ATP- and Polyphosphate-mediated Stimulation of pp60c-src Kinase Activity in Extracts from Vascular Smooth Muscle*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The Amerian Soiety for Biohemistry and Moleular Biology, In Vol. 264, No. 18, Issue of June 25, pp ,1989 Printed in U. S. A. ATP- and Polyphosphate-mediated Stimulation of pp60-sr Kinase Ativity in Extrats from Vasular Smooth Musle* (Reeived for publiation, August 9, 1988) Joseph Di Salvo$, Donetta Gifford, and Anastasia Kokkinakis From the Department of Physiology and Biophysis, University of Cininnati College of Mediine, Cininnati Ohio Reently, we reported that pp60' kinase ativity alled v-sr. pp60-ax is enoded by the protoonogene -sr. was present in adult bovine oronary arterial smooth Although the ellular funtions of pp6oc~' are unknown, musle and showed that the ativity of the enzyme in several reports have suggested that it may partiipate in in vitro immunopreipitation assays was stimulated regulating signal transdution and ellular differentiation ( fold by ATP (Di Salvo, J., Gifford, D., and 7). In this ontext, Simon et al. (6) reported that high levels Kokkinakis, A. (1988) Biohem. Biophys. Res. Com- of mrna for -sr were expressed in the viseral smooth mun. 153, ). In the present study, ATP-mediated stimulation of ativity was also demonstrated in musle of Drosophila during early embryogenesis whereas low extrats from aorti vasular smooth musle. In onlevels were expressed during later stages of development. trast, no stimulation was apparent in extrats from However, enzymi ativity ofpp6oc- during early or late brain. Stimulation of ativity in vasular preparations development of Drosophila was not reported. In this ontext, was also indued 5triphos- preliminary studies in our laboratory showed that moderate phate (AMP-PNP), a nonmetabolizable analog of ATP, levels of pp60-ax kinase ativity were present in adult bovine and with several other polyphosphates inluding ADP oronary arterial smooth musle (8). A signifiant and interand sodium pyrophosphate. No stimulation ourred in esting feature of the enzyme whih we observed in mammalian response to monophosphates suh as AMP or KHzP04. oronary extrats was that its ativity ould be stimulated As expeted, the speifi ativity of pp60-' in brain more than 20-fold by preinubation of the extrat with ATP. extrats did not hange when the amount of extrated Thus, the potential ativity of oronary pp60~ was as high protein inluded in immunopreipitation mixtures was as the ativity reportedly present in brain (7, 9, 10) and inreased. Unexpetedly, however, the speifi ativprobably as high as the ativity present in ertain transformed ity of the vasular enzyme dereased markedly as the amount of extrated protein subjeted to immunopreells (1-3, 11, 12). Our results also suggested the possibility ipitation was inreased. Following stimulation of that the ativity of vasular pp60~ might be subjet to pp60-' in vasular extrats with ATP, the enzyme regulation by ATP through a mehanism whih did not inbehaved in a fashion similar to pp6oc~' extrated from volve phosphorylation of the enzyme. brain. That is, the enhaned speifi ativity of the This study shows that stimulation of pp60. kinase ativstimulated vasular enzyme did not derease with in- ity also ours in extrats prepared from aorti musularis. reasing amounts of extrated protein. Moreover, mix- Moreover, stimulation of ativity in either oronary or aorti ing experiments in whih vasular smooth musle ex- extrats ours in response to several different polyphostrats were added to brain extrats showed that the phates. The data strongly suggest that vasular smooth musle musle extrats ontained a fator whih inhibited probably ontains an inhibitor of pp6oc- and that the ation pp60' kinase ativity. This inhibition was bloked of the inhibitor an be bloked by polyphosphates. The puwhen the mixed extrats were immunopreipitated in tative inhibitor does not appear to be present in erebral the presene of ATP, or when inhibitory extrat was treated with trypsin. Taken together, these data sugextrats suggesting that a novel mehanism for modulating gest that pp60- kinase ativity in vasular tissue the ativity of pp6oc-' might exist in adult vasular smooth may be subjet to a novel regulatory mehanism in- musle. volving an inhibitory protein fator whih an be nul- MATERIALS AND METHODS lified by polyphosphates. Preparation of Tissue Extrats-Extrats were prepared from adult pp60 is a 60-kDa phosphoprotein whih exhibits tyrosine kinase ativity and is assoiated with the ytoplasmi fae of the plasma membrane (reviewed in Refs. 1-3). This enzyme is the ellular ounterpart of pp60-sr, the tyrosine kinase whih is enoded by the onogene of the Rous saroma virus * This study was supported by Grant HLB from the National Institutes of Health. The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked aduertisernent in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. $ To whom orrespondene should be addressed Dept. of Physiology and Biophysis, University of Cininnati College of Mediine, 231 Bethesda Ave., Cininnati, OH bovine brain, oronary arteries, and aorti musularis. Only the erebral ortex was used for the preparation of brain extrats. Coronary arteries were disseted from fresh bovine hearts, freed of adhering onnetive tissue, and kept on ie (13). The leaned seg- ments were slit longitudinally and the intimal layer was rubbed vigorously with a otton swab to remove endothelial ells (14). Similarly, fresh aortae were rinsed in saline at 4 C, ut along their length, and plaed on ie. After removing attahed onnetive tissue, the intimal layer ontaining aorti endothelial ells was stripped from the underlying layer of musularis (15). Experiments with either oronary or aorti preparations were performed only with the medial layer of smooth musle. Preliminary experiments showed that tissue samples whih were frozen and pulverized under liquid NP ould be stored at -70 C for at least 3 months without any apparent hange in the basal ativity of pp60 or in the responsiveness of the enzyme to polyphosphates. Aordingly, segments of frozen tissue ( mg) were homoge

2 10774 Vasular pp60- Kinase Ativity nized at 4 C in 5 volumes of buffer onsisting of 20 mm MOPS,' ph 7.0, 1% sodium deoxyholate, 5% Nonidet P-40 (v/v), 0.1% SDS, 1 mm EDTA, 100 pg/ml N-p-tosyl-L-lysine hloromethyl ketone (Sigma), 100 pg/ml phenylmethylsulfonyl fluoride (Sigma), 100 pg/ ml N-tosyl-L-phenylalanine hloromethyl ketone (Sigma), and 100 pg/ml aprotinin (Sigma). The homogenates were kept on ie for 30 min and entrifuged for 30 min at 15,000 rpm (Sorvall, SS-34 rotor, 4 C). Eah supernatant was transferred to a preooled Eppendorf tube (1.5 ml), stored on ie for an additional 15 min, and entrifuged for 5 min at maximum speed in a Bekman Mirofuge at 4 C. The protein ontent of the supernatant was determined aording to the method of Bradford using bovine serum albumin as standard (16). The onentration of protein was adjusted to 8 mg/ml by dilution of the supernatant in homogenization buffer immediately before assaying the preparation for kinase ativity. Immunopreipitation of pp6fthe method used for assessing pp60 kinase ativity in immunopreipitates prepared from tissue extrats was adapted from proedures desribed by Courtneidge (11) and Bolen et al. (12). These proedures utilized a monolonal antibody (mab 327), developed by Lipsih et al. (17), whih reognizes pp60' and does not alter its enzymi ativity. Immune omplexes were formed in a mixture (350 pl) onsisting of mg of solubilized tissue protein ( p1of extrat adjusted to 8 mg of protein/ml), 5 p1 of the appropriate dilution of mab 327 (Onogene Sienes) and an amount of homogenization buffer ( pl) to bring the total volume to 350 pl. After inubation of the mixtures at 4 C overnight, immune omplexes were preipitated by adding 10 pl of 10% formalin-fixed Staphyloous aureus (Pansorbin, Calbiohem) whih had been preoated with rabbit antimouse immunoglobulin (Zymed). Mixtures were stored on ie for 30 min and then entrifuged to sediment the immunopreipitates. Eah immunopreipitate was washed 3 times with 500 pl of a solution (RIPA/DTT), onsisting of 50 mm Tris, ph 7.5, 150 mm NaCl, 1% Triton, 1% sodium deoxyholate, 0.1% SDS, and 1 mm DTT, followed by 2 washes with 500 &of 20 mm, Tris ph 7.5, 150 mm NaC1, and 1 mm DTT (TBS/DTT). The effets of ATP and other ompounds on pp60' kinase ativity were assessed before and after immunopreipitation of tissue extrats. In the former ase, the ompound of interest was inluded in the buffer added to the immune omplex mixture (see above). For assessments of enzymi ativity after immunopreipitation, immune omplexes were prepared in the usual way (i.e. in the absene of test ompounds). However, the proedure for washing immunopreipitates was altered. The preipitates were washed 2 times with 500 p1 of RIPA/DTT, 3 times with of TBS/DTT ontaining the desired onentration of test ompound, and 2 times without the ompound. Control samples washed in the same way but without added ompound were inluded in eah series of experiments of this kind. Determination ofpp6@-kinase Atiuity-Kinase assays were performed in a reation mixture (20 pl) ontaining the washed immunopreipitate, 20 mm MOPS, ph 7.0, 0.5 mg/ml a-asein (Sigma), 5 mm MgC12, and 20 p~ ATP supplemented with pci of [r-p] ATP (Du Pont-New England Nulear). The speifi ativity of the ATP was 1.1 X lo3 to 2.2 X lo3 pm/pmol. Reations were started by adding ATP, and stopped after 15 min of inubation at 30 C by adding 10 pl of Laemmli SDS buffer and heating in a boiling water bath for 3 min (8). After ooling the mixture to room temperature, a 25-pl aliquot was used for SDS eletrophoresis on a 7.5% polyarylamide gel (18). The gel was stained with Coomassie Blue, destained, dried, and plaed on a sheet of Kodak XRPl film for autoradiography at room temperature (8). Phosphorylation was expressed as femtomoles of 32P inorporated into substrate per min/mg of tissue protein inluded in immunopreipitation mixtures. Radioative bands on a given eletrophoreti gel were loated by plaing the dried gel over its orresponding autoradiogram. The desired bands were ut from the gel and subjeted to liquid sintillation spetrosopy for assessment of inorporated radioativity (8, 11, 15, 19). In some experiments rabbit skeletal musle enolase (Boehringer Mannheim, 0.5 mg/ml), whih had been pretreated at low ph (20), was used as an exogenous substrate in plae of asein. Standard proedures were used for determination of the K,,, for enolase, the K, The abbreviations used are: MOPS, 4-morpholinepropanesulfoni aid; AMP. PNP, 0,y-imidoadenosine 5triphosphate; DTT, dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaeti aid; mab 327, anti-sr monolonal antibody 327; SDS, sodium dodeyl sulfate; TBS, Tris-buffered saline. for ATP in kinase assay mixtures, and for the determination of V, for either autophosphorylation of pp60e.r or phosphorylation of enolase. RESULTS Influene of ATP and Other Polyphosphates on pp6@. Kinase Atiuities-In aordane with our earlier studies (8), immunopreipitation of oronary arterial pp60' in the presene of 1 mm ATP resulted in 20-fold stimulation of kinase ativity (Table I). Interestingly, ATP-mediated stimulation of ativity was also apparent in extrats from aorti vasular smooth musle. In ontrast, no stimulation of ativity by ATP ourred in extrats prepared from bovine brain. Though not shown, eletrophoreti analysis of aid hydrolysates prepared from 32P-labeled aseins and pp6oc- showed that only tyrosine was phosphorylated during the kinase assays (12). Stimulation of ativity in both oronary and aorti extrats was refleted by enhaned autophosphorylation of pp60, inreased phosphorylation of al- and a2-aseins, and by enhaned phosphorylation of several endogenous substrates whih were present in immunopreipitates (Table I, Fig. 1). These endogenous substrates were of M, 97,000, 83,000, 45,000,42,000, and 26,500. The extent of stimulation attained with either endogenous or exogenous substrates was dependent upon the onentration of ATP and inreased progressively as the onentration of ATP inluded in immunopreipitation mixtures was inreased (Fig. I). Surprisingly however, stimulation of ativity in preparations from vasular smooth musle also ourred when the ATP in the immunopreipitation mixture was replaed with either of several polyphosphates inluding different nuleotide triphosphates, ADP, or sodium pyrophosphate (Table 11). The extent of stimulation was omparable and greatest with ATP, ADP, AMP. PNP, and CTP, intermediate with GTP and UTP, and moderate but least with pyrophosphate. No stimulation of ativity ourred in response to monophosphates suh as AMP or KH2P04, indiating that more than one phosphate group was required for enhanement of ativity, and that other strutural features of the polyphosphate ould influene the extent of stimulation. It is noteworthy that polyphosphates also were effetive in stimulating pp60~' kinase ativity even after immunopreipitation of the enzyme (Fig. 2). That is, when immunopreipi- TABLE p~6@-'~ kinase atiuity of tissue extrats immunopreipitated in the presene and absene of ATP Tissue extrats were prepared, immunopreipitated with anti-sr monolonal antibody 327, and assayed for pp60- kinase ativity as desribed under Materials and Methods. Immunopreipitates were formed in the presene (+ATP) or absene (-ATP) of 1 mm ATP, and they ontained 2.4 mg of extrated protein for either oronary or aorti preparations and 1.2 mg of protein for brain preparations. Values are the mean 2 S.E. for 12 oronary experiments, 16 aorti experiments, and 6 brain experiments. Substrate Aorta Coronary r Kinase ativity Brain fmol 3ZP/min/mg pp60 -ATP 0.7 f & f 1.3 +ATP 16.4 & f 1.6 al-casein -ATP 0.6 k f k 1.4 +ATP 15.7 t f f 1.6 aa-casein -ATP f 2.0 +ATP f f 2.1

3 Vasular Ativity IPP 60 UC v FIG. 2. Influene ofwashing aorti immunopreipitates with different organi and inorgani phosphates on pp60 kinase ativity. Eah immunopreipitate was prepared in the abquots of the extrat ontaining 2.4 mg of solubilized protein were sene of ATP or other phosphatesfrom aliquots (300 PI) of the same aorti extrat ontaining 2.4 mg of solubilized protein. The immuimmunopreipitated with anti-sr monolonal antibody 327 in the presene of differentonentrations of ATP as desribed under nopreipitates were washed in the absene of phosphates (ontrol, Materials and Methods. pp6oe.' and the exogenous substrates, 0,- lane I ), or 2 times with RIPA/DTT, 3 times with TBS/DTT ontainasein and az-asein, are identifiedat the right. Arrows at the right ing thedesired phosphate, and2 times with TBS/DTT in the absene point to endogenous substrates of M,97,000, 83,000, 45,000, 42,000, of phosphate. Assays were performed as desribed under MAterials and 26,500 (top to bottom). Control ativity in the immunopreipitate and Methods. Thephosphates were: 1 mm ATP (lane 2 ) ; 1 mm lane 1. The onentrations AMP.PNP (lane 3 ) ; 1 mm G T P (lane 4 ) ; 1 mm CTP (lane 5); 1 mm prepared in the absene of ATP is shown in of ATP were: 0.05 mm (lane 2); 0.1 mm (lane 3); 0.25 mm (lane 4 ) ; U T P (lane 6 ) ; 1 mm ADP (lane 7); 10 mm sodium pyrophosphate (lane 8 ) ; 25 mm KHzP04, ph7.4 (lane 9 ). 0.5 mm (lane 5); 1 mm (lane 6 ) ;5 mm (lane 7). FIG. 1. Autoradiogram showing pp60 kinase ativity in an extrat from bovine oronary arterial smooth musle. Ali- TABLE I1 (Fig. 2, Table 11). Similarly, ADP and pyrophosphate, ompounds whih annot phosphorylate proteins, also stimulated enzymi ativity. Moreover, the stimulatoryresponse to ATP persisted even when highonentrations of EDTA and EGTA were inluded inimmunopreipitationmixturestohelate divalent ations whih are usually required for a phosphorylation reation. For example, the mean value for autophosphorylation of pp60~in 4 aorti extrats immunopreipitated in the absene of ATP was only 1.1 f 0.4 fmol of 32P/ min/mg. In keeping with our earlier findings, autophosphorylation inreased to 16.9 f 1.2 fmol of P/min/mg after the Kinase ativity same extrats were immunopreipitated in thepresene of 1 Phosphate pp6oc n,-casein n2-casein mm ATP. Similar high levels were obtained for stimulated autophosphorylation in immunopreipitates prepared with an fmol 32P/min/mg ATP solution ontaining 10 mm EDTA (15.6 f 1.3 fmol of None (ontrol) 0.9 f f f 0.4 ATP 19.9 f 1.2 P/min/mg), 10 mm EGTA (17.4 f 1.4 fmol of 32P/min/mg), 22.3 f f 1.7 ADP 19.7 f f f 1.4 or a mixture of 10 mm EDTA and 10mM EGTA (16.4 f 1.3 AMP 1.1 f f f 0.5 fmol of P/min/mg). In ontrast, inlusion of the helating AMP-PNP 19.0 f f f 1.4 agents in the kinase assay mixtures ompletely prevented GTP 20.9 f f f 1.3 autophosphorylation of pp60 as well as phosphorylationof CTP 31.4 f f f 1.6 endogenous and exogenous substrates. Aordingly, divalent UTP 12.6 f f f 1.4 PPi 9.4 f f f 1.4 ations apparently were required for phosphorylation of prokhzpo4 1.1 f f f 0.6 teins in the assays for kinase ativity, but the ations were not required for ATP-mediated stimulationof ativity. tates whih were prepared in theusual way (i.e. in the absene Influene of Stimulation on K,,, for Substrates and V, for of added polyphosphates) were treated at 4 C with wash Phosphorylation during Kinase Assays-The data in Fig. 3 solutions ontaining any of several polyphosphates, marked show that stimulationof vasular pp60 kinase ativity also ourred when a-asein was replaed with enolase, an oftenstimulation ofpp60 kinase ativity ourred. Thus, the used substrate for the enzyme (1,4,5, 7, 11, 19, 20). Sine the mehanism underlying stimulation of ativity appeared to involve a polyphosphate-mediatedinrease in the speifi stimulatory response was demonstrable with different endogwith different exogenous substrates ativity of pp60'., rather thanmore effiient immunopreip- enoussubstratesand (Figs. 1-3), it probably was largely asribable to an enzymeitation of the enzyme. Several lines of evidene suggested that the apparentpoly- direted mehanism. However, this does not prelude the phosphate-mediated inrease in the speifi ativity of possibility that a substrate-direted mehanism also partiipp60 ould not be asribed to a proess involving phos- pates in the stimulatoryresponse. Fig. 3 also shows that pronouned stimulation of kinase phorylation of pp60 or other proteins in the immunopreipitates.thus,enhanedkinaseativity was indued by ativity was evident over a wide range of ATP onentrations AMP. PNP,a nonmetabolizable analog of ATP whih annot used in the kinase assay mixtures. Analysis of the data reserve as a phosphate donor in phosphotransferase reations vealed that the K,,,of pp60 for ATP was essentially the pp6wamkinase ativity in aorti immunopreipitates prepared in the presene of different phosphates Extrats of bovine aorti musularis ontaining 2.4 mg of solubilized protein were immunopreipitated with anti-sr monolonal antibody in the presene of different phosphates andassayed for pp6oc' IrC kinase ativity as desribed under Materials and Methods. The onentration of eah phosphate was 5 mm. Control immunopreipitates were prepared from the same extrats in the absene of any added phosphate. Values are the mean f S.E. for four experiments. Similar results were obtained in immunopreipitates from oronary arterial smooth musle.

4 Vasular Ativity A. ontrol - B. ativated A L - PP enolase FIG. 3. Influene of different onentrations of ATP in assay mixtures on pp60'' kinase ativity. Aliquots (300 pl) of a oronary extrat ontaining 2.4 mg of protein were immunopreipitated and assayed for kinase ativity employing rabbit skeletal musle enolaseas exogenous substrate (see Materials and Methods). A (ontrol)shows ativities obtained with immunopreipitates prepared in the absene of unlabeled ATP, whereas B (atiuated)shows results obtained with immunopreipitates prepared in the presene of unlabeled 1 mm ATP. Theonentrations of labeled ATP studied in assay mixtures were: 1.25 mm (lanes 1 and 1 ' ) ; 2.5 mm (lanes 2 and 2'); 5.0 mm (lanes 3 and 3'); 10 mm (lanes 4 and 4 ' ) ; 20 mm (lanes 5 and 5'); 30 mm (lanes 6 and 6'); 40 mm (lanes 7 and 7'). The speifi ativity of ATP was maintained onstant at lo3pm/pmol. - Protem (m3) A T P I ow 0 a0 I same before (9 p ~ and ) after immunopreipitation with un. like fashion, thek,,, for enolase was labeled ATP (12 p ~ ) In Proteln ) afteratp-mediated aboutthesame before (2.4 p ~ and (mgl stimulation of enzymi ativity (3.7 pm). In ontrast, v, for ATP either autophosphorylation of pp60 or phosphorylation of r enolase inreased markedly by about 20-fold. Thus, the V,,, C~ for autophosphorylation ofpp60 under basal onditions was only about 1 fmol of 32P/min/mg, and the V,,, for phosphorylation of enolase was of the samelow order of magnitude PP 60 (1.4 fmol of 32P/min/mg). However, following stimulation of the enzyme, the V,,, for inorporationof 32Pinreased to about 32 fmol of 32P/min/mg for autophosphorylation and to about y1 36 fmol of 32P/min/mg for phosphorylation of enolase. 12 Relationships between Volume of Extrats Subjeted to Immunopreipitation and pp6@- Kinase Atiuities-The relationship between basal ativity ofpp60 and amount of Protein fmgl extrated protein subjeted to immunopreipitationwas difatp ferent for brain and vasular extrats (Fig. 4). Under basal onditions enzymi ativity in brain extrats was proportional 4. pp60 kinase ativity as a funtion of the amount to the amount of extrated protein inluded in immunopre- of FIG. tissue protein ( mg) inluded in immunopreipitaipitation mixtures, and the speifi ativity of the enzyme tion mixtures ontainingextrats from bovine brain ( A ),orwas essentially onstant (Fig. 4A). For example, total ativity onary artery ( B ),and aorti musularis (C). For eah amount of protein tested, immunopreipitates were prepared in the absene measuredwith respet toautophosphorylation ofpp60 of ATP to measure basal ativity (first lane in eah braket), and in using a load of 0.8 mg of protein was 17 fmol of P/min, the presene (+) of either 1 mm (seond lane of braket), or 5 mm whereas the ativitywas about %fold higher (56 fmol of P/ (third lane of braket) to measure ATP-stimulated ativity. Arrows min) when 2.4 mg of extrated protein was inluded in the at theright of eah panel point to endogenous substrates of M,97,000, immunopreipitation mixture. Speifi ativities under these 83,000, 45,000, 42,000, and 26,500 (top to bottom). pp60 and exogonditions were, respetively, 21.3 and 23.3 fmol of P/min/ enous substrates (a,and r2-asein) are also identified at the right. mg. In ontrast, basal ativity in oronary and aorti extrats were proportional to the amount of protein added to immudid not inrease with inreasing protein. Instead, basal ativnopreipitation mixtures. In oronary extrats, the propority either remained unhanged (oronary, Fig. 4B) or 'de- tionality between ATP-stimulated ativity and protein load reased progressively (aorta, Fig. 4C) with inreasing protein was apparent with both1 and 5 mm ATP. Total ativity with load. Aordingly, the speifi ativity of pp60'. in vasular respet to autophosphorylation ofpp6oc.' measured after extrats dereased as the amount of extrated protein used stimulation of the oronary enzyme during immunopreipifor immunopreipitation was inreased. tation of 0.8 mg of extrated proteinwas 13 fmol of P/min. As expeted, ATP at either1 or 5 mm did not alter ativity When the amountof extrated proteinsubjeted to immunoin brain extrats (Fig. 4), whereas ativities in vasular ex- preipitation was inreased %fold, autophosphorylation intrats were stimulated markedly (Fig. 4, B and C). Moreover, reased to 46 fmol of P/min (Fig. 4C). The speifi ativity of the stimulated enzyme was 16 fmol of P/min/mg at the unlikevasularbasalativities,atp-stimulatedativities - I -

5 Vasular Kinase Ativity low protein load and 19 fmol of 32P/min/mg at the high load. Although stimulation of ativity in aorti extrats inreased progressively with low to moderate protein loads ( mg, Fig. 4C), stimulation with 1 mm ATP was no longer present at the highest protein load (2.4 mg), and it was blunted in response to 5 mm ATP. Colletively, these data suggested that vasular extrats ontained an inhibitor of pp60' kinase whih ould be bloked by treatment with polyphosphates. pp6@- Kinase Ativities in Mixtures of Extrats from Different Tissues-To gain further insight into the possible presene of a polyphosphate-sensitive inhibitor of pp60- kinase ativity in vasular extrats, we studied enzyme ativities in mixtures of extrats from different tissues. In aordane with earlier observations (Fig. 4A), immunopreipitation in the presene of ATP did not alter ativity in brain extrats, whereas aorti ativity was markedly enhaned (Table 111). However, basal ativity was profoundly suppressed in the mixture of aorti and brain extrats (Table 111, row 3a). This suppression was prevented when the mixed extrat was subjeted to immunopreipitation in the presene of ATP. Suppression of ativity was also prevented when mixed immunopreipitates whih were prepared in the absene of ATP were treated with a wash solution ontaining ATP. Similar results were obtained with mixtures of brain and oronary extrats, but no suppression of ativity ourred in mixtures of brian and atrial extrats (Table 111). Thus, autophosphorylation of pp60- in atrial extrats was unaltered by immunopreipitation in the presene of 5 mm ATP. Autophosphorylation in immunopreipitates from a mixture of brain and atrial extrats losely approximated the sum of ativities attained in brain and atrial extrats alone indiating that the ativities were additive. In ontrast, autophosphorylation in mixed brain and oronary preparations (7.1 fmol of P/min/mg), though greater than that in oronary alone (1.1 fmol of 32P/min/mg) was less than 1h of the sum of ativities in brain alone (24.2 fmol of 32P/min/mg) and oronary alone (1.1 fmol of 32P/min/mg). However, when the mixed brainoronary extrats were immunopreipitated in the presene of 5 mm ATP, autophosphorylation now attained a value (32.4 fmol of 32P/min/mg) whih approahed the sum of ativities TABLE I11 Influene of ATP on pp6p kinase ativities in immunopreipitates from extrats of bovine brain, oronary, aorta, and atrium, and in mixed extrats of brain and oronary, brain and aorta, and brain and atrium pp60' kinase ativity was assessed in immunopreipitates from brain only (row 1, 0.8 mg protein), oronary only (row 2, 2.4 mg of protein), aorta only (row 3, 2.4 mg of protein), and atrium only (row 4, 2.4 mg of protein). Ativities were also determined in immunopreipitates prepared from mixtures of extrats of oronary and brain (row 2a), aorta and brain (row 3a), and atrium and brain (row 4a). Immunopreipitates were olleted in the presene (+) and absene (-) of 5 mm ATP. Values are means from three experiments. pp60' kinase ativity Assay ondition pp60 a]-casein a,-casein fmol 32P/rnin/rng 1. Brain only Coronary only a. Coronary + brain Aorta only a. Aorta + brain Atrium only a. Atrium + brain TABLE IV Influene of dialysis and inubation of aorti extrats with DNase, RNase, and trypsin on inhibition of pp6v. kinase ativity in brain extrats pp60-sx kinase ativity was assessed in immunopreipitates from brain only (row 1, 0.8 mg of protein), aorta only (row 2, 2.4 mg of protein), and in immunopreipitates prepared in mixtures in brain and aorti extrats. In the mixed immunopreipitates, the aorti extrat was added to the brain extrat after: (a) storage for 4 h at 4 C (row 3); (b) dialysis against 4 liters of 20 mm MOPS, ph 70, at 4 C (row 4); digestion with 10 pg/ml DNase and 10 pg/ml RNase for 30 min at 37 C (row 5); () digestion with 7.5 pg/ml trypsin for 30 min at 37 C following by addition of 100 pg of soybean trypsin inhibitor. Immunopreipitates were olleted in the presene (+) and absene (-) of 5 mm ATP. Values are the mean of two experiments. pp6w kinase ativity Assay ondition pp60' a2-casein al-casein --I /mol SzP/rnin/mg 1. Brain only Aorta only Brain + aorta Brain +dialyzed aorta Brain + DNase/RNase aorta 6. Brain + trypsinized aorta attained in eah extrat alone. As shown in Table 111, omparable results were obtained with respet to phosphorylation of al- and u2-asein. Taken together, these data also suggested that vasular extrats ontained an inhibitor of pp60' whih ould be bloked by ATP. The effiay of the aorti inhibitory fator was unaltered following dialysis or inubation of the extrat with DNAse and RNAse (Table IV). In ontrast, inubation with trypsin destroyed inhibitory ativity. Following trypsinization, the aorti extrat no longer inhibited pp60' kinase ativity in mixed aorti-brain immunopreipitates. The kinase ativity attained in suh mixtures was the same in immunopreipitates prepared with and without ATP, a result whih probably reflets proteolysis of the aorti inhibitory fator. However, although kinase ativity was greater than that measured in extrats from brain alone or aorta alone, it did not attain the high values attained in ontrol mixtures of brian and aorta that were immunopreipitated in the presene of ATP. This may be attributable to onomitant trypsin-indued proteolysis of the aorti pp60 and its putative inhibitor. DISCUSSION Several lines of evidene developed in this study suggest the interesting hypothesis that the kinase ativity of pp60'x in mammalian vasular smooth musle may be modulated by a polyphosphate-sensitive inhibitory fator, whih is probably protein in nature. First, the ativity of the enzyme in extrats from either oronary or aorti vasular smooth musle is stimulated about 20-fold by organi and inorgani polyphosphates (Figs. 1-4, Tables 1-111). In ontrast, no stimulation of ativity ours when polyphosphates are replaed with monophosphates or in extrats from brain or atrium treated with either mono- or polyphosphates (Tables I and 111). Seond, total ativity whih is measured in vasular extrats is either unhanged or dereased as the amount of extrat analyzed is inreased, whereas the total ativity in brain extrats inreases with the amount of extrat assayed (Fig. 4). Third, ativity attained in a mixture of extrats from brain and atrium is omparable to the sum of the ativities measured in the brain extrat alone and in the atrial extrat alone

6 10778 Vasular pp60-' Kinase Ativity (Table 111). However, ativity attained in a mixture of vasular and brain extrats is only about one-third of the sum of the ativities for separate brain and vasular extrats. Fourth, the tein represent different polypeptides whih are ovalently or nonovalently linked, or different domains of a single moleule whih exhibit atalyti and regulatory funtions, annot pronouned inhibition ofpp6w. kinase ativity in mixed be dedued from the urrently available data. Further studies extrats ontaining vasular protein is prevented when the mixture is immunopreipitated in the presene of polyphosphate, or when the mixed immunopreipitate is washed with polyphosphate. Lastly, the aorti inhibitor of pp60- is probably a protein beause its inhibitory ativity was virtually abolished after inubation with trypsin, whereas it was unaltered following dialysis or inubation with DNase and RNase (Table IV). are required to distinguish between these possibilities. The funtion of mammalian pp60' is unknown. Reent studies with Drosophila suggest that pp60~' is involved in regulating embryogenesis of viseral smooth musle (6). However, studies in this and other laboratories also show that moderate to high levels of the enzyme are present in adult mammalian tissue (8, lo), suggesting that pp60' may funtion in diverse ellular mehanisms. Moreover, it is now lear Colletively, these data suggest that vasular smooth musle that isozymi forms of pp60 exist. Thus, a tissue-speifi ontains a protein whih markedly inhibits pp60- kinase form of the enzyme with high speifi ativity exists in neural ativity. Moreover, the effiay of the inhibitor an be mod- tissues (21). The results of our study suggest that tissueulated by polyphosphates suh that resultant kinase ativity an be varied over 20-fold. These data are onsistent with the hypothesis that pp60- and inhibitor protein o-preipitate speifi mehanisms for regulating the ativity of the enzyme also exist. Possibly, the regulation of pp60 kinase ativity by the inhibitory mehanism desribed in this report partiiduring the immunopreipitation reation. In this setting, pates in modulating arterial signal transdution and ontrameasured basal kinase ativity is low. However, inlusion of tility. polyphosphate in immunopreipitation mixtures, or washing preipitates with polyphosphate, bloks the ation of the Aknowledgment-We are grateful to A. Lutz for typing the maninhibitor protein and thereby results in a large inrease in usript. kinase ativity. Thus, although the basal ativity in vasular REFERENCES extrats is omparable to the low value present in atrial 1. Jove, R., and Hanafusa, H. (1987) Annu. Reu. Cell Biol. 3,31-56 extrats, the expressed ativity an be inreased to the high 2. Sefton, B. M., and Hunter, T. (1986) Caner Suru. 6, levels present in extrats from brain and ertain transformed 3. Bishop, J. M. (1985) Cell 42, ells (9-11, 12, 19). This broad potential for modulating the 4. Barnekow, A., and Gessler, M. (1986) EMBO J. 6, ativity of the enzyme may play a signifiant role in regulating 5. Rohrshneider, L. R. (1986) in Onogenes and Growth Control the funtion(s) of pp60-' in uiuo. (Kahn, P., and Graf, T., eds) pp , Springer-Verlag, Berlin 6. Simon, M., Dress, B., Kornberg, T., and Bishop, J. M. (1985) Cell Beause the inhibitor does not appear to be present in atrial 42, or brain extrats, its distribution and funtion may be tissue- 7. Brugge, J., Cotton, P., Lustig, A., Yonemoto, W., Lipsih, L., speifi. In this ontext, reent studies in our laboratory show Coussens, P., Barrett, J. N., Nonner, D., and Keane, R. W. that the inhibitor and polyphosphate-mediated stimulation of (1987) Genes & Deu. 1, pp60 kinase ativity is demonstrable in extrats from whole 8. Di Salvo, J., Gifford, D., and Kokkinakis, A. (1988) Biohem. lung and type I1 pulmonary epithelial ells.' However, no Biophys. Res. Commun. 153, Brugge, J. S., Cotton, P. C., Queral, A. E., Barett, J. 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Physiol. 248, C550-C556. kinetis showing a marked inrease in V,,, for phosphorylation, 15. Silver, P. J., and Di Salvo, J. (1979) J. Biol. Chem. 254, and no hange in K,,, for substrates (Fig. 3). It is possible that Bradford, M. M. (1976) Anal. Biohem. 72, the polyphosphates interat diretly with the inhibitory pro- 17. Lipsih, L. A,, Lewis, A. J., and Brugge, J. S. (1983) J. Virol. 48, tein omponent to render it less effetive. It is also possible that the polyphosphates at diretly on the atalyti ompo- 18. Laemmli, U. K. (1970) Nature 227, nent to render it less suseptible to inhibition. In either ase, 19. Cooper, J. A., and King, C. S. (1986) Mol. Cell. Biol. 6, whether the atalyti omponent and putative inhibitory pro Cooper, J. A., Esh, F. S., Taylor, S. S., and Hunter, T. (1984) J. Biol. Chem. 269, J. Di Salvo, D. Gifford, and A. Terzian, Exp. Lung Res., submitted 21. Martinez, R., Prevot-Mathey, B., Bernards, A., and Baltimore, for publiation. D. (1987) Siene 237,