Phosphatidylinositol 3-kinase and its novel product ...

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 264, No. 34, Issue of December 5, pp. 20181-201&1,1989 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication Phosphatidylinositol 3-Kinase and Its Novel Product, Phosphatidylinositol 3-Phosphate, Are Present in Saccharomyces cerevisiae” (Received for publication, June 26, 1989)

Kurt R. Auger$., Christopher L. Carpenterg, Lewis C. Cantley, and Lyuba Varticovski From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 021I 1 and the $Hematology-Oncology Unit, Massachusetts General Hospital, Boston, Massachusetts 02114

The metabolism of polyphosphoinositides has been shownto be an important factor in controlling the proliferation of Saccharomyces cerevisiae. The monophosphate form of phosphatidylinositol has been assumed to be phosphatidylinositol 4-phosphate (PI-4P). Recent evidence from our laboratory has established that a phosphatidylinositol (PI) kinase, which phosphorylates the D-3 position of the inositol ring (PI 3-kinase), is associated with many activated proteintyrosine kinasesand may play an important role in the signaling of cell proliferation (Auger, K. R., Serunian, L. A., Soltoff,S. P., Libby, P., and Cantley, L. C. (1989) Cell 57,167-175). To determine the evolutionary conservation of this enzymatic activity, we investigated of yeast its presence in yeast.In vitro PI kinase assays cell homogenates demonstrated that PI 3-kinase activity was present. Preliminary biochemical characterization of the activity suggestedthat it was quite different from the mammalian enzyme yet catalyzed the same reaction, i.e. phosphorylating the D-3 hydroxyl position of the inositol ring of phosphatidyl-myo-inositol. [3H]Inositollabeling of intact yeast cells withthe subsequent extraction, deacylation,and high performance liquid chromatography analysis of the lipids demonstrated that PI-3-P was as abundant as the PI-4-P isomer. The conservation of this enzymatic activity from yeast to man suggests that it has an important functional role in the cell cycle.

Polyphosphoinositides and theirmetabolites have been implicated as mediators of hormone responses, growth factor stimulation, cell cycle progression, and oncogenic transformation in many eukaryotic systems including simple organisms suchas yeast (1-3). Until recently, phosphatidylinositol (PI)’ phosphorylation was thought to involve only two en* This research was supported by National Institutes of Health Grant GM 36624. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-956-6744. Fax: 617-956-0445. ’The abbreviations used are: PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP,, phosphatidylinositol bisphosphate; PI-3-P, phosphatidylinositol 3-phosphate; PI-4-P, phosphatidylinositol 4-phosphate; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate;

zymes. PI-4 kinase phosphorylates the D-4 position of the inositol ringto produce phosphatidylinositol 4-phosphate(PI4-P). PI-4-Pmay then be phosphorylated by PI-4-P 5-kinase to produce phosphatidylinositol 4,5-bisphosphate (PI-4,5-P*). PI-4,5-Pz is the precursor of the two second messengers, inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and diacylglycerol. We have recently identified another PI kinase that phosphorylates the D-3position on the inositol ring to produce phosphatidylinositol3-phosphate (PI-3-P) (4, 5). This enzyme, phosphatidylinositol 3-kinase (PI 3-kinase), was discovered because of its specific association with activated proteintyrosine kinases in normal and transformed cells (6). Recent evidence also suggests that other polyphosphoinositides with a phosphateat the D-3 position of the inositol ringare present after stimulation of the cell: namely, phosphatidylinositol 3,4bisphosphate (PI-3,4-P2) (7) and phosphatidylinositol trisphosphate (PIP3) (7, 8). The function of these polyphosphoinositides in cell activation and proliferation remains unresolved; however, it is clear that these products are not in the direct pathwayfor generating the second messenger Ins-1,4,5-

PB. Polyphosphoinositides have been shown to be an essential component of the membranes of the yeast Saccharomyces cereuisiae (9) and seem to play a necessary role in cell proliferation (3). We looked for PI 3-kinase activity inthe yeast S. cerevisiue and for the presence of PI-3-P in [3H]inositollabeled cells. Here we report the presence of PI 3-kinase activity in yeast cell homogenates. We also report the surprising finding thatPI-3-P is asabundantasPI-4-Pas determined by HPLC analysis of deacylated lipids from yeast labeled i n uiuo. MATERIALS ANDMETHODS

Yeast Culture, Harvest, and Fractionation-A protease-deficient strain of S. cerevisiae (A/cu, leu 2/leu 2, trp l/trp 1, ura 352/ura 352, prb 1-1122/prb 1-1122, prc 1-407/prc 1-407, pep 4-3/pep 4-3) was obtained from Dr.FredWinston(Genetics Department, Harvard Medical School, Boston, MA) and grown in YEPD (1%yeast extract, 2% peptone, and 1%dextrose) to saturation in an overnight culture at 28 “C. Thecells were harvested by centrifugation and washed with lysis buffer (100 mM KCI, 15 mM HEPES (pH 7.5), 3 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 pg/ml leupeptin, and 10% glycerol). The cells were resuspended in cold lysis buffer (1 g/5 ml) and passed through a French press two times at 18,000p.s.i. The lysate was cleared by centrifugation at 2,000 X g for 10 min at 4 “C. The cleared lysate was then centrifuged at 150,000 X g for 45 min. The supernatant was collected and designated as the “cytosolic fraction,” and the pellet was resuspended in lysis buffer and designated as “membranes.” The samples were quick-frozen in a dry ice/ethanol bath and stored at -70 “C until used. PI Kinase Assays-PI kinase reactions were performed as described previously (7, 10). Briefly, the yeast fraction was diluted so that final protein concentration was 80 pg/ml in a 50.~1assay volume. Sonicated PI 3-kinase,phosphatidylinositol3-(hydroxy) kinase; PI 4-kinase, phosphatidylinositol 4-(hydroxy) kinase; PI-4-P 5-kinase, phosphatidylinositol4-phosphate5-(hydroxy) kinase; Ins-1,4,5-P3, inositol 1,4,5-trisphosphate; inositol-1,4-P,, inositol 1,4-bisphosphate; PI-3,4P,, phosphatidylinositol 3,4-bisphosphate; PIP3, phosphatidylinositol trisphosphate; HEPES, 4-(hydroxyethyl)-l-piperazineethanesulfonic acid;EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic a c i d HPLC, high performance liquid chromatography; gPI-3-P, glycerophosphoinositol3-phosphate; gPI-4-P, glycerophosphoinositol 4phosphate.

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Phosphatidylinositol 3-Phosphate

Yeast Is Present in

lipid substrate was added to the extract for a final concentration of TABLE I 0.2 mg/ml of which 25% (by weight) of the lipid was phosphatidylDistribution of PI 3-kinaseand PI 4-kinasein Saccharomyces serine and thereaction was initiated by addition of 10 mM MgCI2and cerevisiae 100 p~ ATP ([r-”P]ATP at 20 pCi/reaction: (Du Pont-New England S. cereuisiae were grown, harvested, fractionated, and assayed as Nuclear, 3000 Ci/mmol)) in 20 mM HEPES (pH 7.5). The reaction of three indedescribed in the text. These data are representative was incubated a t room temperature for5 minandstoppedand pendent experiments. extracted with 80 pl of 2 M HCI and 160 p1 of methano1:chloroform Total PI kinase PI 3-kinase PI 4-kinase (1:l). The assays were linear for a t least 8 min under these assay prnollrninlrng protein conditions. The organic phase was collected and analyzed by either thin layer chromatography or HPLC. Membrane 30 6.9 23 Thin LayerChromatography-Samples were separated by thin 5.9 0.46 Cytosol 6.4 layer chromatography in either acidic or basic conditions on oxalatetreated silica gel plates (E. Merck). The acidic condition provides good resolution of the highly phosphorylated polyphosphoinositides and is composed of l-propanol:2 M acetic acid (13:7, v:v) (7). Routine 3000 300 separations were done in basic a solvent system of chloroform:methanol:2.2 M ammonium hydroxide (9:7:2). Lipid standards were obtained from Sigma (PI-4-P and PI-4,5-P2) and Avanti Polar Lipids (PI) and co-chromatographed with the samples on the plates. 2500 250 The standards were visualized by exposing the dried plates to iodine vapor. The plates were subjected to autoradiography to visualize the reaction products. Deacylation and HPLC Analysis-J2P-Labeled reaction products 2000 200 E ”s were analyzed as described (5, 7). Deacylation of the lipids was done V n oil a Partiswith methylamine reagent and the products separated e % z : n a n phere SAX HPLC column (Whatman). PPl3P P

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RESULTSANDDISCUSSION

PI and PIP kinaseactivities were detected in both the cytosolic and membrane fractionsof S. cereuisiue cell lysates (Fig. 1 and Table I). The assay conditionswere performed to optimize the ability to detect PI 3-kinase activity as determined from mammalian cells. In the cytosolic fraction, these

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FIG. 2. HPLC analysis of the products of a PI kinase reaction performed on the membrane fraction with exogenous PI. The reaction, lipiddeacylation, and HPLC analysis were done as described under “Materials and Methods.” A [3H]gPI-4-P standard wasco-injected with the 32P-labeled sample for the analysis. The filled diamonds represent the “P-labeled sample and solid the line the “-labeled gPI-4-P (gPI4P) standard.

activities were dependent on the addition of exogenous PI and PI-4-P (data not shown). However, PIP and PIP2 products could be produced in the membrane fraction in the absence of exogenous lipids (Fig. 1, lane a). Addition of exogenous mammalian PI and PI-4-P to the assay increased the amount of PIP and PIP2produced (Fig. 1, lunes b and c). Solubilization of the membranes with1%Nonidet P-40 followed by dilution of the sample into the assay reduced the amount of PIP and PlP2 produced in the absence of exogenous substrate (Fig. 1, a b c d e f g h lune e ) but had little effect on the total PIP and PIP2 formed +NP40 -NP40 in the presence of exogenous PI and PI-4-P(Fig. 1, compare FIG. 1. Thin layer chromatography separation of the prodlunes b and c to lanes f and g). When PI-4,5-P2was added as ucts of PI, PIP, and PIP2 kinasereactions using s. cereuisiae a substrate to either the membranes (Fig. 1, lanes d and h ) or membranes. In lanes a-d, assays took place in the absence of the cytosolic fraction, no higher phosphorylated products were detergent Nonidet P-40. In lanes e-h, the membraneswere solubilized detected. Thus, S. cereuisiae cell lysates have enzymatic acin 1% Nonidet P-40 for 2 h prior to assay. Lanes a and e have no tivities that phosphorylate mammalian PI and PI-4-P. added lipid substrate. Lanes b and f were assayed in the presenceof 0.15 mg/ml exogenous PI, lanes c and g in the presence of 0.15 mg/ HPLC analysis of the products of the PI kinase reaction ml PI-4-P, and lanes d and h in the presence of 0.15 mg/ml PI-4,5indicated that both PI-3-P and PI-4-P were generated. The Pp.PI, PIP, and PIPpstandards were co-chromatographed in separate [“’PpIPIP produced from the membrane fraction with exogelanes, visualized by Ip vapor, and are indicated by circles. The slower nous PI was deacylated and co-injected with ‘H-labeled demigrating radioactivity co-migrates with inorganic phosphate. The acylated PI-4-P(gPI-4-P)standard. As shownin Fig. 2, solvent system used forthis analysiswas the acidic mixture described approximately 70% of the “’P-labeled product co-migrated under “Materials andMethods.”

Phosphatidylinositol 3-Phosphate Is Present in Yeast

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1000 with the [3H]gPI-4-P standard.However, 30% of the product co-migrated with the gPI-3-P standard (prepareddescribed as ( 5 ) ) . A small amount of the PI 3-kinase activity was also detected in thecytoplasmic fraction (Table I). Because mammalian PI was used as the substrate and the deacylation BOO product has migration properties identical to authentic PI-3P, it appears thatS. cereuisiue, like mammalian cells, has an activity that phosphorylates PI at the D-3 position of the inositol ring. In addition, the amountof PI 3-kinase in yeast U membranes is much higher than we have detected in mam600 2 E n m malian membranes.' The PI3-kinase andPI 4-kinase activities in the membrane fraction were comparedtothemammalian enzymeswith ECD respect to adenosine and detergent sensitivity. The mouse 400 fibroblast P I 4-kinase is dramatically activated by presentation of PI in non-ionic detergents such as Nonidet P-40while P I 3-kinase is almostcompletely inhibited under these assay conditions (4).Assaying P I kinase activity in yeast membranes using Nonidet P-40/PImicelles as the substrate caused , 200 a dose-dependent inhibition of both the PI 4-kinase and PI 3-kinase (data not shown). The mammalian enzymes can also be distinguished by the ability of adenosine to inhibit PI 4kinase extensively with only minor effects on PI3-kinase (4). However, both yeast enzymes were only inhibited 15%by 240 0 0.0 ptM adenosine. Similiar resultsfor the cytosolic activities were 4333098765 4 1 444432 observedfor bothNonidetP-40andadenosine(datanot Elution Time (minutes) shown).Thus,unlikethemammalian enzymes,non-ionic detergents and adenosine do not distinguishbetween the yeast FIG. 3. S. cereuisiue were labeled in vivo with [3H]inositol.Lipids P I 4- and PI 3-kinaseactivities. were extracted and HPLC analysis of the deacylated lipids was S. cerevisiae were labeled in vivo with [3H]inositol (10 pCi/ performed as described in the text. The total amount of 3H-labeled ml) for36 h in inositol-free medium. The lipids were extracted PIP recovered in this experiment was 1941 dpm of which 1040 dpm gPI-3-P. The filled diamonds represent the [3H]inositol-labeled in methano1:HCl and chloroform as described (11).The sam- was sample and the solid line the 32P-labeledgPI-3-P standard. ple was deacylated and analyzed by anion exchange HPLC with a 32P-labeled gPI-3-P standard. As illustrated in Fig. 3, PI-3-P is as abundant as PI-4-P in the organic extract of S. cerevisiae membranes and reported to be P I 4-kinase based cerevisiae. The [32P]gPI-3-P standard co-elutes precisely with on the finding that only inositol 1,4-bisphosphate (Ins-l,4a [3HJinositol-labeledcompoundextractedfromtheyeast P2)was produced by phospholipase Ctreatment of the product membranes. The gPI-4-P elutes soon after the gPI-3-P as (18). However, we (19) and others (20) have shown that a described previously ( 5 ) . This is in contrast to mammalian variety of phospholipases C fail to hydrolyze PI-3-P. Phoscells that have been investigated where PI-3-P constitutes pholipase C hydrolysis of a mixture of PI-4-P and PI-3-P only 3-10% of the PIP( 5 , 7, 12). The increased ratio of PI-3- would likely produce only Ins-1,4-P2,so the presence of P I 3P to PI-4-P in the intact yeast cells correlates with the greaterkinase activity would not be detected. Thus, although phosamount of P I 3-kinase activity in the membrane fraction of pholipase C hydrolysis of the product isuseful for the identiyeast comparedto mammalian cells. fication of PI-4-P from purified enzyme preparations, this The demonstration that PI-3-P is present in S. cereuisiue technique can not be utilized to determine the ratioof PI 3suggests that previous reports implicating polyphosphoinosi- kinase and PI 4-kinase activities in impure preparations. Our tide metabolism inyeast cell proliferation need to be re- results indicate that S. cerevisiae contain both types of P I evaluated. Glucose and sterol regulation of growth involves kinase. activation of the PI pathway (13, 14). CAMP,which in yeast We find it interesting that yeast have such a high PI-3-P is controlled by RAS ( E ) , has beenshown to activate PI to PI-4-P ratio relative to mammalian cells. Although the kinase (16) and to be involved in the progression of the cell quantity of the lipid does not necessarily reflect its imporcycle. This was assumed tobe P I 4-kinase but the questionof tance,it is clear thatPI-3-Psynthesishas been as well which P I kinase is activated now needs to be addressed. A conserved evolutionarilyas the synthesis of PI-4-P. The physrecent report indicates thatestrogen controls the entry of S. iological significance of PI-3-P is presently unclear, yet its cerevisiue into G1 early in the cell cycle by increasing the abundance in S. cereuisiue and the genetic manipulation camRNA for adenylate cyclase and consequently increasing the pable in yeast may facilitate a better understanding of the level of CAMP (17). This estrogen effect is probably also tied function of this lipid. into the RAS-CAMP-PI kinase system to promote cell prolifREFERENCES of the abilityof a monoclonal eration (17). In addition, reports antibody against PI-4,5-P2 to inhibit cell growth must now be 1. Whitman, M., and Cantley, L. (1988) Biochim. Biophys. Acta 948,327-344 reconsidered (3). The possibility that this antibody cross2. Fleischman, L. F., and Cantley, L. (1988) Am. J . Physiol. 255, reacts with novel polyphosphoinositides that containa phosC531-C535 phate in the3-hydroxyl position of the inositol ring needs to 3. Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikhwa, T. be examined.A P I kinase was recentlypurified from S. (1988) Nature 333, 188-190

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* K. R. Auger and C. L. Carpenter, unpublished observations.

4. Whitman, M., Kaplan, D., Roberts, T., and Cantley, L. (1987) Biochem. J. 247, 165-174 5. Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley,

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L. (1988) Nature 3 3 2 , 644-646 6. Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D.C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 5 0 , 1021-1029 7. Auger, K. R., Serunian, L. A,, Soltoff, S. P., Libby, P., and Cantley, L. C . (1989) Cell 57, 167-175 8. Traynor-Kaplan, A. E., Harris, A. L., Thompson, B. L., Taylor, P., and Sklar, L. A. (1988) Nature 3 3 4 , 353-356 9. Nikawa, J., Kodake, T., and Yamashita, S. (1987) J. Biol. Chem. 262,4876-4881 10. Whitman, M., Kaplan, D. R., Schaffhausen, B., Cantley, L., and Roberts, T. M. (1985) Nature 3 1 5 , 239-242 11. Holland, K. M., Homann, M. J., Belunis, C. J., and Carman, G . M. (1988) J. Bacteriol. 170, 828-833 12. Stephens, L., Hawkins, P. T., and Downes, C. P. (1989) Biochem. J. 259,267-276 13. Kaibuchi, K., Miyajima, A., Arai, K., and Matsumoto, K. (1986)

Is Present in Yeast Proc. Natl. Acad. Sci. U. S.A. 83,8172-8176 14. Dahl, J. S., and Dahl, C. E. (1985) Biochem. Biophys. Res. Commun. 133,844-850 15. Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J., Matsumoto, K., and Wigler, M. (1985) Cell 40, 27-36 16. Kato, H., Uno, I., Ishikawa, T., and Takenawa, T. (1989) J. Biol. Chem. 264,3116-3121 17. Tanaka, S., Hasegawa, S., Hishinuma, F., and Kurata, S. (1989) Cell 57,675-681 18. Belunis, C. J., Bae-Lee, M., Kelley, M. J., and Carman, G. M. (1988) J.Biol. Chem. 263,18897-18903 19. Serunian, L.A., Haber, M. T., Fukui, T., Kim, J. W., Rhee, S. G., Lowenstein, J. M., and Cantley, L. C. (1989) J.Biol. Chem. 264,17809-17815 20. Lips, D. L., Majerus, P. W., Gorga, F. R., Young, A. T., and Benjamin, T. L. (1989) J. Biol. Chem. 264, 8759-8763