journal of vol. 269, no. 7, of pp. 5241-5248, 1994 1994 ...vvvvvvvvvv vvvvvvvvvv cultured aortic...

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

Vol. 269, No. 7, Issue of February 18, pp. 5241-5248, 1994 Printed in U.S.A.

A Specific Inhibitor of Phosphatidylinositol 3=Kinase, 2-(4-Morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002)"

(Received for publication, July 12, 1993, and in revised form, September 28, 1993)

Chris J. MahosS, William F. Matter, Kwan Y. Hui, and Raymond F. Brown From Cardiovascular Research, Lilly Research Laboratories, Indianapolis, Indiana 46285-0403

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by asso- ciating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an ICm of 1.3 pg/ml (3.8 p ~ ) ; inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC,, values in the range of 1.7-8.4 pg/ml. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (ICso > 30 pg/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)- 8-phenyl-4H-l-benzopyran-4-one (also known as 2-(4- morpholinyl)-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (ICao = 0.43 p g / d 1.40 p ~ ) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity rela- tionship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in met-Leu- Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.

PtdIns13-kinase is an enzyme that acts as a direct biochemi- cal link between a novel phosphatidylinositol pathway and a number of proteins containing intrinsic or associated tyrosine kinase activities, including the receptors for PDGF (1-3), insu-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t To whom correspondence should be addressed. "el.: 317-276-

bromodeoxyuridine; DMEM, Dulbecco's modified Eagle's medium; EGF, The abbreviations used are: PtdIns, phosphatidylinositol; BrdU,

epidermal growth factor; fMLP, N-formyl-Met-Leu-Phe; HPLC, high performance liquid chromatography; M A P , microtubule associated protein; MES, 2-(N-morpholino)-ethanesulfonic acid; NGF, nerve growth factor; TLC, thin layer chromatography.

7891;Fm: 317-276-9722.

lin (4, 5), and colony-stimulating factor-1 (6, 7), and the products of oncogenes v-src (81, v-yes (81, and v-abl(91, as well as the polyomavirus middle T antigen/pp60~-~" complex (10). Growth factor stimulation of the associated tyrosine kinases results in phosphorylation of the 85-kDa subunit of PtdIns 3-kinase; whereas the mechanism for activation of PtdIns 3-kinase is not well understood, it is thought that phosphorylation of PtdIns 3-kinase is important for activation of PtdIns 3-kinase activity and the subsequent mitogenesis observed in stimulated cells (11). However, PtdIns 3-kinase activity has also been identified with G-protein-associated receptors such as the thrombin receptor in platelets (12-15) and the formyl peptide receptor in neutrophils (16,17), both of which are found in non-proliferating cells. Stimulation of neutrophils with formyl peptide results in activation of PtdIns 3-kinase independent of tyrosine phosphorylation (18, 19), suggesting an alternative pathway for PtdIns 3-kinase activation in association with G-proteins.

PtdIns 3-kinase transfers the terminal phosphate of ATP to the D-3 position of PtdIns, PtdIns-4-monophosphate, or PtdIns- 4,5-bisphosphate to yield the products PtdIns-3-P, PtdIns-3,4- P2, or PtdIns-3,4,5-P,, respectively (20). The biological role of PtdIns 3-kinase or its products has not been established. It is clear that these novel products are not directly involved in the traditional pathway for generating the second messenger inositol-1,4,5-trisphosphate or for generating substrates for phos- pholipase C (21, 22). The kinase has been suggested to play a role in cell proliferation and/or motility in response to growth factors and chemotactic agents (20, 23, 24). For example, the production of PtdIns-3,4,5-P3 is concomitant with changes in actin polymerization in neutrophils stimulated with formyl peptide (171, and changes in actin cytoskeletal structure also occur during mitogenesis (25-27). Furthermore, these cytoskel- eta1 changes are mediated in part by PtdIns-4,5-P2, which has been demonstrated to bind to profilin, gelsolin, and villin and therefore promote actin polymerization (28-30). Thus, a pos- sible role for PtdIns-3,4,5-P3 may be to induce actin polymerization in response to mitogenic or chemotactic stimuli.

Effective inhibitors of PtdIns 3-kinase may help to define the role of PtdIns 3-kinase and its products in cells. The bioflavinoid quercetin was previously shown to effectively inhibit PtdIns 3-kinase with an ICEo of 1.3 pg/ml(3.8 p ~ ) (31); however, quercetin has also been demonstrated to inhibit other kinases, including PtdIns 4-kinase (32) and several tyrosine and serine/ threonine kinases (33-37). Using quercetin as a model compound, several chromones were synthesized and evaluated for their ability to inhibit PtdIns 3-kinase. One such compound, 2-(4-morpholinyl)-8-phenylchromone (2-morpholino-8-phenyl- 4H-l-benzopyran-4-one, LY294002) was found to be a selective inhibitor of PtdIns 3-kinase with a 2.7-fold greater potency than quercetin. LY294002 completely abolished PtdIns 3-kinase activity against purified PtdIns 3-kinase as well as against PtdIns 3-kinase in fMLP-stimulated intact neutrophils. The compound was also effective in inhibiting the proliferation of rabbit aortic smooth muscle cells in response to injury in a

5241

Chromones Inhibit Phosphatidylinositol 3-Kinase

v v v v v v v v v v

v v v v v v vvvv

cultured aortic segment model. LY294002 had no inhibitory effect against several ATP-requiring enzymes, including PtdIns 4-kinase, EGF receptor tyrosine kinase, c-src, MAP kinase, S6 kinase, diacylglycerol kinase, protein kinase A, protein kinase C, and ATPase. LY294002 had no effect on PDGF receptor- mediated tyrosine phosphorylation in primary cultures of rabbit aortic smooth muscle cells, including phosphorylation of the 85-kDa subunit of PtdIns 3-kinase. Furthermore, inhibition of PtdIns 3-kinase was very sensitive to substitutions in the morpholine ring; analogs of LY294002 featuring single-atom re- placements lost their ability to inhibit PtdIns 3-kinase effectively. LY294002 is the first compound to act as a specific inhibitor of PtdIns 3-kinase and may be useful in investigating the functional and regulatory mechanisms mediated by PtdIns 3-kinase.

EXPERIMENTAL PROCEDURES

Materials

[Y-~~PIATP (6000 Ci/mmol), PtdIns-4-phosphate (inositol WH(N), 10 Ci/mmol), and F'tdIns-4,5-bisphosphate (inositol 2-3H(N), 10 Ci/mmol) were products of New England Nuclear. PtdIns, PtdIns-4-P, and PtdIns- 4,5-P2 were purchased from Sigma. Flow-Scint IV liquid scintillator was obtained from Radiomatic Instrument and Chemical Co., Inc. (Meriden, CT). Milli-Q water (Millipore Corp., Bedford, MA) was used for all aqueous solutions. All other reagents were of the highest quality com- mercially available.

Methods Synthesis of PtdZns 3-Kinase Inhibitors-2-Methylthiochromones (2-

methyl thio-4H-l-benzopyran-4-ones) were prepared by methods reported by Bantick and Suschitzky (38). Refluxing these compounds in n-butyl alcohol with the appropriate amine (e.g. morpholine) and a catalytic amount of acetic acid, followed by evaporation of the butanol in Vacuo and recrystallization from an appropriate solvent (Table I) af- forded 2-dialkylaminochromones (Fig. 1). In the case of LY294002, the starting 2-hydroxy-3-phenylacetophenone was prepared from commer- cially available 3-phenylsalicylic acid by treatment with methyl lithium as described by Rubottom and Kim (39).

Purification of PtdZns 3-Kinase from Bovine Brain--Two bovine brains (wet weight -900 g) were obtained from a local abattoir within minutes of slaughter, packed on ice, and homogenized within 1 h. Brains were trimmed of excess fat and blood vessels and then homogenized using a Tekmar Tissuemizer at 4 "C in 20 m~ Tris, pH 8.3, containing 250 m~ sucrose, 6 m~ P-mercaptoethanol, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, 0.4 m~ phenylmethylsulfonyl fluoride, and 1 m~ MgC1,.

Following centrifugation for 60 min at 19,000 x g, the pH of the supernatant (-1200 ml) was lowered to 5.75 using dropwise addition of 1 M acetic acid at 4 "C. After stirring for an additional 15 min at 4 "C, the solution was centrifuged 60 min at 13,500 x g . Supernatant was dis- carded. Pellets were resuspended in buffer A (20 rn Tris, pH 8.3, containing 6 m~ P-mercaptoethanol, 0.1 rn EGTA, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, and 1 m~ MgC1.J and loaded onto a Fast Flow Q Sepharose column (300 ml) at a flow rate of 5 mumin at 4 "C. After loading, the column was washed with 3 volumes of buffer A containing 0.1 M KCl, and the kinase was then eluted with a linear gradient of buffer A, 0.1 M KC1 to buffer A, 0.6 M KC1 at 3 d m i n over 7 volumes.

Fractions were assayed for PtdIns kinase activity using 10 pl of fraction and PtdIns as substrate as described below. PtdIns 4-kinase, which eluted in the breakthrough, was retained without any further purification; PtdIns 3-kinase eluted at approximately 0.3 M KCl. The PtdIns 3-kinase pool was subjected to a 40% ammonium sulfate pre- cipitation. Following centrifugation (60 min at 13,500 x g), pellets were resuspended in buffer B (10 m~ potassium phosphate, pH 7.4, containing 6 m~ P-mercaptoethanol, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, and 1 m~ MgCI,) and loaded onto a 50-ml fast-flow hydroxylappatite column (Calbiochem) at 2.5 mumin. The column was washed with 150 ml of buffer B until the Azso base line reached zero and the kinase was then eluted with a linear gradient of 10-320 m~ potassium phosphate, pH 7.4, at 1 mumin over 450 min.

Active fractions were pooled and then loaded at 3 d m i n onto a Monos column (8 ml) (Pharmacia LKB Biotechnologies Inc.) equili- brated in buffer C (50 m~ MES, pH 6.2, containing 6 m~ 6-mercaptoethanol, 0.1 m~ EGTA, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, and 1 m~ MgCl,). PtdIns 3-kinase was eluted with a linear gradient of 0-0.4 M

Chromones Inhibit Phosphatidylinositol 3-Kinase 5243

FIG. 1. Synthesis scheme for chromones.

a. MeLi. THF. TMSCI b. potassium t-butoxide, CS2, benzene c. NaH, CH3l d. HNRR'. butanol, acetic acid

KC1 in buffer C over 120 min. In assaying fractions, two pools of PtdIns 3-kinase activity were routinely found; the bulk of the activity was found in the flow-through (presumably due to the high phosphate concentration during sample loading), while about 20% of the activity was eluted in the gradient. Whereas the material in the gradient had con- siderable PtdIns 4-kinase activity, essentially no PtdIns 4-kinase activity was associated with the PtdIns 3-kinase eluted in the flow-through. Therefore, the MonoS flow-through was concentrated by tangential flow filtration on a Mini-Ultrasette Omega 50 K membrane (Filtron, Inc., Northborough, M A ) and diluted in buffer C to lower the conductivity. The material was then reloaded onto the MonoS column using the above conditions. The PtdIns 3-kinase bound to the column during the wash and was eluted in the gradient as above. The pooled PtdIns 3-kinase was found to contain 95% PtdIns 3-kinase activity (and 5% PtdIns 4-kinase).

The MonoS column pool was diluted with buffer A and chromatographed on MonoQ (1 ml) and eluted with a gradient of 0-0.4 M KC1 in buffer A. The final pool was assayed for PtdIns 3-kinase and PtdIns 4-kinase activity. The final product was found to contain >99% PtdIns 3-kinase activity.

Purification of Qrosine-phosphorylated PtdIns 3-Kinase-PtdIns 3-kinase that was phosphorylated on tyrosine residues by PDGF-receptor was isolated as follows. Swiss 3T3 cells were grown in media and stimulated with 10 ILM PDGF as previously described (40). Cell lysates were subjected to immunoprecipitation with a monoclonal antiphosphotyrosine antibody (Upstate Biologicals, Inc., Lake Placid, N Y ) and the antibody-antigen complex immobilized with protein A-Sepharose (Sigma). PtdIns 3-kinase was eluted from the immune complex by washing the immunopellet with 10 m~ Tris buffer, pH 7.6, containing 100 m~ NaCl, 400 p~ sodium orthovanadate, 1 m~ phenylmethylsulfonyl fluoride, 1 m~ EGTA, 40 pg/ml leupeptin, 20 pg/ml aprotinin, 200 p~ adenosine, and 5 m~ phenylphosphate. The purified enzyme in the phosphorylated state was collected by centrifugation.

PtdIns Kinase Assays-PtdIns 3-kinase activity was measured as previously described (31). Inhibitor candidates were initially dissolved in dimethyl sulfoxide and then diluted 10-fold with 50 mM HEPES buffer, pH 7.5, containing 15 m~ MgCI, and 1 m~ EGTA. Ten pl of this solution was incubated with enzyme (9 p1) and PtdIns (5 pl of a 2 mg/ml stock solution in 50 m HEPES buffer, pH 7.5, containing 1 m~ EGTA). The final reaction mixture contained 100 p~ inhibitor and 3% dimethyl sulfoxide (v/v). This concentration of dimethyl sulfoxide had no effect on RdIns 3-kinase activity; control reaction mixtures contained 3% dimethyl sulfoxide (v/v) without inhibitor. Reactants were preincubated 5 min at room temperature and then the enzyme reaction was started upon addition of 1 pl of [y-32P]ATP (2 mCi/ml, 500 p~ stock solution; 0.08 mCi/ml, 20 p~ final concentration). The reaction was allowed to proceed 10 min a t room temperature with frequent mixing, after which time the reaction was quenched by addition of 40 pl of 1 N HC1. Lipids were extracted with addition of 80 pl of CHCI&leOH ( l : l , v/v). The samples were mixed and centrifuged, and the lower organic phase was applied to a silica gel TLC plate (EM Science, Gibbstown, NJ), which was developed in CHCI,/MeOH/H,O/NH,OH (45:35:8.5:1.5, v/v). Plates were dried and the kinase reaction visualized by autoradiography. The PtdIns 3-monophosphate region was scraped from the plate and quantitated using liquid scintillation spectroscopy with Readyprotein (Beck- man Instruments, Inc., Fullerton, CA) used as the scintillation mixture. The level of inhibition for LY294002 and analogs was determined as the percentage of 32P/counts/minute compared with controls,

PtdIns 4-kinase activity was measured in a similar manner except that the enzyme reaction mix contained 0.05% Nonidet P-40. The crude PtdIns 4-kinase was obtained from the breakthrough of the FastFlow Q column above. PtdIns 3-kinase was completely inactive in the presence of Nonidet P-40 (41), while PtdIns 4-kinase retained full activity (42).

Alternatively, products of the PtdIns 3-kinase reaction were con- firmed by HPLC (43). Phospholipids were deacylated in methylamine reagent and separated using a Whatman Partisphere SAX anion-exchange column as previously described (44). A Radiomatic model A-140 Flo-Onemeta on-line rafiioactivity detector was used to monitor the

deacylated [32P]enzyme products; deacylated [3HlPtdIns 4-monophosphate was added as an internal standard.

Kinetic Studies:ATPDependence-Bovine brain PtdIns 3-kinase was incubated as described above in the presence of 1-20 p~ LY294002 while simultaneously varying the ATP concentration from 1 to 20 1.1~ (0.08 mCi/ml final concentration); phosphatidylinositol was held con- stant at 0.4 mg/ml. The reaction volume was 25 pl. The kinase reaction was allowed to take place for 10 min at room temperature, after which time the reaction was stopped by addition of 1 N HCl as above. Lipid extraction and thin layer chromatography were performed as described above. Quantitation was achieved by liquid scintillation counting of the [32PlPtdIns 3-monophosphate as described above.

Preparation of Cultured Rabbit Aortic Segments-Thoracic aortas were obtained from adult male rabbits and placed in chilled DMEM containing 0.1 mg/ml gentamicin. f i r removal of the adventitia, the aorta was cut into 4-mm segments. Uniform mechanical injury was performed on each segment by clamping a hemostat to a region of the aorta along its axis. Both untreated control and treated segments were incubated for 1 week in 10 ml of DMEM (containing 10% fetal bovine serum, 0.05 mg/ml gentamicin, and 10 p~ BrdU) at 37 "C and 7% COP either in the presence or absence of LY294002. The segments were fed every 2 days with fresh media and drug. All segments were then fixed in Methyl Carnoy's furative, embedded in parafin, and cut into 4-5-pm thick sections. Tissue sections were immunostained with monoclonal antibody to BrdU (451, and diaminobenzedine was used as a chromogen, resulting in a dark brown precipitate at the site of the reaction. All tissues were counterstained with hematoxylin. Random tissue sections were stained with smooth muscle-specific actin to confirm the specificity of BrdU labeling for proliferating smooth muscle nuclei (46). The number of BrdU-stained nuclei were quantified by direct counting. The sampling region for quantification was limited to the nuclei in the intima and media delineated by the limits of the perturbed area. Per- cent BrdU uptake was determined as a ratio of the number of BrdU- stained nuclei in drug-treated tissue uersus untreated control.

Inhibition of PtdIns 3-Kinase Activity in Human Neutrophils- Neutrophils were isolated from freshly drawn, heparin-treated human blood as previously described (18). Neutrophils were labeled with [32Plorthophosphate, preincubated in the presence or absence of 50 p~ LY294002, and stimulated with 10 n M fMLP for 60 s as previously described (18, 47). Lipids were extracted and analyzed by TLC and autoradiography, as well as by HPLC of the deacylated phosphatidylinositides (18). Migration of the neutrophil phosphatidylinositides was compared with enzymatically derived products of bovine brain PtdIns 3-kinase (18).

Assays of Other ATP-requiring Enzymes-Protein lunase C was assayed as previously described using a mixture of equal amounts of recombinant protein kinase Ca and protein kinase Cp expressed in St9 insect cells using recombinant baculovirus; histone was employed as substrate (48). In vitro protein tyrosine kinase was measured using EGF receptor membrane preparations with poly(Glu, Ala, Tyr 6:3:1) as substrate (Sigma) (49,501. In uiuo protein tyrosine kinase activity was determined using antiphosphotyrosine immunoprecipitations of PDGF- BB-stimulated cultured rabbit aortic smooth muscle cell lysates using methods previously described (40); cells were treated with 50 p~

LY294002 for 10 min prior to addition of PDGF. Protein kinase A activity was determined by the ability of the protein kinase A catalytic subunit to phosphorylate phospholamban in rat cardiac sarcoplasmic reticulum membrane preparations as previously described (51). Cyto- solic MAP kinase and S6 kinase assays were performed as previously described, using myelin basic protein and 3R peptide derived from 40 S ribosome as substrates, respectively (52). Diacylglycerol kinase (Calbio- chem) was assayed as described by Preiss et al. (53) using dioleoylglyc- erol as substrate. Rabbit kidney ATPase activity (Sigma) was assayed according to methods supplied by the manufacturer; activity was determined colorimetrically using Taussky-Shorr reagent (54). Protein-tyrosine kinase ( ~ 6 0 " " ~ ) (Oncogene Science, Uniondale, N Y ) was assayed according to methods supplied by the manufacturer using RaytideTM (Oncogene Science, Uniondale, N Y ) as substrate.

5244 Chromones Inhibit Phosphatidylinositol 3-Kinase

PIP- , ' 9

Origin - I

FIG. 2. Inhibition of PtdIns 3-kinase by chromones. Bovine brain PtdIns 3-kinase was incubated with 100 pr inhibitor, 0.4 mglml phosphatidylinositol, and 20 PM [y-"PIATP (0.08 mCi/ml) for 10 min as described under "Experimental Procedures." Reaction was analyzed by autoradiography following thin layer chromatography. ["PlPtdIns-3- monophosphate was quantitated using liquid scintillation counting.

RESULTS

Inhibition of PtdZns 3-Kinase by Chromones-Compounds were initially tested for their ability to inhibit PtdIns 3-kinase a t a concentration of 30 pg/ml (approximately 100 p). Using thin layer chromatography to assess enzyme activity (Fig. 2), LY294002 completely abolished enzymatic activity, whereas other compounds partially inhibited the enzyme to varying de- grees. Chromones showing any inhibitory activity were then tested at concentrations ranging from 0.05 to 30 pg/ml to de- termine ICs0 values (concentration for 50% inhibition). LY294002 was the most potent inhibitor of PtdIns 3-kinase, with an ICs0 value of 0.43 pg/ml(1.40 p~), while the ICs0 values for other chromones ranged from 5 to 33 PM, demonstrating the ability of these compounds to inhibit PtdIns 3-kinase (Table I).

Znhibition of PtdIns 4-Kinuse-The most potent chromone inhibitor of PtdIns 3-kinase activity, LY294002, was tested at a concentration of 30 pg/ml for its ability to inhibit PtdIns 4-kinase. As indicated in Fig. 3, activity comparable to control was found for PtdIns 4-kinase in the presence of LY294002; liquid scintillation counting shows that 98% of the control PtdIns 4-kinase activity is present in the sample treated with LY294002. In contrast, PtdIns 3-kinase was completely inhibited by LY294002 a t 30 pg/ml; less than 1% of the control PtdIns 3-kinase activity in present in the sample treated with LY294002. In addition, PtdIns 3-kinase is shown to be completely inactivated in the presence of 0.05% Nonidet P-40, whereas the PtdIns 4-kinase is fully activated. Therefore, while LY294002 is a potent inhibitor of PtdIns 3-kinase, it does not inhibit RdIns 4-kinase.

Kinetic Studies: ATP Dependence-PtdIns 3-kinase activity was assayed at various concentrations ofATP in the presence of increasing concentrations of LY294002. As indicated in Fig. 4, Lineweaver-Burk analysis demonstrates that LY294002 be- haves as a competitive inhibitor for the ATP binding site of PtdIns 3-kinase. AK, value of 1.60 p is obtained from a plot of the slopes in the Lineweaver-Burk plot against concentration of LY294002 (Fig. 4, inset).

Inhibition of nrosine-phosphorylated PtdZns 3-Kinase-The phosphorylation state of PtdIns 3-kinase did not affect ability of LY294002 to act as an inhibitor of the enzyme. Phospho-PtdIns 3-kinase, isolated from antiphosphotyrosine immunoprecipitations of PDGF-stimulated 3T3 cells, was completely inhibited by 100 PM LY294002, which is comparable to results obtained by inhibiting purified bovine brain PtdIns 3-kinase with 100 p LY294002 (Fig. 5). These results are consistent with the idea that LY294002 inhibits PtdIns 3-kinase irrespective of the

- Ptdlns-P

- Origin

FTG. 3. Inhibition of PtdIns 4-kinase and PtdIns 3-kinase by LY294002. Crude bovine brain PtdIns 4-kinase or purified bovine brain 3-kinase were incubated with 100 p~ LY294002,0.4 mg/ml phosphatidylinositol, and 20 PM [y-"P]ATP (0.08 mCi/ml) for 10 min as described under "Experimental Procedures." PtdIns 4-kinase was assayed in the presence of 0.05% Nonidet P-40. Reaction was analyzed by autoradiography following thin layer chromatography, and products were quantitated using liquid scintillation counting.

/ /

n " . 0.0 0.2 0.4 0.6 0.8 1 .o

l/[ATP] (pM - 1 )

FIG. 4. LineweaveIcBurk plot of the inhibition of PtdIns 3-kinase with LY294002. Inhibition was by 0 PM (O), 1 p~ (0). 5 PM (W), 10 p~ (A), or 20 p~ (0) LY294002 against varying concentrations of ATP. The Ki for LY294002 was determined from a plot of the slopes obtained from the Lineweaver-Burk plot a t each concentration of LY294002 (inset ).

phosphorylation state of the enzyme. Inhibition of Other ATP-requiring Enzymes-Table I1 sum-

marizes the effects of LY294002 on other ATP-requiring enzymes, including representative protein tyrosine kinases, protein serinelthreonine kinases, lipid kinases, and rabbit kidney ATPase. Data are presented as percent activity remaining in samples treated with 50 PM LY294002 compared with untreated control; at the indicated concentration, PtdIns 3-kinase activity in the presence of LY294002 is less than 1% of control. Whereas quercetin, the initial chromone tested against PtdIns 3-kinase, was active against PtdIns 4-kinase (IC50 = 1.8 pg/ml = 5.3 p ~ ) (32), EGF receptor tyrosine kinase (IC5o = 26 p) ( 5 9 , and protein kinase C (IC5o = 82 p) (36), LY294002 did not inhibit any of these proteins. In addition, LY294002 did not inhibit cytosolic MAP kinase or S6 kinase, rabbit kidney ATPase, protein kinase A, diacylglycerol kinase, or p60""" a t concentrations 35-fold greater than the ICso of PtdIns 3-kinase. These data further substantiate that LY294002 is a selective


“Ptdlns 3-P

1 -origin

Brain Ptdlns3K Ptdlns3K PtdlnstK Ptdlns3K

Brain 3T3 3T3

+LY294002 +LY294002 FIG. 5. Inhibition of tyrosine-phosphorylated PMIns 3-kinase

by LY294002. PtdIns 3-kinase isolated from antiphosphotyrosine im- munoprecipitates from platelet-derived growth factor-stimulated Swiss 3T3 cells was incubated with 100 PM LY294002, 0.4 mg/ml phosphatidylinositol, and 20 p~ [Y-~~PIATP (0.08 mCi/ml) for 10 min as described under “Experimental Procedures.” Reaction was analyzed by autoradiography following thin layer chromatography.

TABLE I1 Inhibition of other ATP-requiring enzymes by LY294002

Representative serinelthreonine kinases, tyrosine kinases, lipid kinases, and ATPase were assayed in the presence of LY294002 as described under ‘Experimental Procedures.” None of these enzymes were significantly inhibited by 50 p~ LY294002. In contrast, PtdIns 3-kinase was completely inactivated at this concentration of inhibitor (<I% activity remaining compared with control).

ATP-requiring enzyme

o/r activity remaining SE at 50 PM LY294002

Protein Ser/”hr kinases Protein kinase C Protein kinase A MAP kinase S6 kinase

Protein tyrosine kinases EGF receptor c-src kinase

Lipid kinases PtdIns 4-kinase Diacylglycerol kinase

ATPase Rabbit kidney ATPase

102 f 15 112 r 11

105 25 92 14

127 14 100 f 3

98 r 4 108 = 9

113 5 1

inhibitor of PtdIns 3-kinase and does not show any significant inhibition of selected tyrosine kinases, serinelthreonine kinases, lipid kinases, or ATPases when tested at doses that completely abolish PtdIns 3-kinase activity.

Inhibition of PtdIns 3-Kinase Activity in Human Neutrophils-Isolated human neutrophils, labeled with [32Plorthophosphate, were incubated 5 min in the presence or absence of 50 p~ LY294002 and then stimulated with 10 nM fMLP for 60 s. Labeled phospholipids were extracted and analyzed by thin layer chromatography (Fig. 6A ). Migration of the products was compared with [32P]PtdIns-3P, [“2P]PtdIns-3,4- Pz, and [32PJPtdIns-3,4,5-P3 produced using purified bovine brain PtdIns 3-kinase; standard PtdIns, PtdIns-4-P, and Pt- dIns-4,5-Pz were co-chromatographed and visualized by I2 vapor. Stimulation of neutrophils with fMLP resulted in rapid

production of PtdIns-3,4,5-P,, which migrated below PtdIns-Pz and was absent in unstimulated neutrophils (18). However, the spot corresponding to PtdIns-3,4,5-P3 was missing in neutrophils treated with LY294002. HPLC analysis following chemical deacylation of the phosphatidylinositols further demonstrated that LY294002 blocked PtdIns 3-kinase activity in neutrophils (Fig. 6B). Whereas PtdIns-3,4-Pz and PtdIns- 3,4,5-P3 were present in neutrophils stimulated with WLP, these products were absent in cells treated with LY294002. In addition, whereas unstimulated neutrophils contained a basal level of PtdIns-3-P (which was also elevated in fMLP-stimulated neutrophils), this product was also absent in LY294002- treated neutrophils. Finally, there was no effect of LY294002 on levels of PtdIns-4-P and PtdIns-4,5-Pz, and LY294002 had no effect on cell viability as determined using 3-4,5-dimethyl- thiazol-2-yl-2,5-diphenyltetrazolium bromide. Therefore, LY294002 inhibited PtdIns 3-kinase activity in fMLP-stimulated human neutrophils without cell toxicity. These data dem- onstrate that LY294002 was effective in inhibiting PtdIns 3-kinase in whole cells.

Inhibition of Smooth Muscle Cell Proliferation in Cultured Rabbit Aortic Segments-Fig. 7 summarizes the effect of LY294002 on BrdU uptake by the smooth muscle cells in the experimentally damaged regions of the rabbit aorta in culture. The PdtIns 3-kinase inhibitor prevented BrdU uptake in a dose-dependent manner, suggesting the effect of LY294002 is specific. Microscopic examination of tissue sections did not re- veal visual evidence of tissue necrosis at any of the concentrations studied. Control culture studies using known cytotoxic compounds showed characteristic tissue death indiscrimi- nantly across the entire tissue (data not shown). Under experimental assay conditions in which the perfusion of the drug into the tissue was limited by diffusion, the ICso of LY294002 for inhibiting BrdU uptake was 32 p ~ . These data suggest that LY294002 blocks proliferation of smooth muscle cells without cell death, and further suggest that an inhibitor of PtdIns 3-kinase may act as an antiproliferative agent.

PDGF-stimulated Z’yrosine Phosphorylation in Cultured Rabbit Aortic Smooth Muscle Cells-Primary cultures of rabbit aortic smooth muscle cells were preincubated with 50 p~ LY294002 for 10 min prior to stimulation with 10 nglml PDGF- BB. Following cell lysis, a monoclonal antibody against phos- photyrosine was used to measure differences in protein phosphorylation between unstimulated and PDGF-treated cells. Several proteins were phosphorylated on tyrosine in response to PDGF that were absent in unstimulated cells (Fig. 8). Among the labeled bands present following SDS-polyacrylamide gel electrophoresis were those corresponding in molecular mass to the autophosphorylated receptor (185 kDa) and the regulatory subunit of PtdIns 3-kinase (85 kDa). PDGF-stimulated cells treated with LY294002 do not show any differences in protein phosphorylation when compared with PDGF-stimulated cells in the absence of inhibitor, demonstrating that LY294002 had no inhibitory effect on PDGF receptor tyrosine phosphorylation. It is noteworthy to point out that phosphorylation of the 85-kDa subunit of PtdIns 3-kinase is not affected by LY294002. These data, as well as other data presented in this paper, dem- onstrate that LY294002 specifically inhibits PtdIns 3-kinase but does not inhibit other tested protein or lipid kinases in whole-cell and cell-free systems.

DISCUSSION

The association of PtdIns 3-kinase with a number of growth factor and chemotactic receptors suggests that the enzyme may be involved with mitogenic and/or chemotactic responses in cells. The products of PtdIns 3-kinase (PtdIns 3-monophosphate, PtdIns-3,4-bisphosphate, and PtdIns-3,4,5-trisphos-

5246

A

i ri 0)

0 u)

(0

aJ E

Chromones Inhibit Phosphatidylinositol 3-Kinase

B

- Ptdlns - P

- Ptdlns - P,

- Origin

200 A

E, k Y 0

0

Elution Time (min)

neutrophils were incubated in the absence or presence of 50 pw LY294002 for 5 min and then stimulated with 10 nM fMLP for 60 s. Phospholipids FIG. 6. The ability of LY294002 to inhibit PtdIns 3-kinase in fMLP-stimulated human neutrophils. [3zP10rthophosphate labeled

were extracted and separated as previously described (18). A, thin layer chromatography. Migration of the phosphatidylinositols was compared with PtdIns-3-P, PtdIns-3,4-Pz, and PtdIns-3,4,5-P, produced by bovine brain PtdIns 3-kinase; standards of PtdIns, PtdIns-4-P, and PtdIns-4,5-Pz were chromatographed and visualized by Iz vapor. B , HPLC analysis of deacylated PtdIns-phosphates. PtdIns-phosphates were extracted from TLC plates, chemically deacylated with methylamine, and subjected to anion-exchange HPLC analysis using an on-line radiochemical detector as described under "Experimental Procedures." Deacylated [:'H]PtdIns-4-P and [3H1PtdIns-4,5-Pz were used as internal standards.

phate) are not found in resting cells but are produced within seconds in cells stimulated by growth factors or neutrophils stimulated with fMLP (10, 17, 56). Mutants of the PDGF-receptor that lack the PtdIns 3-kinase-binding site fail to have associated PtdIns 3-kinase activity, and cells expressing these mutant receptors fail to show increased DNA synthesis and cell division (20,231. These data support a role for PtdIns 3-kinase in mitogenesis, although the exact role has yet to be determined. Specific inhibitors of PtdIns 3-kinase would not only be useful as potential antiproliferative agents, but would also aid in elucidating the biological role of the enzyme and its products.

Quercetin, a naturally occurring bioflavinoid, inhibits PtdIns 3-kinase activity with an IC5o = 1.3 pg/ml (3.8 p.d (31) but is not a specific inhibitor of PtdIns 3-kinase in that it also inhibits PtdIns 4-kinase (32), as well as tyrosine and serinekhreonine kinases (33-37). Quercetin was therefore viewed as the lead compound to develop agents intended to specifically inhibit PtdIns 3-kinase without affecting other PtdIns and protein kinases.

Previous findings with quercetin suggested that PtdIns 3-kinase inhibition is greatly influenced by the pattern of substitution on the flavinoid ring, with the 2- and 3-positions being

dihydroxyphenyl group at the 2-position of the chromone ring was replaced with a morpholine moiety. In addition, the hydroxyl groups at the 3-, 5-, and 7-positions of quercetin were deleted. The resulting compound, LY292223 (2-(4-morpholinyl)- chromone), inhibited PtdIns 3-kinase with an ICso value of 5 w, which is very similar to that of quercetin. Fused ring analogs did not improve potency, but the placement of a phenyl moiety at the 8-position of the chromone ring resulted in the most potent compound of the series, LY294002 (2-(4-morpholinyl)-8-phenylchromone) (Table I).

In order to characterize the specificity of LY294002, the compound was also tested against PtdIns 4-kinase. While PtdIns 3-kinase was completely inhibited by LY294002 at a concentration of 30 pg/ml (100 m), PtdIns 4-kinase was not affected a t the same concentration (Fig. 3). LY294002 a t a concentration of 50 p~ did not inhibit the EGF receptor tyrosine kinase or protein kinase C (Table 11), which are other targets of quercetin (36, 55). In addition, LY294002 did not inhibit other representative ATP-requiring enzymes, including protein kinases, lipid kinases, and ATPase (Table 11). Although LY294002 and quercetin are both competitive inhibitors for the ATP-binding site of PtdIns 3-kinase (Fig. 4), LY294002 does not inhibit several

most essential (31). For example, the isoflavinoid genistein did other ATP-requiring enzymes, in contrast to quercetin. not inhibit PtdIns 3-kinase, while the degree of inhibition with LY294002 also had no effect on PDGF receptor-mediated tyro- flavinoids such as quercetin was not only sensitive to the sub- sine phosphorylation in cultured rabbit aortic smooth muscle stituent at the 2-position but also the locations of the hydrogen cells (Fig. 8), including phosphorylation of the 85-kDa subunit bonding groups (hydroxyls) on its 2-substituent. In an effort to of PtdIns 3-kinase. Therefore, LY294002 appears to be a selec- convey specificity of the inhibitor against PtdIns 3-kinase, the tive inhibitor of PtdIns 3-kinase.


100

80

60

40

20

0 0 20 40 60 80 100

[294002] (pM) FIG. 7. Dose-response curve showing the ability of LY294002 to

prevent BrdU uptake by the smooth muscle cells in mechani- cally perturbed regions of the intima and media of cultured rabbit aortic segments. Segments were cultured for 7 days in medium containing 10 mM BrdU in the presence or absence of LY294002 as described under “Experimental Procedures.” BrdU-labeled nuclei were identified by immunostaining and counted. Percent BrdU uptake was determined as a ratio of the number of BrdU-stained nuclei in drug- treated tissue uersus unstimulated control. Values are mean * standard error (n = 4).

- 205 - 116.5 APtdlns 3K - 80

- 49.5

FIG. 8. Antiphosphotyrosine immunoprecipitations. Primary cultures of rabbit aortic smooth muscle cells were placed in DMEM containing 0.1% bovine serum albumin overnight and then treated for 10 min in fresh medium containing either 50 p~ LY294002 or dimethyl sulfoxide (control); final dimethyl sulfoxide concentration for all samples was 1.6% (v/v). Cells were then stimulated * 10 ng/ml platelet- derived growth factor-BB for 5 min. Cell lysates were immunoprecipi- tated using an antiphosphotyrosine monoclonal antibody (Upstate Bio- logicals, Lake Placid, N Y ) as described previously (40). Following labeling with [Y-~~PIATP, phosphoproteins were analyzed by SDS-polyacrylamide gel electrophoresis on a Novex (San Diego, CA) 8% gel. Prestained molecular weight markers (Bio-Rad): 205 ma, myosin; 116 kDa, P-galactosidase; 80 kDa, bovine serum albumin; 49.5 kDa, oval- bumin.

A significant feature of the ability of LY294002 to inhibit PtdIns 3-kinase is the effect of single atom substitutions in the morpholine ring (Fig. 9). The simple replacement of the oxygen with sulfur, hydroxymethyl, methylene, or nitrogen causes a dramatic decrease in the efficacy of these compounds against PtdIns 3-kinase. For example, the 4‘-sulfur heteroatom in LY297978 gives a compound that inhibits 50% of the PtdIns 3-kinase activity at 100 p ~ , while methylene or amine groups essentially do not inhibit PtdIns 3-kinase at the same concen-

2-Substituted-8-Phenyl Chromones

pQ 0

\

X Serial Number Ptdlns 3-Kinase

0 294002 <I %

S 297978 50

CH-OH 298619 81

C-H, 297962 94

N-H 30351 1 99

* %Activity Remaining @ 100 pM

FIG. 9. The effect of single atom substitution in the morpholino ring of LY294002. Bovine brain F‘tdIns 3-kinase was incubated with 100 PM inhibitor, 0.4 mg/ml phosphatidylinositol, and 20 p~ [y-”PIATP (0.08 mCi/ml) for 10 min as described under ”Experimental Proce- dures.” Reaction was analyzed by autoradiography following thin layer chromatography. [32P]F’tdIns-3-monophosphate was quantitated using liquid scintillation counting and is presented as percent activity remaining compared with control in the absence of inhibitor.

tration. Interestingly, LY298619, which contains a 4’-hydroxymethyl substituent, is a very poor inhibitor of PtdIns 3-kinase, especially since quercetin contains a hydroxyl group in this same position. The morpholine ring is expected to be in the chair conformation rather than a planar configuration such as the dihydroxyphenyl group of quercetin, which may affect both hydrogen bonding as well as steric fit. In addition to the strict requirement for a 4“oxygen heteroatom in the morpholine ring, structurally related classes of compounds such as quinolones, quinazolones, and quinazolines did not inhibit PtdIns 3-kinase, whereas substitutions on positions 5-8 of the chromone ring did not result in such drastic differences in activity. For example, LY292223, which is LY294002 without the 8-phenyl substituent, was still a relatively potent inhibitor of PtdIns 3-kinase. While structural information about the ATP-binding domain of PtdIns 3-kinase has not yet been reported, these data suggest a very specific binding site involving the morpholine and pyran rings of these inhibitors. Thus, it is apparent that the chromone ring system is necessary for a PtdIns 3-kinase inhibitor, that inhibition is particularly sensitive to substituents at the 2-position of the chromone ring, and that inhibition is less sensitive to substituents at positions 5-8 of the chromone ring.

In addition to inhibiting purified PtdIns 3-kinase, LY294002 was also efficacious in whole-cell assays. LY294002 completely blocked PtdIns 3-kinase activity in fMLP-stimulated human neutrophils (Fig. 6). PtdIns-3,4,5-P3 was not detected in TLC analysis of the phosphatidylinositols extracted from stimulated human neutrophils treated with LY294002, and further HPLC analysis demonstrated that the other products of PtdIns 3-kinase (PtdIns-3-P and PtdIns-3,4-P2) were not found as well. Interestingly, whereas LY294002 blocks fMLP-mediated events in the neutrophil, it has no effect in inhibiting phorbol 12- myristate 13-acetate-stimulated events (PtdIns 3-kinase is not activated by phorbol 12-myristate 13-acetate). The effects of

5248 Chromones Inhibit Phosphatidylinositol 3-Kinase

the PtdIns 3-kinase inhibitor LY294002 on fMLP- and phorbol 12-myristate 13-acetate-mediated events in neutrophils will be described later.' LY294002 also prevented proliferation of smooth muscle cells in response to mechanical injury of cultured rabbit vascular segments (Fig. 7). Proliferation, as measured by uptake of BrdU, was inhibited in a dose-dependent manner without exhibiting cell toxicity. These data demon- strate that a PtdIns 3-kinase inhibitor may act as an antiproliferative agent and further substantiate a role for PtdIns 3-kinase in cell proliferation.

F'tdIns 3-kinase is a dimeric enzyme consisting of a 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Whereas the products of the enzyme, PtdIns 3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3, have been identified, the cellular events regu- lated by the enzyme and its substrates remain an enigma. It has been postulated that PtdIns-3,4,5-P3 may induce cytoskel- eta1 reorganization in cells, perhaps either by direct interaction with cytoskeletal proteins or by regulating a protein kinase cascade that ultimately regulates assembly of microtubules, such as MAP kinase activation in EGF- and nerve growth factor-stimulated PC12 cells (57). The identification of a potent and specific inhibitor of PtdIns 3-kinase provides a valuable tool in which to study the cellular role of the enzyme and may also lead to the development of therapeutic agents for the treatment of proliferative diseases.

Acknowledgments-We thank William B. Lacefield for his support, encouragement, and many helpful insights. We also thank Chuck Fkidy for performing the cultured aortic segment assays, George Boder, Ter- esa Burke, and Lori Eichelberger for performing the EGF receptor tyrosine kinase assays, Judi Schelm and Paul Simpson for neutrophil isolation, and Todd Wiernicki and Dan Wood for providing cultured rabbit aortic smooth muscle cells. We also extend our gratitude to Bent- ley Cheatham (Joslin Diabetes Center) for performing the assays against MAP kinase and S6 kinase, to Curtis Ashendel (Purdue Uni- versity) for in vitro testing of LY294002 against protein kinase C, to Jim Thomas and Roger Roudebush for investigating the inhibition of ~ 6 0 " - " ~ , to Nancy Bowling for performing the protein kinase A assays, and to Alexis Traynor-Kaplan (UCSD) for helpful suggestions and dis- cussions.

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journal of vol. 269, no. 7, of pp. 5241-5248, 1994 1994 ...vvvvvvvvvv vvvvvvvvvv cultured aortic...

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