Failure of 1-Oleoyl-2-acetylglycerol to Mimic the Cell-differentiating Action of 12-0-Tetradecanoylphorbol 13-Acetate in HL-60 Cells*

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The Americn Society of Biologicl Chemists, Inc Vol. 260, No. 26, Issue of November 15, pp Printed in &%A. Filure of 1-Oleoyl-2-cetylglycerol to Mimic the Cell-differentiting Action of 12-0-Tetrdecnoylphorbol 13-Acette in HL-60 Cells* Stoshi Ymmoto, Hiromi Gotoh, Eriko Aim, nd Ryuichi Kt0 From the Deprtment of Phrmcology, School of Medicine, Keio University, Tokyo 160, Jpn (Received for publiction, June 5, 1985) Tretment of humn promyelocytic leukemi cells pprent dissocition constnt of TPA for its specific receptor (HL-60 cells) with 12-0-tetrdecnoylphorboll3-ce- of HL-60 cells (7). Furthermore, chnges in protein phosphotte (TPA) results in terminl differentition of the ryltion specificlly induced by TPA hve lso been shown to cells to mcrophge-like cells. Tretment of the cells occur in HL-60 cells (8-10). Therefore, it seems possible tht with TPA induced mrked enhncement of the phos- the initil event of TPA-induced HL-60 cell differentition is phoryltion of 28- nd 67-kD proteins nd decrese none other thn the ctivtion of protein kinse C by this in tht of 75-kD protein. When the cells were gent. treted with dicylglycerol, Le. 50 pg/ml l-oleoyl-2- Nishizuk nd his co-workers (5, 11, 12) hve proposed cetylglycerol (OAG), similr chnges in the phosphoryltion of 28-, 67-, nd 75-kD proteins were liketht, under physiologicl conditions, protein kinse C is cwise observed, indicting tht OAG ctully stimultes tivted by dicylglycerols which re trnsiently produced from protein kinse C in intct HL-60 cells. OAG (1-100 phosphoinositides by phospholipse C ctivity, nd tht TPA pgfml),whichweused, ctivted prtilly purified is ble to substitute for dicylglycerol (3). In intct cell sysmouse brin protein kinse C in concentrtion-de- tems, synthetic dicylglycerol, such s OAG, ctully stimupendent mnner. Tretment of HL-60 cells with 10 nm ltes protein kinse C nd mimics certin TPA-induced cel- TPA for 48 h cused n increse by bout 8-fold in lulr responses (13-19). We hve previously reported tht cellulr cid phosphtse ctivity. Although signifi- OAG mimics TPA ction nd induces ornithine decrboxylse cnt increse in cid phosphtse ctivity ws induced by OAG, the effect ws scnt compred to tht of TPA (47% tht of TPA). After 48-h exposure to 10 nm TPA, bout 95% of the HL-60 cells dhered to culture dishes. On the contrry, tretment of the cells either with OAG (2-100 pgfml) or phospholipse C filed to induce HL- 60 cell dhesion. C" ionophore A23187 filed to ct synergisticlly with OAG. In ddition, hourly or bihourly cumultive ddition of OAG for 24 h lso proved ineffective to induce HL-60 cell dhesion. Our present results do not imply tht protein kinse C ctivtion is inessentil for TPA-induced HL-60 cell differentition, but do demonstrte tht protein kinse C ctivtion is not the sole event sufficient to induce HL-60 cell differentition by mens of this gent. TPA,' the potent tumor promoter, hs been shown to induce terminl differentition of the humn promyelocytic cell line HL-60 into mcrophge-like cells (1, 2). There is considerble body of evidence tht demonstrtes n intimte coupling between TPA ction nd the ctivtion of protein kinse C, nd further indictes tht protein kinse C is receptor of tumor-promoting phorbol esters nd tht some of the ctions of TPA re medited through the ctivtion of this enzyme (3-5). In fct, in HL-60 cells, high level of protein kinse C hs been demonstrted (6). The concentrtion of TPA required for induction of differentition coincides with the * The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked "udvertisement" in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. ' The bbrevitions used re: TPA, 12-0-tetrdecnoylphorbol13- cette; OAG, 1-oleoyl-2-cetylglycerol; SDS, sodium dodecyl sulfte; HEPES, 4-(2-hydroxyethyl)-l-piperzineethnesulfonic cid; MeZSO, dimethyl sulfoxide; EGTA, ethylene glycol bis(p-minoethyl ether)-n,n,n,'n'-tetrcetic cid. ctivity in isolted epiderml cells (20). If protein kinse C ctivtion is n initil event in HL-60 cell differentition following TPA stimultion, OAG would mimic the cell-differentiting ction of TPA. Thus, we investigted the question of whether OAG is ble to induce the differentition of HL- 60 cells s TPA does. EXPERIMENTAL PROCEDURES Mterils-The following gents were purchsed from the compnies indicted TPA, phospholipse C (from Clostridiumperfringens), t--phosphtidyl-l-serine, ATP, histone (Type 111-SI, nd phenylmethylsulfonyl fluoride from Sigm; Ir-32P]ATP (10.5 Ci/mmol) from New Englnd Nucler; A23187 from Clbiochem-Behring; nd r3'p] H3POl (crrier-free), from Amershm Corp. OAG ws kindly provided by Kken Chemicl Compny Ltd., Tokyo, Jpn. Protein Kinse C Assy-Prtilly purified mouse brin protein kinse C ctivity ws determined ccording to the methods of Ashendel et l. (21) nd Couturier et l. (22) with slight modifictions. Brin ws homogenized in 3 volumes (w/v) of 20 mm Tris-HC1 buffer (ph 7.4) contining 10 mm EDTA, 2 mm EGTA, nd 2 mm phenylmethyisulfonyi fluoride with polytron homogenizer, nd the homogente ws centrifuged t 10,000 X g for 10 min t 4 "C. The superntnt obtined ws ultrcentrifuged t 105,000 X g for 60 min t 4 "C. The resultnt cytosol frction ws loded onto 1.5 X 14-cm column of Sephdex G25 equilibrted with the bove homogenizing buffer. The mcromoleculr frction ws used for the ssy of protein kinse ctivity. Protein kinse ctivity ws determined by incubting 2.5 nmol of [y3'p]atp (3 X X lo6 cpm), 30 pg of histone, 500 nmol of mgnesium cette, 6.25 pmol of 2-mercptoethnol, vrious mounts of CCI,, 3.1 pg of phosphtidylserine nd 1-2 pg of protein of prtilly purified cytosol extrct in the presence or bsence of either OAG or TPA in 125 pl of 20 mm Tris-HC1 (ph 7.4) buffer. In the cse of zero C2, 125 nmol of EGTA ws dded to the rection mixture insted of CC12. The rection ws strted by the ddition of [T-~'P]ATP. Incubtion ws crried out t 37 "C for 3 min, then the rection ws stopped by trnsferring 50-pl liquots of the incubtion mixture onto 2.5-cm squre pieces of Whtmn cellulose phosphte pper (P81). The pieces of pper were wshed four times with deionized wter, twice with cetone, nd once with petroleum ether. The rdioctivity on ech piece of pper ws determined by scintilltion counting

2 Dicylglycerol nd HL-60 Cell Differentition Protein Phosphoryltion-HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% het-inctivted fetl clf serum. Just before use, HL-60 cells were wshed twice with P04-free Locke's solution of the following composition: 142 mm NC1; 5.6 mm KC1; 3.6 mm NHC03; 2 mm CC12; 1.2 mm M&12 supplemented with 5 mm HEPES (ph 7.4) nd 2 mg/ml glucose. Cells (lo6 cells/0.3 ml/tube) were first incubted in P04-free Locke's solution t 37 "C for 30 min. Therefter, 0.1 ml of ["P]H3P0, in P04-free Locke's solution ws dded to the cell suspension t finl concentrtion of 0.25 mci/ml nd the cells were further incubted for 20 min. The cells were then exposed to the indicted concentrtions of TPA or OAG for 10 or 30 min. At the end of the incubtion, hlf the volume of SDS buffer (187.5 mm Tris, 9% SDS, 6% mercptoethnol, nd 15% glycerol, ph 6.7) ws dded to the cell suspension. Therefter the mixture ws boiled for 5 min nd 50-pl liquots were subjected to SDS-polycrylmide gel electrophoresis. SDS-polycrylmide gel electrophoresis ws performed ccording to the method of Lemmli (23) using 8% slb gels with 1.5-mm thickness. After electrophoresis, gels were fixed, stined with Coomssie Brillint Blue, nd destined. For utordiogrphy, gels were dried on filter ppers nd exposed to x-ry films (Fuji RX). Cell Adhesion Assy-HL-60 cells ( lo5 cells) were seeded in 1.ml of RPMI 1640 medium supplemented with 10% het-inctivted fetl clf serum in 16-mm dimeter wellof plstic multidishes. In experiments shown in Tble 11, the cells (2 X lo5 cells) were cultured in 2 ml of the bove medium in plstic culture tubes. TPA, OAG, nd A23187 were first dissolved in MezSO nd dded to the cells (with finl concentrtion of MezSO lwys less thn 0.3%). Me2S0 t concentrtion up to 0.5% showed no detectble effects on cell growth or on cell dhesion induced by TPA. The suspended cells nd dhesive cells were counted 24 or 48 h fter the ddition of vrious gents s described previously (7). Cell vibility ws estimted by trypn blue exclusion test. Acid Phosphtse Actiuity-HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% het-inctivted fetl clf serum either with OAG or TPA for 24 or 48 h. Cellulr cid phosphtse ctivity ws determined by monitoring the sodium fluoridesensitive p-nitrophenol formtion from p-nitrophenyl phosphte c- cording to the method of Schnyder nd Bggiolini (24). Protein concentrtion ws determined by the method of Lowry et l. (25). RESULTS Activtion of Protein Kinse C by OAG-Prtilly purified cytosol frction of mouse brin homogente ws used for the ssy of protein kinse C ctivity. Protein kinse C ws ctivted in the presence of C2 plus phosphtidylserine nd lso ctivted in the presence of TPA plus phosphtidylserine but without C2 (Fig. 1). Stimultory effect of TPA on protein kinse C, which ws observed in the presence of phosphtidylserine, ws evident only under the presence of none or smll mount ofcc12. Therefore, the effect of OAG on protein kinse C ws investigted in the bsence of C2. As shown in Fig. 1, OAG ctivted the enzyme ctivity in concentrtion-dependent mnner. With pg/ml OAG, protein kinse ctivity reched level somewht similr to tht ttined by 30 nm TPA (Fig. 1). At this concentrtion, TPA induced the mximl stimultion of protein kinse C ctivity (dt not shown). Chnges in Protein Phosphoryltion following Tretment of HL-60 Cells with TPA or OAG-HL-60 cells were prelbeled with 32P04 nd were treted either with TPA or OAG for 30 min. Therefter the cells were lysed nd subjected to SDSpolycrylmide gel electrophoresis. A representtive utordiogrphy is presented in Fig. 2. Tretment of the cells with TPA induced remrkble enhncement of the phosphoryltion of proteins with pprent moleculr weights of 28,000 nd 67,000 in greement with the previous observtions (8-10). When the cells were treted with 50 pg/ml OAG, distinct enhncement of the phosphoryltion of 28- nd 67-kD proteins ws lso observed (Fig. 2). Another ltertion induced by TPA ws the decrese in phosphoryltion of the 75-kD protein. This is lso consistent with our previous findings e 'r "-; OAG (pg/ml) 2J FIG. 1. Activtion of protein kinse C by OAG. Cytosol frction of mouse brin homogente ws prtilly purified by gel filtrtion (Sephdex G25) nd the mcromoleculr frction ws used s crude enzyme preprtion. Protein kinse ctivity ws determined in the presence of 3.1 pg/125 pl phosphtidylserine, 125 nmol/ 125 pl EGTA nd either 30 nm TPA (closed squre) or vrious concentrtions of OAG (closed circle) s described under "Experimentl Procedures." Open circle denotes the ctivity in the bsence of both TPA nd OAG. (10). Decrese in the phosphoryltion of the 75-kD protein ws lso induced by OAG (Fig. 2). Chnges in the phosphoryltion of 28-, 67-, nd 75-kD proteins induced by OAG were detected within 10 min fter the ddition of this gent (dt not shown). Filure of OAG to Induce HL-60 Cell Adhesion-After 48-h exposure to 10 nm TPA, bout 95% of the cells dhered to plstic dishes (Tbles I nd 11). Inhibition of cell growth ws lso observed by treting the cells with 10 nm TPA (Tbles I nd 11). These results re consistent with erlier published findings (1, 2,7). On the contrry, tretment of the cells with vrious concentrtions of OAG (2-100 pg/ml) filed to induce HL-60 cell dhesion (Tble I). OAG t 100pg/ml cused obvious reduction in cell vibility. Effects of clcium ionophore A23187, nd A23187 plus OAG on HL-60 cell dhesion were lso exmined. A23187 lone ( PM) did not induce cell dhesion (Tble I). With A23187 t concentrtion of 0.5 p~ or more, n obvious reduction in cell vibility ws observed. Combined tretment of the cells with OAG nd A23187 lso filed to induce HL-60 cell dhesion (Tble I). Although the combined tretment of the cells with 50 pg/ml OAG nd 0.03 p~ A23187 cused moderte retrdtion of cell growth, cell dhesion ws not induced. Simultneous ddition of 50 pg/ml OAG nd 0.1 p~ A23187 cused distinct reduction in cell vibility. At present it cnnot be denied tht OAG is ineffective becuse of its reltively rpid metbolism compred to TPA. Therefore, the effect of hourly or bihourly cumultive ddition of OAG ws lso investigted. As clerly shown in Tble IT, cumultive ddition of OAG gin filed to induce HL-60 cell dhesion. Sixty to eighty per cent of HL-60 cells becme dherent by treting the cells with 10 nm TPA for 24 h. Therefore, the number of suspended nd dhesive cells ws lso counted (concerning the experiments shown in Tbles I nd 11) t 24 h fter the strt of the experiments. Agin, cell dhesion ws not observed by dicylglycerol tretment (dt not shown). Filure of Phospholipse C to Znduce HL-60 Cell Adhesion- Effect of exogenous phospholipse C (1-100 milliunits/ml) on

3 14232 Dicylglycerol nd HL-60 Cell Differentition A B TABLE I C Effects of OAG, A23187, nd OAG plus A23187 on HL-60 cell dhesion HL-60 cells (lo5 cells/l ml/well) were cultured with or without vrious concentrtions of the indicted gents. Cell numbers were counted 48 h fter the ddition of the gents. Vlues re mens of duplicte determintions. Similr types of experiments were repeted twice nd the results obtined were reproducible. % of dhesive = (dhesive/totl) X " Adhesive Number of cells X 10.0 TPA, 10 nm OAG, 2 pg/ml 8.5 OAG, 10 pg/ml 8.6 OAG, 25 pg/ml 8.5 OAG, 50 pg/ml OAG, 100 pg/ml" < kd 67 kd 95 TPA, 10 nm1.9 A23187,O.ol p M A23187,0.03 p M A23187, 0.1 p M A23187,0.5 p M A23187,o.oI LLM OAG, 50 pg/ml A23187,0.03 p M OAG, 10 pg/ml A23187,0.03 p MC0.03 OAG, 50 pg/ml A23187,o.l pm OAG, 50 pg/ml". :_ - Suspended % ' of dhesive TABLE I1 Effect of cumultive ddition of OAG on HL-60 cell dhesion HL-60 cells (2 X lo5 cells/2 ml/culture tube) were cultured with or without the indicted gents. OAG ws cumultively dded to the medium throughout the first 24-h experimentl period s indicted in the Tble. The cell number ws counted 48 h fter the strtof the experiments. Vlues re mens of duplicte determintions. Similr types of experiments were repeted three times ndthe results obtined were reproducible. % of dhesive = (dhesive/totl) X 100. Agents dded 28 kd FIG. 2. Effects of TPA nd OAG on protein phosphoryltion in HL-60 cells. HL-60 cells (IO6 cells/tube) were incubted in Podfree Locke's solution t 37 "C for 30 min. Then, 32P04ws dded t 0.25 mci/ml nd cells were incubted for 20 min. Therefter the cells were treted either with 30 nm TPA ( A ) or 50 pg/ml OAG (C)t 37 "C for 30 min. Control cells ( B ) were treted with vehicle (0.1 % Me2SO).After incubtion, the cells were lysed nd subjected to SDSpolycrylmide gel electrophoresis (8% slb gel). HL-60 cell dhesion ws lso exmined (Tble 111).Phospholipse C filed to induce HL-60 cell dhesion. Phospholipse C t 100 milliunits/ml cused obvious reduction in cell vibility. Effect of OAGon Cellulr Acid Phosphtse Activity-Tretment of the HL-60 cells with 10 nm TPA for 24 h induced significnt increse in cellulr cid phosphtse ctivity (Tble IV). However, cells treted with 50 pg/ml OAG for 24 h showed no increse in thistype of ctivity. After 48 h of TPA tretment, cellulr cid phosphtse ctivity incresed by Wsh TPA, 10 nm d 5 pg/m1/2 h X 11' 10 pg/ml/2 h X 11 5 pg/ml/h X 23' 10 pg/ml/h X 23 ()b Wsh (-Y Number of cells" X lo-' '% of dhesive Wsh () Wsh (-) c ' 0.4' Totl vible cell number. *Twenty-four hours fter the strt of experiments, the medium ws chnged to new one which contins no OAG. Therefter, the cells were cultured for nother 24 h. Medium chnge ws not performed. Initil loding dose. 'Twenty-three hourly or 11 bihourly doses following the initil dose.

4 TABLE I11 Effect of phospholipse C on HL-60 cell dhesion See footnote to Tble I sus- % of pended Adhesive dhesive Dicylglycerol nd HL-60 Cell Differentition Number of cells X IO" 12.5 <I TPA, 10 nm < >96 Phospholipse C, 1 milliunit/ml 10.0 <I Phospholipse C, 10 milliunits/ml 7.2 <I Phospholipse C, 100 milliunits/ml" 0.7 TABLE IV Increse in cid phosphtse ctivity by OAG nd TPA in HL-60 cells HL-60 cells were cultured with the indicted concentrtions of OAG or TPA. Cellulr cid phosphtse ctivity (5 X IO4 or IO5 cells) ws ssyed 24 nd 48 h fter the ddition of the bove gents. Acid phosphtse ctivity ws determined by detecting the sodium fluoride-sensitive 4-nitrophenol formtion from 4-nitrophenylphosphte. Ech vlue represents men f S.E. (n = 6). 4-Nitrophenol formed 24 h 48 h crmol/h/mgprotein 0.58 f f 0.02 TPA, 10 nm 1.30 f 0.05" 5.15 f 0.44" OAG, 50 pg/ml 0.61 f f 0.04" "p < 0.01 versus none. pproximtely 8-fold. In contrst, tretment of the cells with 50 pg/ml OAG for 48 h induced significnt, lthough very slight, increse in cellulr cid phosphtse ctivity. DISCUSSION TPA induced enhncement of the phosphoryltion of 28- nd 67-kD proteins nd decrese in the phosphoryltion of 75-kD protein in HL-60 cells. Our previous experiments reveled tht the ltertions of the phosphoryltion of 67- nd 75-kD proteins re specific for TPA nd re not induced by other biologiclly inctive phorbol derivtives (LO) nor by other gents (lo), such s 1,25-dihydroxyvitmin Ds, retinoic cid, nd Me2S0, which re known to induce HL-60 cell differentitions (26-29). We lso observed n enhnced phosphoryltion of 28-kD protein by TPA. It is highly likely tht this 28-kD protein corresponds to 27-kD protein stted by Feuerstein nd Cooper (8, 9). They demonstrted tht the enhnced phosphoryltion of 27-kD protein is specific for TPA nd tht inctive derivtives of phorbol esters fil to mimic the effect of TPA (8). Thus, it is conceivble tht increses in phosphoryltion of 28- nd 67-kD proteins re due to direct ctivtion of protein kinse C by TPA. The decrese in the phosphoryltion of 75-kD protein lso seems to be indirectly medited by protein kinse C (10). OAG ( pg/ml), which we used in the present studies, ctivted prtilly purified mouse brin protein kinse C to n extent somewht similr to tht of 30 nm TPA. In ddition, OAG induced distinct enhncement in the phosphoryltion of 28- nd 67-kD proteins nd decrese in the phosphoryltion of 75-kD protein, suggesting tht OAG ctully stimultes protein kinse C in intct HL-60 cells s TPA does. HL-60 cells undergo differentition to mcrophge-like cells in the presence of TPA. TPA-induced differentition hs been chrcterized by ttchment of the treted cells to culture dish surfces, n increse in the percentge of phgocytizing cells, inhibition of cell growth, nd n increse in cid phosphtse ctivity (1, 2, 30). If ctivtion of protein kinse C is n initil event in HL-60 cell differentition fter TPA stimultion, OAG would indubitbly mimic the celldifferentiting ction of TPA. Retrdtion of cell growth, which ws obvious in TPA-treted HL-60 cells, ws lso observed in cells treted with reltively high concentrtions of OAG or OAG plus A Since higher concentrtions of the bove gents cused reduction in cell vibility, it seems possible tht the inhibition of cell growth induced by these gents is due to their cytotoxic effects. Approximtely 95% of the cells becme dherent 48 h fter the ddition of TPA, wheres OAG (2-50 pglml) filed to induce HL-60 cell dhesion. Although OAG induced sttisticlly significnt increse in cellulr cid phosphtse ctivity, the effect ws scnt compred to tht of TPA (less thn 7% tht of PA). OAG t 50 pg/ml induces mximl effects in pltelets (13), neutrophils (15), mst cells (L6), or epiderml cells (20). It hs been reported tht in certin cell systems, C2* ionophore cts synergisticlly with TPA or dicylglycerols (13, 16, 31). In HL-60 cells, however, OAG filed to ct synergisticlly with A The possibility tht OAG is ineffective becuse of its reltively rpid metbolism compred to TPA needs to be considered. However, the fct tht the hourly or bihourly cumultive ddition of OAG lso filed to induce HL-60 cell dhesion denies the bove possibility. OAG stimultes the phosphoryltion of 28- nd 67-kD proteins just s TPA does, but induces only slight increse in cellulr cid phosphtse ctivity nd lso fils to induce HL-60 cell dhesion. Although dt re not presented, nother synthetic dicylglycerol, dicprylin (obtined from Sigm nd lso from Nu-Chek prep.), which competitively binds to the TPA receptor (32) nd mimics TPA ction (18), lso incresed the phosphoryltion of 28- nd 67-kD proteins but filed to induce HL-60 cell dhesion. These experimentl results indicte tht protein kinse C ctivtion is not the sole event sufficient to initite signl to induce HL-60 cell differentition. Exogenously dded phospholipse C my produce endogenous dicylglycerols from membrne phospholipids nd, s consequence, my ctivte protein kinse C. The filure of phospholipse C to induce HL-60 cell dhesion lso supports the bove contention. In conclusion, our present results clerly show tht OAG induces chnges in protein phosphoryltion just s TPA does. However, HL-60 cell dhesion, which is normlly observed fter ddition of TPA, ws not induced by OAG. Exogenous phospholipse C lso filed to induce HL-60 cell dhesion. The results do not imply tht protein kinse C ctivtion is inessentil for TPA-induced HL-60 cell differentition, but do demonstrte tht protein kinse C ctivtion is not the sole event sufficient to induce HL-60 cell differentition by mens of TPA. REFERENCES 1. Hubermn, E., nd Cllhm, M. F. (1979) Proc. Ntl. Acd. Sei. U. S. 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