-src in mediting ngiotensin II-induced EGFR trnsctivtion nd ERK ctivtion. In ddition, we evluted whether cytosolic phospholipse A 2 (cpla 2 ) nd rchidonic cid might ct s meditors of ngiotensin II signling in these proximl tuulr cells. Herein, we demonstrte for the first time tht ngiotensin II induces ERK phosphoryltion nd ctivtion in primry cultures of rit proximl tuule cells vi n AT 2 -induced ctivted c-src nd its SH2 domin ssocition with the EGFR. In ddition, our dt demonstrte tht rchidonic cid mimics nd medites the effects of ngiotensin II, independent of eicosnoid iosynthesis. To our knowledge, the ility of rchidonic cid to ctivte c-src nd induce inding of the EGFR to glutthione S-trnsferse (GST) fusion protein contining the c-src SH2 domin to modulte EGFR trnsctivtion nd ERK ctivtion is entirely novel nd hs not een reported previously. Thus, this my serve s new signling prdigm, since rchidonic cid nd other ftty cids re linked to the tyrosine kinse pthwy, n effect tht my extend to other cell types. RESULTS Angiotensin II-induced Src tyrosine phosphoryltion We recently reported tht rchidonic cid induces c-src ctivtion in time- nd dose-dependent mnner in rit proximl tuule cells. 14 Considering tht rchidonic cid mimics nd medites the iologicl functions of ngiotensin II y the AT 2 -receptor sutype in these sme cells, 5,6 we determined whether ngiotensin II ctivted c-src. As shown in Figure 1, ngiotensin II (1 mmol/l) significntly incresed c-src phosphoryltion in time-dependent mnner. Mximum effect ws oserved t 5 min nd the response declined t 15 3 min, ut ws still significnt, nd then returned to sl levels y 6 min. c-src phosphoryltion y ngiotensin II ws concentrtion dependent, in tht it significntly incresed t 1 7 mol/l ngiotensin II for 5 min nd remined similr nd higher t 1 6 nd 1 5 mol/l of ngiotensin II (Figure 1). Therefore, susequent ssys of ngiotensin IIinduced c-src phosphoryltion utilized 1 6 mol/l ngiotensin II stimultion for 5 min. These results were confirmed with n in vitro kinse ssy. A significnt increse ( % compred to controls) in phosphoryltion of n Srcspecific sustrte peptide (ctivity) y rchidonic cid, in mnner similr to ngiotensin II (263748%), ws oserved, which prlleled the increse in c-src phosphoryltion (Figure 2). In ddition, the Src fmily kinse inhiitor PP2 significntly reduced c-src phosphoryltion y rchidonic cid nd ngiotensin II in dose-dependent mnner (Figure 3 nd, respectively). Similr inhiition ws demonstrted y trnsfection of proximl tuule cells with n Src dominnt-negtive mutnt cdna (Figure 2). In ddition, PD123319, selective AT 2 -receptor locker, lso Angiotensin II (min) c-src [py 418 ] c-src c- Src phosphoryltion c-src [py 418 ] c- Src phosphoryltion Angiotensin II (min) Angiotensin II (log M) rogted the phosphoryltion of c-src. Losrtn (Lor) nd cndesrtn (Cnd), two selective AT 1 -receptor lockers, did not lter ngiotensin II-elicited ctions (Figure 4). Pretretment of proximl tuulr cells with 17-octdecynoic cid (1 mmol/l), potent inhiitor of oth o-hydroxylse nd epoxygense enzyme ctivity, indomethcin (5 mmol/l), cyclooxygense inhiitor, or nordihydroguiretic cid (1 mmol/l), lipoxygense inhiitor, did not ffect rchidonic cid nd ngiotensin II-induced c-src ctivtion (dt not shown). By nlogy, ngiotensin II-induced c-src ctivtion ws totlly rogted y the cpla 2 inhiitors methyl rchidonyl fluorophosphte (MAFP) (1474%, Po.1) nd mepcrine (17711%, Po.1), wheres hloenol lctone suicide sustrte, potent irreversile inhiitor of C 2 þ -independent phospholipse A 2 (PLA 2 ), did not interfere with ngiotensin II-induced c-src ctivtion (Figure 4). Collectively, these results demonstrte tht Angiotensin II (log M) c-src 5 Figure 1 Angiotensin II-induces c-src ctivtion in time- nd dose-dependent mnner in rit proximl tuule cells. () Serum-strved proximl tuule epithelil cells were exposed to either ngiotensin II for the indicted times or () vrious concentrtions of ngiotensin II ( 1 mmol/l) for 5 min. The cells were then lysed nd equl mounts of proteins were used for Western lotting. c-src ws evluted y Western lot nlysis using n nti-phosphorylted c-src (py 418 )-specific ntiody (upper pnel). The sme lots were stripped nd reproed with control nti-c-src ntiody, showing equl loding nd confirming the signl detected with the phospho-ntiody (lower pnels). The utordiogrph shows representtive Western lot. Br grphs represent intensities of phospho-c-src quntified y scnning densitometry of lots nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with one-wy nlysis of vrince (ANOVA) followed y Newmn Keuls multiple-comprison test. Sttisticl significnce: Po.1 versus unstimulted control cells Kidney Interntionl (6) 69,

3 LD Alexnder et l.: Archidonic cid induces c-src ctivtion o r i g i n l r t i c l e Src ctivity Archidonic cid (15 μmol/l) Empty vector PP2 (1 μmol/l) c-src DN Figure 2 Archidonic cid nd ngiotensin II increses c-src kinse ctivity. Rit proximl tuulr epithelil cells were grown to confluency nd serum strved for 48 h. The cells were pretreted with PP2 (1 mmol/l) or trnsfected with control plsmid cdna (empty vector) or dominnt-negtive c-src K298M (c-src DN), nd were mde quiescent with serum-free medium for 24 h. They were then stimulted with rchidonic cid (15 mmol/l) or ngiotensin II (1 mmol/l) for 5 min. Activtion of c-src kinse ws detected s descried in Mterils nd methods. Vlues re represented s men7s.e.m. of three independent experiments nd re expressed s percent reltive to control with unstimulted s 1%. Sttisticl comprisons were mde with Student s t-test or one-wy nlysis of vrince (ANOVA), followed y Newmn Keuls multiple-comprison test. Sttisticl significnce: Asterisks indicte significnt effect of rchidonic cid or ngiotensin II tretment compred with the non-stimulted-empty vector control cells (Po.1). þþ Po.1, þþþ Po.1 indicte significnt effect of PP2 or c-src DN construct compred with the rchidonic cid- nd ngiotensin II-stimulted cells. ngiotensin II-induced c-src ctivtion in kidney epithelium occurs vi AT 2 receptor-dependent signling pthwys nd tht ngiotensin II evokes rchidonic cid relese y stimulting cpla 2, ut not independent phospholipse A 2 in rit proximl tuule cells. Moreover, rchidonic cid-induced c-src ctivtion ppers to e direct nd independent of eicosnoid iosynthesis. Angiotensin II nd rchidonic cid ctivte extrcellulr signl-regulted kinse vi c-src-dependent phosphoryltion of the EGFR Erlier studies, y this lortory, demonstrted tht ngiotensin II nd rchidonic cid stimulted phosphoryltion of EGFR in cultured proximl tuule cells. 13 Likewise, EGFR trnsctivtion ppers to e meditor of ngiotensin II nd rchidonic cid signling, leding to ERK ctivtion. To exmine the role of c-src ctivtion in EGFR trnsctivtion nd ERK ctivtion y ngiotensin II nd rchidonic cid, cells were pretreted either with the Src fmily kinse inhiitor PP2 or trnsiently trnsfected with dominnt-negtive mutnt of c-src. Blockde of Src fmily kinses using PP2 significntly decresed rchidonic cid- nd ngiotensin II-induced ERK ctivtion in dosedependent mnner (Figure 5 nd, respectively). PP2 tretment, however, did not ffect EGF-induced EGFR tyrosine phosphoryltion nd ERK ctivtion (Figure 6 nd, respectively). Similrly, trnsfection of proximl tuule cells with dominnt-negtive c-src did not ffect the ility of EGF to stimulte EGFR tyrosine phosphoryltion (Figure 7) : p-src [py 418 ] Totl Src Archidonic cid (15 μmol/l): Src phosphoryltion Src phosphoryltion Nonstimulted control Archidonic cid (15 μmol/l) +++ nd ERK ctivtion (Figure 7c), ut significntly reduced ngiotensin II- nd rchidonic cid-induced c-src ctivtion, EGFR tyrosine phosphoryltion nd ERK ctivtion (Figure 7 c, respectively). By nlogy, pretretment with the selective EGFR ntgonist AG1478 gretly reduced ngiotensin II-, rchidonic cid-, nd EGF-induced EGFR tyrosine phosphoryltion (Figure 8), ut hd no effect on the cpcity of ngiotensin II nd rchidonic cid to induce the ctivtion of c-src (Figure 8). In ddition, EGF tretment ppered to hve no effect on c-src ctivtion (Figures 7 nd 8, respectively). Moreover, mong the memers of the Src fmily kinses, only c-src ws specificlly coimmunoprecipitted with the EGFR (Figure 9 nd ). In ddition, rchidonic cid-induced c-src ssocition with the EGFR DMSO : p-src [py 418 ] Totl Src : Nonstimulted control ++ ** ** DMSO Figure 3 Effects of Src fmily kinse inhiitor PP2 on rchidonic cid- nd ngiotensin II c-src ctivtion. Rit proximl tuulr epithelil cells were grown to confluency nd serum strved for 48 h. Quiescent cells were incuted with or without PP2 (1, 1, nd 2 mmol/l) for 3 min, followed y exposure to () rchidonic cid (15 mmol/l) or () ngiotensin II (1 mmol/l) for 5 min. The cells were then lysed nd equl mounts of proteins were resolved y 1% SDS-PAGE nd immunolotted with n nti-phospho-c-src polyclonl ntiody (p-src (py 418 ), upper lots). The sme lots were stripped nd reproed with n nti-c-src ntiody to demonstrte equl protein loding (lower lots). The utordiogrph shows representtive Western lot. (, ) Densitometric nlysis of the experiments is reported. Br grphs represent intensities of phospho-c-src quntified y scnning densitometry of lots nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with one-wy nlysis of vrince (ANOVA) followed y Newmn Keuls multiple-comprison test. Sttisticl significnce: Asterisks indicte significnt effect of PP2 tretment compred with the rchidonic cid- nd ngiotensin II-stimulted cells ( ** Po.1; Po.1). þþ Po.1; þþþ Po.1 indicte significnt effects of rchidonic cid nd ngiotensin II tretment compred with the non-stimulted (DMSO-treted) control cells. Kidney Interntionl (6) 69,

4 o r i g i n l r t i c l e LD Alexnder et l.: Archidonic cid induces c-src ctivtion Angiotensin II Losrtn + + Cndesrtn + + PD p-erk 1/2 Archidonic cid (15 μmol/l) p-erk 1/2 p-c-src ERK 1/2 ERK 1/2 Src c-src phosphoryltion p-c-src Src c-src phosphoryltion 35 3 Angiotensin II Lor Cnd PD MAFP (1 μmol/l) + + HELSS (25 μmol/l) + + Mepcrine (5 μmol/l) CONT ws locked y pretretment of the cells with the Src fmily kinse inhiitor PP2 (Figure 9) nd y trnsient expression of dominnt-negtive c-src (Figure 9). Thus, we hve demonstrted for the first time tht, mong the memers of the Src fmily kinses, only c-src specificlly coimmunoprecipitte with the EGFR. Collectively, these dt suggest tht c-src is involved in ngiotensin II- nd rchidonic cidinduced trnsctivtion of the EGFR nd ERK ctivtion, nd +++ MAFP +++ HELSS MEP Figure 4 Effects of ngiotensin II-receptor ntgonist on ngiotensin II-induced c-src phosphoryltion. Rit proximl tuulr epithelil cells were grown to confluency nd serum strved for 48 h. () Quiescent cells were incuted with or without losrtn (Lor,1 mmol/l), cndesrtn (Cnd,1 mmol/l) or PD (PD,1 mmol/l) for 6 min, followed y exposure to ngiotensin II (1 mmol/l) for 5 min or () incuted with or without 1 mmol/l MAFP or 25 mmol/l hloenol lctone suicide sustrte (HELSS) for 3 min or 5 mmol/l mepcrine for 5 min, nd then exposed to 1 mmol/l ngiotensin II for 5 min. The cells were then hrvested nd equl mounts of proteins were resolved y 1% SDS-PAGE nd immunolotted with n nti-phospho-c-src polyclonl ntiody (p-src (py 418 ), upper lots). The sme lots were stripped nd reproed with n nti-c-src ntiody to demonstrte equl protein loding (lower lots). The utordiogrph shows representtive Western lot. Br grphs represent intensities of phospho-c-src quntified y scnning densitometry of lots, nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of t lest three independent experiments. Sttisticl significnce: Po.1 versus nonstimulted control with the sme group; þþþ Po.1, þþ Po.1; Lor, Cnd, PD, MAFP, HELSS or mepcrine (MEP) versus ngiotensin II-stimulted cells. ERK 1/2 phosphoryltion Archidonic cid (15 μmol/l) ERK 1/2 phosphoryltion Figure 5 Effects of Src fmily kinse inhiitor PP2 on rchidonic cid- nd ngiotensin II-induced ERK phosphoryltion. Rit proximl tuulr epithelil cells were grown to confluency nd serum strved for 48 h. Cells were pretreted with PP2 (1, 1 or 2 mmol/l) for 3 min, followed y () rchidonic cid (15 mmol/l) or () ngiotensin II (1 mmol/l) for 5 min. The cells were then lysed nd totl cellulr lystes (2 mg) were immunolotted with ntiodies to ERK 1/ 2 phospho-threonine 22 /tyrosine 24 (perk 1/2; upper pnel). The sme lots were stripped nd reproed with control nti-erk1/2 ntiody showing equl loding (lower pnel). The utordiogrph shows representtive Western lot. Br grphs represents the intensities of phospho-erk 1/2 quntified y scnning densitometry of lots nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with one-wy nlysis of vrince (ANOVA) followed y Newmn Keuls multiple-comprison test. Sttisticl significnce: Po.1 versus stimulted cells fter PP2 tretment. EGF (1 ng/ml): + + PP2 (1 μmol/l): + + IB: PY2 EGFR phosphoryltion EGF (1 ng/ml) NS PP2 (1 μmol/l) EGF (1 ng/ml): + + PP2 (1 μmol/l): + + p-erk /2 ERK 1/2 ERK 1/2 phosphoryltion EGF (1 ng/ml) NS PP2 (1 μmol/l) Figure 6 Src fmily kinse inhiitor hs no effect on EGFinduced EGFR tyrosine phosphoryltion nd ERK ctivtion. Rit proximl tuulr epithelil cells were grown to confluency nd serum strved for 48 h. Cells were pretreted with PP2 (1 mmol/l) for 3 min, followed y EGF (1 ng/ml) for 5 min. The cells were then lysed nd totl cellulr lystes were () immunoprecipitted (.5 mg protein) with n nti-egfr (15) rit polyclonl ntiody followed y lotting with nti-phosphotyrosine (PY2) ntiody (upper pnels), or () immunolotted (2 mg) with ntiodies to ERK 1/2 phospho-threonine 22 /tyrosine 24 (perk 1/2; upper pnel). The sme lots were stripped nd reproed with control nti-egfr or nti-erk1/2 ntiodies showing equl loding (lower pnels). The utordiogrph shows representtive Western lot. Br grphs represent intensities of phospho-egfr or phospho-erk 1/2 quntified y scnning densitometry of lots, nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with Student s t-test. Sttisticl significnce: NS versus stimulted cells fter PP2 tretment Kidney Interntionl (6) 69,

5 LD Alexnder et l.: Archidonic cid induces c-src ctivtion o r i g i n l r t i c l e c EGF (1 ng/ml): + + Archidonic cid (15 μmol/l): + + : + + p-src [Try 418 ] Totl c-src EGF (1 ng/ml): + + Archidonic cid (15 μmol/l): + + : + + IB: PY2 EGF (1 ng/ml): + + Archidonic cid (15 μmol/l): + + : + + p-erk 1/2 ERK 1/2 + c-src DN + c-src DN + c-src DN Figure 7 Angiotensin II nd rchidonic cid ctivte ERK vi c-src-dependent phosphoryltion of the EGFR in rit proximl tuule cells. Rit proximl tuulr epithelil cells were grown to 8% confluency, trnsfected with empty vector (pcmv5) or dominnt-negtive c-src K298M (c-src DN), nd susequently mde quiescent with serum-free medium for 24 h. Cells were then incuted either in the presence or sence of ngiotensin II (1 mmol/l), rchidonic cid (15 mmol/l) or EGF (1 ng/ml) for 5 min. The cells were then lysed nd equl mounts of proteins were sujected to immunolot nlysis with polyclonl ntiodies tht specificlly recognize the phosphorylted forms of () c-src or (c) ERK 1/2, or () were immunoprecipitted with n nti-egfr (15) rit polyclonl ntiody followed y lotting with nti-phosphotyrosine (PY2) ntiody (upper pnels). The sme lots were stripped nd reproed with control nti-c-src, nti-erk 1/2, or nti-egfr ntiodies showing equl loding (lower pnels). The utordiogrphs represent the results of single experiment, which ws crried out t lest three times. is consistent with c-src eing upstrem of EGFR tyrosine phosphoryltion nd ERK ctivtion. Archidonic cid induces inding of EGFR with the c-src homology 2 domin GST fusion protein We evluted whether there ws n ssocition of the EGFR with the c-sh2 domin. Here cell lystes were immunoprecipitted with c-src GST SH2 fusion protein nd susequently immunolotted with monoclonl nti-egfr ntiodies. As shown in Figure 1, rchidonic cid induced inding of EGFR with the c-src SH2 domin fusion protein in time-dependent mnner tht ws significnt t 5 min, mximl t 15 min, nd returned to sl levels y 6 min. This effect ws significnt t 5 mmol/l nd reched plteu y 15 mmol/l (Figure 1). Angiotensin II stimulted n ssocition of c-src SH2 domin with the EGFR in mnner similr to rchidonic cid (Figure 1c nd d). In ddition, PP2 inhiited rchidonic cid- nd ngiotensin II-induced ssocition of EGFR with the c-src SH2 domin GST fusion protein y 6475% (Po.1) nd 672% (Po.1), respectively, while hving no effect on EGF-induced ssocition of EGFR with the c-src SH2 domin of GST fusion EGF(1 ng/ml): + + Archidonic cid (15 μmol/l): + + : + + IB: PY2 EGF (1 ng/ml): + + Archidonic cid (15 μmol/l): + + : + + p-src [Try 418 ] Totl c-src AG1478 AG1478 Figure 8 Effects of EGFR kinse inhiitor AG1478 on ngiotensin II- nd rchidonic cid-induced EGFR tyrosine phosphoryltion nd c-src ctivtion in rit proximl tuule cells. Rit proximl tuulr epithelil cells were grown to confluency nd serum strved for 48 h. Cells were pretreted with AG1478 (5 nmol/l) for 6 min, followed y ngiotensin II (1 mmol/l), rchidonic cid (15 mmol/l) or EGF (1 ng/ml) for 5 min. The cells were then lysed nd (.5 mg protein) were () immunoprecipitted with n nti-egfr (15) rit polyclonl ntiody followed y lotting with nti-phosphotyrosine (PY2) ntiody, or equl mounts of proteins were used for Western lotting. The lots were proed with () rit polyclonl nti-phosphorylted c-src (py 418 ) (upper pnels). The sme lots were stripped nd reproed with control nti-egfr or nt-c-src ntiodies showing equl loding (lower pnels). The utordiogrph represents the results of single experiment, which ws crried out t lest three times. Archidonic cid (min): PP2 (1 μmol/l): IP: c-src IP: c-src Archidonic cid (min): c-src DN: IP: c-src IP: c-src Figure 9 Archidonic cid-induced EGFR ssocition with c-src is dependent of c-src. Serum-strved proximl tuulr cells were either pretreted with () PP2 (1 mmol/l) or () trnsfected with control plsmid or dominnt-negtive c-src K298M (c-src DN constructs) serum restricted for 48 h, followed y stimultion with rchidonic cid (15 mmol/l) for 5 min. The cells were then lysed nd totl cellulr lystes (.5 mg protein) were immunoprecipitted with nti-c-src monoclonl ntiody, followed y lotting with nti-egfr polyclonl ntiody (upper pnels). The memrne ws stripped nd reproed with n nti-c-src monoclonl ntiody (ottom pnels). The utordiogrphs represent the results of single experiment, which ws crried out t lest three times. protein (Figure 11). Similrly, overexpression of dominntnegtive c-src totlly rogted rchidonic cid- nd ngiotensin II-induced ssocition of EGFR with the c-src SH2 domin GST fusion protein, while hving no effect on EGFinduced ssocition of EGFR with the c-src SH2 domin GST fusion protein (Figure 11). Collectively, these dt demonstrte tht in erly pssged kidney epithelil cells, rchidonic cid- nd ngiotensin II-induced trnsctivtion Kidney Interntionl (6) 69,

6 o r i g i n l r t i c l e LD Alexnder et l.: Archidonic cid induces c-src ctivtion c Archidonic cid (min) Associted EGFR Angiotensin II (min) Archidonic cid (min) d Archidonic cid (μmol/l) Associted EGFR Angiotensin II (log M) Archidonic cid (μmol/l) * Associted EGFR Angiotensin II (log M) Associted EGFR ** Angiotensin II (log M) Figure 1 Archidonic cid- nd ngiotensin II-induced time- nd dose-dependent ssocition of Src SH2 domin with the EGFR. Serum-strved proximl tuulr cells were () treted either without or with 15 mmol/l rchidonic cid or (c) 1 mmol/l ngiotensin II for the indicted times or incuted with vrious concentrtions of () rchidonic cid or (d) ngiotensin II for 5 min t 371C. Cells were lysed nd immunoprecipitted (IP) with GST-Src SH2-Sephrose s descried in Mterils nd methods. Precipitted proteins were resolved y SDS-PAGE nd immunolotted (IB) with polyclonl nti-egfr ntiody (upper pnels). The memrne ws stripped nd reproed with n nti-c-src monoclonl ntiody (ottom pnels). Comprle mounts of proteins were detected in immunoprecipittes of ll the ove experiments. The utordiogrphs represent the results of single experiment, which ws crried out t lest three times. Br grphs represent intensities of ssocited EGFR quntified y scnning densitometry of lots nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with one-wy nlysis of vrince (ANOVA) followed y Newmn Keuls multiple-comprison test. Sttisticl significnce: Po.1, **Po.1, *Po.5 versus unstimulted control cells. of the EGFR occur vi inducing the ssocition of EGFR with the SH2 domin of the Src protein, nd tht the tyrosine phosphoryltion of c-src is involved. Archidonic cid induces ssocition of EGFR with the c-src homology 2 domin GST fusion protein independent of eicosnoid iosynthesis We hve previously demonstrted tht unsturted longchin free ftty cids such s linoleic nd linolenic cid potently ctivted memers of the MAP-kinse superfmily 15 in rit proximl tuule cells. As shown in Figure 12, stimultion of proximl tuule cells with 5,8,11,14-eicostetrynoic cid (ETYA), non-metolized nlog of rchidonic cid, which locks rchidonic cid oxidiztion y cting s flse sustrte, 16,17 induced ssocition of the EGFR with the c-src SH2 domin GST fusion protein y % (Po.1), s compred with control. Two polyunsturted ftty cids, linoleic cid (C18:2), nd linolenic cid (C18:3), lso significntly incresed ssocition of the EGFR with the c-src SH2 domin GST fusion protein y 24717% nd 23723%, respectively, s compred to control. By contrst, neither the monounsturted ftty cid plmitoleic cid (C16:1) nor the two sturted ftty cids, steric (C18), nd rchidic cid (C2), hd ny effects on the ssocition of the EGFR with the c-src SH2 domin GST fusion protein. Agin, the ction of rchidonic cid ws not ffected y specific inhiitors of cyclooxygense-, lipoxygense-, nd cytochrome P45-dependent pthwys (dt not shown), which is consistent with the notion tht the effects of rchidonic cid or ETYA on the ssocition of the EGFR with the c-src SH2 domin GST fusion protein re specific effects nd re therefore not dependent on n rchidonic cid metolite. Specificity of inding of the EGFR with the c-src SH2 domin versus SH3 domin ws demonstrted, since rchidonic cid did not induce ssocition of the EGFR with the c-src SH3 domin GST fusion protein, n effect tht ws similr to ngiotensin II (Figure 13). DISCUSSION In prior reports, we demonstrted tht ngiotensin II nd rchidonic cid rpidly ctivte plethor of kinses, including the EGFR tyrosine kinse, p21rs, nd memers of the MAP kinse superfmily, prticulrly ERK, c-jun NH 2 - terminl kinse, nd p38 MAPK, in rit proximl tuule cells. 6,13,15,18 However, the molecules involved in the signling re not yet fully chrcterized. This report descries the mechnism of trnsmemrne signling tht is involved in 1828 Kidney Interntionl (6) 69,

7 LD Alexnder et l.: Archidonic cid induces c-src ctivtion o r i g i n l r t i c l e PP2 (1 μmol/l): EGF (1 ng/ml): + + : + + Archidonic cid (15 μmol/l): + + Associted EGFR 5 4 EGF (1 ng/ml) PP2 (1 μmol/l) Associted EGFR Archidonic cid (15 μmol/l) c-src DN: EGF (1 ng/ml): + + : + + Archidonic cid (15 μmol/l): + + Archidonic cid (15 μmol/l) EGF (1 ng/ml) csrc DN Figure 11 Assocition of the EGFR with Src SH2 domin is dependent on c-src. Serum-strved proximl tuulr cells were either pretreted with () PP2 (1 mmol/l) or () they were trnsfected with control plsmid or dominnt negtive c-src K298M (c-src DN constructs, serum restricted for 48 h. Cells were then incuted either in the presence or sence of rchidonic cid (15 mmol/l), ngiotensin II (1 mmol/l), or EGF (1 ng/ml) for 5 min. Cells were lysed nd immunoprecipitted (IP) with GST-Src SH2-Sephrose s descried in Mterils nd methods. Precipitted proteins were resolved y SDS-PAGE nd immunolotted (IB) with polyclonl nti-egfr ntiody (upper pnels). The memrne ws stripped nd reproed with n nti-c-src monoclonl ntiody (ottom pnels). Comprle mounts of proteins were detected in immunoprecipittes of ll the ove experiments. The utordiogrphs represent the results of single experiment, which ws crried out t lest three times. Br grphs represents intensities of ssocited EGFR quntified y scnning densitometry of lots nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with one-wy nlysis of vrince (ANOVA) followed y Newmn-Keuls multiple comprison test. Sttisticl significnce: þþþ Po.1 indictes significnt effect of PP2 or c-src DN construct compred with the rchidonic cid-, ngiotensin II-, nd EGF-stimulted cells versus unstimulted control cells. ngiotensin II- nd rchidonic cid-induced EGFR tyrosine phosphoryltion nd ERK ctivtion. We report for the first time tht (1) rchidonic cid itself, nd not its metolic metolites, is the direct effector of c-src ctivtion; (2) rchidonic cid, ETYA nd other ftty cids trnsctivte the EGFR vi inducing ssocition of SH2 domin with the EGFR; (3) ngiotensin II- nd rchidonic cid-induced c-src ssocition with the EGFR ppers to e specific for c-src SH2 domin versus SH3 domin nd (4) c-src-medited EGFR trnsctivtion ppers to e criticl component Associted EGFR of oth ngiotensin II- nd rchidonic cid-induced ERK ctivtion in renl proximl tuulr epithelil cells. Severl G protein-coupled receptors, including ngiotensin II, hve een presumed to induce ctivtion of Src; however, the mechnisms hve not een determined. In the present study, ngiotensin II incresed oth c-src phosphoryltion nd ctivity in mnner tht is temporlly consistent with role of c-src in mediting ngiotensin II-induced ERK ctivity. In ddition, there ppers to e n ngiotensin II AT 2 -receptor-medited mechnism for ctivtion of c-src, wherein AT 2 -receptor lockde completely inhiited ngiotensin II-induced c-src ctivtion, wheres AT 1 -receptor lockde did not. This is in discordnce with study in opossum kidney cells demonstrting tht ngiotensin II ctivted c-src vi ctivtion of the AT 1 receptor. 19 Similr requirements for involvement of ngiotensin II-stimulted c-src ctivity hve een demonstrted in smooth muscle cells, 9,2 22 heptocytes, 23 crdic firolsts, 24 nd crdic myocytes. 25 Together, these experiments indicte tht the downstrem ctions of the AT 2 receptor my e highly species dependent (i.e., opossum versus rit) nd/or cell ** * ** * Archidonic ETYA Linoleic Linolenic Plmitoleic Archidonic ETYA Linoleic Linolenic Plmitoleic Steric Archidic * Steric Archidic Figure 12 Archidonic cid-induced ssocition of Src SH2 domin with the EGFR is ftty cid dependent, ut eicosnoid independent. Serum-strved proximl tuulr cells were exposed to equl molr concentrtions (15 mmol/l) of rchidonic cid (C2:4), ETYA (C2:4), linoleic cid (C18:2), linolenic cid (C18:3), plmitoleic cid (C16:1), steric cid (C18), or rchidic cid (C2) for 5 min t 371C. The numers in prentheses indicte the cron chin length followed y the numer of doule onds. Cells were lysed nd immunoprecipitted (IP) with GST-Src SH2-Sephrose s descried in Mterils nd methods. Precipitted proteins were resolved y SDS-PAGE nd immunolotted (IB) with polyclonl nti-egfr ntiody (upper pnel). The memrne ws stripped nd reproed with n nti-c-src monoclonl ntiody (ottom pnel). Comprle mounts of proteins were detected in immunoprecipittes of ll the ove experiments. The utordiogrphs represent the results of single experiment, which ws crried out t lest three times. Br grphs represents intensities of ssocited EGFR quntified y scnning densitometry of lots nd re expressed s percentge reltive to control with unstimulted s 1%. Vlues re represented s men7s.e.m. of three independent experiments. Sttisticl comprisons were mde with one-wy nlysis of vrince (ANOVA) followed y Newmn-Keuls multiple comprison test. Sttisticl significnce: Po.1, *Po.5 versus unstimulted control cells. Kidney Interntionl (6) 69,

8 o r i g i n l r t i c l e LD Alexnder et l.: Archidonic cid induces c-src ctivtion AA Ang II EGF IP: GST fusion protein IP: Src SH2 domin IP: Src SH3 domin Associted EGFR GST c-src SH2 c-src SH3 AA Ang II EGF Figure 13 Archidonic cid-, ngiotensin II-, nd EGF-induced incresed ssocition of EGFR with the c-src SH2 domin, ut not the c-src SH3 domin. Serum-strved proximl tuulr cells were treted in either the sence or presence of rchidonic cid (AA, 15 mm), ngiotensin II (Ang II, 1 mm) or EGF (1 ng/ml) for 5 min t 371C. Cell lystes were immunoprecipitted (IP) with GST-Sephrose, GST-Src SH2- or GST-Src SH3-Sephrose s descried in Mterils nd methods. Precipitted proteins (5 mg) were resolved y SDS-PAGE nd immunolotted (IB) with polyclonl nti-egfr ntiody (upper pnel). The memrne ws stripped nd reproed with n nti-c-src monoclonl ntiody (ottom pnel). Comprle mounts of proteins were detected in immunoprecipittes of ll the ove experiments (dt not shown). A representtive utordiogrm from t lest three independent experiments is shown. Br grphs with error rs represent the men7s.e.m. (n ¼ 3). Po.1 indictes significnt increse in EGFR tyrosine phosphoryltion due to its ssocition with either c-src SH2 or SH3 domin y treted cells versus the sme mtched gonist-stimulted GST cells. type (i.e., vsculture versus epithelil nd primry versus cell lines) specific. In ddition, we present experimentl evidence for n ngiotensin II AT 2 -receptor-medited mechnism for ctivtion of c-src, involving PLA 2 ctivtion nd rchidonic cid relese. Mepcrine nd MAFP, two structurlly unrelted cpla 2 inhiitors, significntly locked ngiotensin II-induced c-src ctivtion, suggesting tht rchidonic cid is involved in ngiotensin II-induced c-src ctivtion. Conversely, inhiition of clcium-independent PLA 2 with hloenol lctone suicide sustrte hd no effect on ngiotensin IIinduced c-src ctivtion, indicting tht c-src ctivtion is dependent of cpla 2 enzyme ctivity in rit proximl tuule cells. Moreover, rchidonic cid itself induced timend dose-dependent phosphoryltion of c-src, supporting the importnce of PLA 2 s meditor of ngiotensin II signling. In ddition, the effects of oth ngiotensin II nd rchidonic cid on c-src phosphoryltion were independent of eicosnoid iosynthesis in tht inhiitors of cyclooxygense, lipoxygense, nd cytochrome P45 isoenzyme were without effect. Collectively, these oservtions document mechnism of ngiotensin II-induced c-src ctivtion, medited y cpla 2 -dependent relese of rchidonic cid independent of production of epoxy derivtives of rchidonic cid. Angiotensin II-induced ERK ctivtion in mny other cell types requires n Src-dependent ctivtion of the EGFR This supports the ssumption tht c-src cts upstrem of the EGFR. In the present study, we showed tht stimultion of proximl tuule cells with ngiotensin II resulted in significnt EGFR tyrosine phosphoryltion nd its ssocition with c-src. Moreover, rchidonic cid mimicked these effects in mnner similr to ngiotensin II. In ddition, oth ngiotensin II- nd rchidonic cid-induced EGFR tyrosine phosphoryltion were uniformly inhiited y the Src fmily kinse inhiitor PP2 nd y trnsient overexpression of dominnt-negtive mutnt of c-src, therey suggesting tht the ctivtion of c-src might e importnt for the ctivtion of ERK in proximl tuule cells exposed to ngiotensin II nd rchidonic cid. By comprison, neither PP2 nor dominnt-negtive c-src effected EGF-induced EGFR tyrosine utophosphoryltion or EGF-induced ERK ctivtion. Interestingly, c-src ctivtion is not inhiited y the EGFR kinse inhiitor AG1478, suggesting tht the EGFR is downstrem of c-src ctivtion. Although rchidonic cid stimulted ctivtion of other memers of the Src fmily kinses, only c-src ws specificlly co-immunoprecipitted with the EGFR. The ctivtion of c-src in proximl tuules y rchidonic cid is novel. This is in strk contrst to those results otined in LLCPK 1 cells, wherey rchidonic cid ws ineffective in inducing EGFR tyrosine phosphoryltion nd c-src ctivtion. However, Chen et l. 3,31 demonstrted tht rchidonic cid induced significnt increses in EGFR tyrosine phosphoryltion nd ssocition with c-src in F87V BM-3 trnsfected cells (regio- nd stereoselective for 14(S), 15(R)-EET) tht ws inhiited y 17-octdecynoic cid, wheres no such effects were oserved in vector-trnsfected cells. Thus, their experimentl results suggested n eicosnoid-dependent tyrosine phosphoryltion of EGFR nd c-src ctivtion in LLCPK1 cells. This too is in discordnce to our studies, wherey we consistently oserved significnt tyrosine phosphoryltion of EGFR nd increses in c-src ctivity with exogenous rchidonic cid independent of eicosnoid iosynthesis. These differences etween the two reports my e due to differences in either culture conditions or cell type specificity. Tken together, our findings provide strong support for the contention tht Src fmily kinses ply n importnt role in mediting EGFR trnsctivtion y rchidonic cid nd ngiotensin II. They lso suggest specific role for c-src in the rchidonic cid nd ngiotensin II stimultion of ERK ctivity, ut they do not exclude contriutions y other Src fmily kinses expressed in proximl tuule cells, prticulrly Fyn nd Yes. Thus, further studies re required to explore the roles, if ny, for these Src fmily kinses s meditors of rchidonic cid- nd ngiotensin II-induced MAP kinse ctivtion. Both the kinse nd the SH2 domin of Src re considered necessry for gonist-induced ctivtion of receptor protein tyrosine kinses Our findings show tht oth ngiotensin II nd rchidonic cid induced the ssocition of the SH2 domin of c-src with the EGFR in dose- nd timedependent mnner tht ws significntly rogted y PP2 or y trnsient overexpression of dominnt-negtive c-src 183 Kidney Interntionl (6) 69,

9 LD Alexnder et l.: Archidonic cid induces c-src ctivtion o r i g i n l r t i c l e mutnt. The effect of ngiotensin II nd rchidonic cid on EGFR tyrosine phosphoryltion nd its ssocition with Src SH2 domin ws similr to the effect of 1 ng/ml EGF. In contrst, the c-src SH3 domin did not interct directly with tyrosine-phosphorylted EGFR. This is in greement with previous study demonstrting tht lysophosphtidic cid, G protein-coupled receptor gonist, induces the ility of the c-src SH2 domin GST fusion protein (ut not the SH3 domin) to precipitte the tyrosine-phosphorylted EGFR. 33 Moreover, the effects were mimicked y ETYA ( nonmetolized stle nlog of rchidonic cid which locks rchidonic cid oxidtion y cting s flse sustrte), 16,17 linoleic cid, nd linolenic cid, suggesting tht downstrem eicosnoid metolites do not medite the effects. Thus, ssocition of Src SH2 domin with the EGFR ppers to e only triggered y polyunsturted, long-cron chin ftty cids (e.g., rchidonic cid, linoleic cid, nd linolenic cid), wheres the ssocition is not ffected y short cron chin (e.g., plmitoleic cid) or sturted (e.g., steric cid nd rchidic cid) ftty cids. The relevnce of the present finding is the fct tht rchidonic cid represents novel mechnism wherey G protein-coupled receptors re linked to receptor nd non-receptor protein tyrosine kinses in epithelil cells. Moreover, these studies re oth timely nd significnt, wherefore they my hve the potentil to define new mechnism for G protein-coupled receptor-induced c-src/egfr-medited downstrem signling, tht is through rchidonic cid, common lipid second messenger. MATERIALS AND METHODS Mterils Archidonic cid ws otined from ICN Phrmceuticls (Cost Ms, CA, USA). (Sr 1 )-ngiotensin II, rdykinin, PD123319, ETYA, nd ftty cids (linoleic, linolenic, oleic, plmitoleic, steric, nd rchidic cids) were otined from Sigm (St Louis, MO, USA). Humn EGF, LipofectAMINE, cell culture medium, nd dditives were otined from Gico BRL Life Technologies (Grnd Islnd, NY, USA). PP2, AG1478, mepcrine, MAFP, hloenol lctone suicide sustrte, horserdish peroxidse-conjugted got ntimouse, nd rit immunogloulin G were otined from Cliochem (L Joll, CA, USA). [g 32 P]ATP ws otined from Dupont/NEN (Boston, MA, USA). c-src (Src 2), Lck (212), Lyn (44) Fyn (15) nd c-yes (3) rit polyclonl ntiodies, protein A-, nd protein A/G Plus-grose eds were otined from Snt Cruz Biotechnology (Snt Cruz, CA, USA). Anti-EGFR sheep polyclonl ntiody, nti-phosphotyrosine (PY2) monoclonl ntiody, nd rit nti-sheep immunogloulin G were otined from Upstte Biotechnology (Lke Plcid, NY, USA). Phospho-specific p44/42 MAP kinse (Thr22/Tyr24 polyclonl ntiody) ws purchsed from Cell Signling Technology (Beverly, MA, USA). Phosphospecific c-src Tyr418 polyclonl ntiody ws purchsed from Biosource Interntionl (Cmrillo, CA, USA), nd glutthione Sephrose 4B eds were otined from Amershm Phrmci Biotech (Pisctwy, NJ, USA). Losrtn (DuP 753) ws the generous gift of Dupont-Merck (White house sttion, NJ, USA). Cndesrtn ws kind gift of AstrZenec (Mölndl, Sweden). Dominnt-negtive c-src K298M ws kind gift from Dr Chung- Ho Chng (Cse Western Reserve University). Cell culture Primry culture of proximl tuule cells were isolted from mle New Zelnd white rits nd mintined in stndrd growth medi s descried previously. 14 Trnsient. trnsfection of dominnt-negtive c-src The cells were trnsfected with 1 mg of plsmid DNA using the Lipofectmine regent ccording to the mnufcturer s instructions. For mock trnsfection, the pcmv5 vector without the cdna inserts ws employed. c-src kinse ssy, immunoprecipittion, nd Western lotting c-src kinse ctivity ws determined y using Src kinse ssy kit (Upstte Biotechnology) ccording to the mnufcturer s protocol. Briefly, 5 mg of protein ws incuted with 1 mg of nti-c-src ntiody 327 for 2 h t 41C, nd the immune complexes were recovered with protein A Plus G Sephrose (Snt Cruz Lortories Inc.). The ctivity ws determined y phosphoryltion of 1 ml Src kinse sustrte peptide (No ) in Src kinse rection uffer nd 1 ml of[g 32 P]ATP. Immunoprecipittion nd Western lotting were performed s descried previously. 14 Protein contents were determined y icinchoninic cid ssy. Sttisticl nlysis The results re expressed s mens7s.e.m. from comined experiments. The dt were evluted y one-wy nlysis of vrince followed y Newmn Keuls multiple-comprison test or y pired Student s t-test when pproprite. The limit of significnce ws P-vlue p.5. 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