A novel pathway for the formation of phosphatidylinositol 3, 4 ...

Communication

THE JOURNAL OF BIOLOGICAL CHEMWXY Vol. 265, No. 36, Issue of December 25, pp. 22086-22089, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

A Novel Pathway for the Formation of Phosphatidylinositol 3,4=Bisphosphate PHOSPHORYLATION OF PHOSPHATIDYLINOSITOL 3-MONOPHOSPHATE BY PHOSPHATIDYLINOSITOL-3-MONOPHOSPHATE 4-KINASE* (Received for publication, June 14, 1990) Kyohei YamamotoS, Andrea Grazianig, Christopher Carpenter& Lewis C. Cantleyg, and Eduardo G. Lapetinag From the SDiuision of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709 and the SDepartment of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Three polyphosphoinositides containing phosphate at the D-3 position of the inositol ring can be generated in vitro by phosphorylation of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate by a phosphatidylinositol-3kinase (Auger, K. R., Serunian L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57,167-175. An alternative pathway for in vivo synthesis of one of these lipids was recently suggested: phosphatidylinositol 3,4-bisphosphate could be produced by phosphorylation of phosphatidylinositol S-phosphate at the D-4 position of the inositol ring (Yamamoto, K., and Lapetina, E. G. (1990) Biochem. Biophys. Res. Commun. 168, 466-472). Here we demonstrate the presence of an enzyme in human platelets that phosphorylates [32P]phosphatidylinositol 3-phosphate to produce [32P]phosphatidylinositol 3,4-bisphosphate. This enzyme is Mg*+-dependent and its apparent molecular mass is approximately 150 kDa as estimated by sucrose gradient centrifugation and gel filtration chromatography. Unlike the major phosphatidylinositol-4-kinase in platelets that is stimulated by the detergent Nonidet P-40, the phosphatidylinositol-3-phosphate 4-kinase is inhibited by Nonidet P-40. Both activities are also differentiated by the action of adenosine. The discovery of this new enzyme raises the possibility that multiple pathways exists for generating D-3 phosphorylated phosphoinositides. Phosphatidylinositol

(PtdIns)’

3-kinase

phosphorylates

at

* 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. ’ The abbreviations used are: PtdIns, phosphatidylinositol; PtJIns(B)P, phosphatidylinositol 3-monophosphate; PtdIns(4)P, phosphatidylinositol I-monophosphate; PtdIns(3,4)P2, phosphatidylinositol3,4-bisphosphate; PtdIns(3,4,5)P, phosphatidylinositol3,4,5trisphosphate; PtdIns 3-kinase, phosphatidylinositol3-kinase; PtdIns 4-kinase, phosphatidylinositol 4-kinase; PtdIns(3)P 4-kinase, phosphatidylinositol-3-monophosphate 4-kinase; GroPIns(S)P, glycerophosphoinositol3-monophosphate; GroPIns(3,4)Pp, glycerophosphoinositol 3,4-bisphosphate; Ins(1,3,4)P3, inositol 1,3,4-trisphosphate; Ins(1,4,5)Pa, inositol 1,4,5-trisphosphate; HPLC, high performance liquid chromatography; HEPES, N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)Jtetraacetic acid.

the D-3 position on the inositol ring of PtdIns, PtdIns 4phosphate (PtdIns(4)P), and PtdIns 4,&bisphosphate to form the novel lipids PtdIns 3-phosphate (PtdIns(3)P), PtdIns 3,4bisphosphate (PtdIns(3,4)P,), and PtdIns 3,4,&trisphosphate (PtdIns(3,4,5)Ps), respectively (l-3). PtdIns 3-kinase was discovered because of its physical association with a variety of activated protein-tyrosine kinases (4-7). Mutational studies of oncogenes and growth factor receptors indicate that this enzyme plays a crucial role in transmitting growth signals (4, 7-10). Levels of PtdIns(3,4)Pz and PtdIns(3,4,5)P3 increase in response to stimulation of cells in culture with platelet derived growth factor (3), insulin (6), epidermal growth factor (ll), and colony-stimulating factor-l (12); these same lipids are elevated in fibroblasts transformed with polyoma middle T (13). These lipids are also produced in nonproliferating cells: formyl peptide stimulates production of PtdIns(3,4)P2 and a PtdInsP3 in neutrophils (14, 15), and thrombin and phorbol ester stimulate production of PtdIns(3,4)P2 in human platelets (16-18). Epidermal growth factor also causes a large increase in PtdIns(3,4)Pz in Leydig tumor cells where it acts as a differentiation factor rather than growth factor (11). The pathway for production of the D-3 phosphorylated phosphoinositides is still unclear. As discussed above, purified PtdIns 3-kinase can produce the three lipids in vitro by direct phosphorylation of the polyphosphoinositides in the canonical pathway. However, the in vivo ratio of PtdIns(3)P to PtdIns(3,4)P2 to PtdIns(3,4,5)P3 varies widely in response to different growth factors and hormones, suggesting multiple and independent steps for control of the pathway. Recently we suggested that the increase in PtdIns(3,4)P2 in platelets could be explained by a stimulation of a PtdIns(3)P 4-kinase rather than stimulation of PtdIns 3-kinase (18). With this study we show that a PtdIns(3)P 4-kinase exists in human platelets and that this enzyme is distinct from the PtdIns 4kinase that synthesizes PtdIns(4)P. EXPERIMENTAL

PROCEDURES

Materials-Phosphatidyl-[2-3H]inositol (5-20 Ci/mmol) was from Amersham Corp. [ 1-3H]Inbs~ol 1,3,4-trisphosphate (lo-30 Ci/mmol), Il-“Hlinositol 1.4.5-trisnhosohate (15-30 Ci/mmol). phosphatidvl-I2‘Hjinbsitol 4-monophosphate (Z-i0 Ci/mmol), ph&phatidyl-[i-?-I] inositol 4,5-bisphosphate (2-10 Ci/mmol), and [r-32P]ATP (30 Ci/ mmol) were from Du Pont-New England Nuclear. Ptdlns and PtdIns(4)P were from Avanti and Sigma, respectively. Partisphere strong anion exchange high performance liquid chromatography (HPLC) column and Silica G nlates were from Whatman. PICO FLOW k for on-line scintillation counting was from Packard Instruments. Sephacryl S-300 was from Pharmacia LKB Biotechnology Inc. Centricon 10 microconcentrator was from Amicon. Methylamine (40%. w/v) was from Eastman Kodak. Sodium orthovanadate was from-Aldrich. NaI04 was from J. T. Baker Chemical Co. Amberlite IR-120(H+j and other organic solvents were from Mallinckrodt. All other reagents were from Sigma or Fisher. Preparation collected with

of Cell Homogenate-Blood 0.1 volume of 3.8% trisodium

from human donors was citrate. Platelets were washed with washing buffer (138 rnM NaCl, 2.9 mM KCl, 5 mM Hepes, 5 mM glucose, 5 mM EDTA, pH 7.4) and finally resuspended in 1 ml of hypotonic buffer (20 mM Tris-HCl, 0.5 mM EGTA, 2 mM sodium

vanadate, 100 pM phenylmethylsulfonyl fluoride, 50 mM 2-mercaptoethanol, 50 pg/ml leupeptin pH 7.4). Human erythroleukemia cells were grown as described previously (19). Cells were washed once in phosphate-buffered saline and resuspended in the hypotonic buffer. Platelets and rapid freezing

22086

human erythroleukemia and thawing and then

cells were centrifuged

lysed by 5 cycles at 800 X g for

of 10

Phosphorylation

of PtdIns(3)P

min to remove unbroken cells. For further fractionation, the resulting homogenate was centrifuged at 160,000 X g for 10 min at 4 “C to separate the cytosol from the particulate fraction. The pellet was washed twice and resuspended in hypotonic buffer and was then used as particulate fraction. Protein concentration was determined according to Bradford (20). Preparation of 32P-Labeled PtdZnsf3)P and PtdZns(3,4JP,-[““PI PtdIns(3)P and [32P]PtdIns(3,4)P2 were made by incubating, respectively, PtdIns and PtdIns(4)P with [-y-32P]ATP and purified PtdIns 3-kinase as described previously (3). The organic extract containing the lipid was stored at -70 “C. “P-labeled PtdIns(3)P or ‘H-labeled PtdIns was dried under nitrogen and resuspended in liposome buffer (50 mM Tris-HCl, 0.1 mM EGTA, pH 7.4); it was then sonicated in a water bath sonicater for 60 min. Incubation and Extraction of Lipids-Fifty ~1 of platelet homogenate or hypotonic buffer was incubated with “P-labeled PtdIns(3)P liposomes (1.0 x lo5 cpm, 400 ng) or 3H-labeled PtdIns liposomes (3 X lo6 cpm, 50 ng) for 20 min at 4 “C in the presence or absence of Nonidet P-40. The reaction was started by adding ATP and MgCl, (final concentration 100 fiM and 10 mM, respectively) in a total volume of 100 ~1. After sucrose gradient centrifugation and gel filtration chromatography (see below), PtdIns(3)P 4-kinase was assayed in 50 ~1 of a reaction mixture containing 32P-labeled PtdIns(3)P (5 X lo4 cpm, 200 ng), 100 pM ATP, 10 mM MgC12, and 25 ~1 of each fraction. PtdIns 4-kinase was assayed in 50 ~1 of reaction mixture containing 300 kg/ml PtdIns liposome, [T-~‘P]ATP (50 fiM and 5 &i), 10 mM MgC&, and 25 ~1 of each fraction. To test the action of adenosine, fractions 5-8 after the sucrose gradient were collected, dialyzed, and concentrated with a Centricon 10 microconcentrator. PtdIns(3)P 4kinase and PtdIns 4-kinase assays were performed as described above except that 50 pM ATP was used. Reactions were started by adding ATP and MgCl,, incubated for 20 min at 30 “C, and lipids were extracted as described previously (16). For PtdIns 4-kinase assay, dried lipids were separated by thin layer chromatography as described previously (21). HLC Analysis of Phospholipids-Phospholipids were deacylated, deglycerated, and separated by HPLC as described previously (16). Sucrose Gradient Centrifugation-A discontinuous sucrose gradient was formed from 3.27 ml of 10% sucrose, 2.45 ml of 20% sucrose, and 3.27 ml of 30% sucrose in hypotonic buffer as described previously (22). Platelet cytosol (0.5 ml) was layered on top of the 10% sucrose, and the gradient was centrifuged at 41,000 rpm for 24 h at 4 “C in an TH-641 rotor (Sorvall). Fractions (950 ~1) were removed and numbered starting from the top of the tube. Each fraction was dialyzed against hypotonic buffer and assayed for PtdIns(3)P 4-kinase and PtdIns 4-kinase. Gel Filtration Chromatography-Platelet cytosol (500 ~1) was loaded onto a Sephacryl S-300 gel filtration column (1 x 40 cm) equibrated with elution buffer (200 mM NaC1,50 mM Tris-HCl, 2 mM sodium vanadate, 100 pM phenylmethylsulfonyl fluoride, 50 mM 2mercapthethanol, 50 rg/ml leupeptin, pH 7.4) at a flow rate of 0.25 ml/min. Fractions (700 ~1) were collected and assayed for PtdIns(3)P 4-kinase and PtdIns 4-kinase. Assay of Type ZZ and Type ZZZ PtdZns 4-Kinases-The type II PtdIns 4-kinase was purified from human red cells as described by Graziani et al.* The type III PtdIns 4-kinase was partially purified from bovine brain as previously described (23). Both enzymes were assayed in the presence of a mixture of approximately 500,000 dpm each of [3H]PtdIns (0.1 mM final concentration) and [3ZP]PtdIns(3)P (0.1 mM final concentration), 0.5 mM ATP, 10 mM Mg&, 50 mM Hepes (pH 7.5), 1 mM EGTA for 1 h at 37 “C!. The type II enzyme assay contained 0.1% Triton X-100 and 10 mM phosphatidylcholine. The type III enzyme assay contained 10 mM phosphatidylserine. The radiolabeled and unlabeled lipids were mixed, dried with N:! gas and sonicated for 10 min in the assay buffer using a bath sonicator before addition to the assay mixture. RESULTS

To test for platelet enzyme activity that phosphorylates PtdIns(3)P at the D-4 position of the inositol ring, we synthesized [32P]PtdIns(3)P. PtdIns was phosphorylated at the D-3 position by purified PtdIns 3-kinase (3) from rat liver and [T-~*P]ATP. [3-32P]PtdIns(3)P was deacylated and [3* A. Graziani, E. L. Ling, G. Endemann, C. Cantley, submitted for publication.

C. L. Carpenter,

and

L.

by a Specific 4-Kinase

32P]glycerophosphoinositol 3-monophosphate (GroPIns(3)P) was analyzed by HPLC (Fig. 1B). Purity of [3-32P]GroPIns(3)P was about 94% based on the HPLC analysis of the deacylated product of [3-32P]PtdIns(3)P ([3-32P]GroPIns(3)P). In seven separate experiments, we observed that the incubation of [32P]PtdIns(3)P with platelet homogenate led to the formation of a product that after deacylation is shown as peak 1 in Fig. 1C. This deacylated product had the same retention time as authentic glycerophosphoinositol 3,4-bisphosphate (GroPIns(3,4)Pz) (Fig. 1, A and C). In order to confirm that peak 1 was GroPIns(3,4)Pz, it was collected, deglycerated, and analyzed by HPLC. After deglyceration, the inositol phosphate produced coeluted with [3H]inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Fig. 2). It also -7 L

A.

zow

loo0

0

B. I

3oGQo

f ~2oooo 8 E E

loom

z 0 0

C. 30000

2ooocl

10000

0

0

lo ELUTION TIME @econd@

FIG. 1. HPLC analysis of deacylated 32P-labeled product from 32P-labeled PtdIns(3)P after incubation with platelet homogenate. A, HPLC elution profile of deacylated [“‘PI PdIns(3,4)P, prepared as described under “Experimental Procedures.” GPZ(4)P, glycerophosphoinositol I-monophosphate; GPZ(4,5)P2, glycerophosphoinositol4,5-bisphosphate; GPZ(3,4)Pz, glycerophosphoinositol 3,4-bisphosphate. B, HPLC elution profile of deacylated [32P]PtdIns(3)P prepared as described under “Experimental Procedures.” GPZ(3)P, glycerophosphoinositol3-monophosphate. C, HPLC elution profile of deacylated lipid after incubating [“‘PI PtdIns(3)P with platelet homogenate as described under “Experimental Procedures.” An extra peak was observed after incubation with platelet homogenate and it was designated as Peak I. Peak 1 coeluted with GroPIns(3,4)P,. Qualitatively similar results were obtained in seven different experiments.

22088

Phosphorylation

-1200

7

- 900

H

- 600

d 2 ii4

- 300

g

of PtdIns(3)P


lo

B.

”

443k158k66k

6-

z SOW 6000

676910 NUMBER

FRACTION

+++

-20

7cxlt

ELUTION TIME (seconds)

FIG. 2. HPLC analysis of the deacylated and deglycerated 32P-labeled product from PtdIns(3)P. Peak 1 from Fig. 1C was collected and deglycerated as described under “Experimental Procedures.” The deacylated and deglycerated sample was subjected to strong anion exchange HPLC, and its elution time was compared with the elution time of [3H]Ins-labeled Ins(1,3,4)P3. The peak of radioactivity from the sample (solid line) is shown along with that from [3H]Ins(1,3,4)P3 standard (broken line), with which it coelutes. This is a representative chromatogram from two different experiments.

coeluted with [32P]Ins(1,3,4)P3, which was made from [“‘PI PtdIns(3,4)Pz (data not shown). We observed that about 531% of PtdIns(3)P was converted to PtdIns(3,4)P2 in different experiments with platelet homogenate. This activity was present in both cytosolic and particulate fractions with a slightly higher percent activity in the cytosolic fraction (data not shown) and it is activated by 2-20 mM M$+ in a concentration dependent fashion (data not shown). The homogenate of human erythroleukemia cells and human red blood cells also resulted in conversion of [32P]PtdIns(3)P to PtdIns(3,4)Pz (data not shown). PtdIns(3)P 4-kinase activity was separated by sucrose gradient centrifugation and gel filtration chromatography (Fig. 3). In both cases the apparent molecular mass was about 150 kDa (Fig. 3, A and B). The peak and migration patterns of PtdIns 4-kinase were distinct but overlapping with those from PtdIns(3)P 4-kinase (Fig. 3, A and B). It was important to know if the activity that phosphorylates PtdIns(3)P to PtdIns(3,4)P2 was the same that converts PtdIns to PtdIns(4)P. Detergents like Nonidet P-40 have been reported to stimulate PtdIns 4-kinase, and this property has been used to differentiate PtdIns 3-kinase from PtdIns 4kinase (22, 24, 25). We determined the effect of different concentrations of Nonidet P-40 on the conversion of [““PI PtdIns(3)P to [32P]PtdIns(3,4)Pz and [3H]PtdIns to [3H] PtdIns(4)P by the same platelet homogenate. The result of a typical experiment is shown in Fig. 4. The conversion of PtdIns to PtdIns(4)P was stimulated by Nonidet P-40, whereas the conversion of PtdIns(3)P to PtdIns(3,4)Pz was inhibited by Nonidet P-40. Also, PtdIns(3)P 4-kinase was more resistant to adenosine inhibition than PtdIns 4-kinase (Fig. 5). Therefore, PtdIns(3)P 4-kinase seems to differ from PtdIns 4-kinase on the basis of the actions of Nonidet P-40 and adenosine. Further evidence that PtdIns(3)P 4-kinase and PtdIns 4kinase are distinct enzymes comes from studies of the type II and type III PtdIns 4-kinases. The purified red cell type II

0' 20

1

30 FRACTION

,\

40 NUMBER

IO 50

FIG. 3. Separation of PtdIns(3)P 4-kinase and PtdIns 4kinase activities of human platelet cytosol. A, sucrose gradient centrifugation. Samples were fractionated by rate zonal centrifugation on lo-30% sucrose gradient. Each fraction (950 ~1) was collected and PtdIns(3)P 4-kinase and PtdIns 4-kinase assays were performed as described under “Experimental Procedures.” The arrows indicate the migration positions of molecular mass weight markers: bovine serum albumin (66 kDa), y-globulin (158 kDa), apoferritin (443 kDa). PtdIns(3)P 4-kinase activity is shown as the radioactivity of PtdIns(3,4)Pz produced by each fraction. B, gel filtration chromatography. Sephacryl S-300 column was eluted at a flow rate of 0.25 ml/ min, and fractions (700 ~1) were collected. Proteins were started to elute from fraction 26 and PtdIns(3)P I-kinase and PtdIns 4-kinase assays were performed as described under “Experimental Procedures.” Protein markers and enzyme activity as in A.

250 r

I

n

0.1

FIG. PtdIns PtdIns different

I

0.5 NP 40 (%)

1 .o

4. Effect of Nonidet P-40 on PtdIns(3)P I-kinase and I-kinase. Liposomes of 32P-labeled PtdIns(3)P or ‘H-labeled were incubated with platelet homogenate in the presence of concentrations of Nonidet P-40 (NP40). All assays were

normalized

to

100%

in the absence of Nonidet P-40. In the absence

of Nonidet P-40, the radioactivities of 32P-labeled GroPIns(3,4)Pp and 3H-labeled GroPIns(4)P were about 12,000 cpm and 1,700 cpm, respectively. The radioactivities of [32P]PtdIns(3)P or [3H]PtdIns added to platelet homogenate were 0.7 x lo5 and 3 x lo5 cpm, respectively. This is representative of two different experiments.

Phosphorylation

of PtdIns(3)P

Pl(3)P 4-mase

0.5 1.0 2.0 ADENOSINE

5.0 (mM)

FIG. 5. Effect of adenosine on PtdIns(3)P 4-kinase and PtdIns 4-kinase. 32P-labeled PtdIns(3)P or PtdIns liposomes were incubated with the concentrated sucrose gradient fractions in the presence of different concentrations of adenosine. All assays were normalized to 100% in the absence of adenosine. In the absence of adenosine, the radioactivity of 32P-labeled GroPIns(3,4)Ps and 32Plabeled PtdIns(4)P were about 4,000 and 30,000 cpm, respectively. Results are representative of three different experiments.

PtdIns 4-kinase was given a mixture of [3H]PtdIns and [“‘PI PtdIns(3)P. After a 60 min incubation at 37 “C in the presence of 10 mM MgClz and 0.5 mM ATP, 12% of the [3H]PtdIns was converted to [3H]PtdIns(4)P, but HPLC analysis of the deacylated lipids revealed no detectable [32P]PtdIns(3,4)P2 (less than 0.5% of added [3H]PtdIns(3)P). Similarly, the bovine brain type III PtdIns 4-kinase converted 20% of the added [3H]PtdIns to [3H]PtdIns(4)P without detectable conversion of [32P]PtdIns(3)P to [32P]PtdIns(3,4)P2. DISCUSSION

It is known that 3-inositol phospholipids are formed by the action of PtdIns 3-kinase (1, 2). This enzyme physically associates with growth factor receptors and oncogenes that have protein-tyrosine kinase activity (4-9). A previous study showed that PtdIns 3-kinase immunoprecipitated from platelet-derived growth factor-treated smooth muscle cells phosphorylates PtdIns(4)P, resulting in formation of PtdIns(3,4)P2 in vitro (2). PtdIns(3,4)Pz is generally not found in unstimulated cells but is produced in uivo in response to growth factors such as platelet-derived growth factor (2), epidermal growth factor (ll), insulin (6), and colony-stimulating factor 1 (12). These 3-inositol phospholipids were not hydrolyzed by the PtdIns-specific phospholipase C that hydrolyzed PtdIns(4,5)P2 (26, 27), which raised the possibility that the lipids themselves, rather than derived inisotol polyphosphates, act as second messengers (26). Our present information shows that an additional pathway for the production of 3-inositol phospholipids might operate in certain cells. This activity, as shown herein, results in the conversion of PtdIns(3)P to PtdIns(3,4)P2. The PtdIns(3)P 4-kinase differs from PtdIns 4-kinase in its response to the detergent Nonidet P-40 and adenosine. Nonidet P-40 stimulated PtdIns 4-kinase but it dramatically inhibited PtdIns(3)P 4-kinase. On the other hand, PtdIns(3)P 4-kinase was more resistant to adenosine inhibition. We have not yet completely separated the PtdIns(3)P 4-kinase of all PtdIns 4-kinase activity, although the peak of both enzyme activities were at different positions on gel filtration and sucrose density gradient centrifugation. We have also found that purified red cell type II and partially purified brain type III PtdIns 4-kinases as well as the purified PtdIns 3-kinase had no significant PtdIns(3)P 4-kinase activity. In addition, other tissues that we investigated did not

22089


have detectable PtdIns(3)P 4-kinase activity in spite of plenty of PtdIns 4-kinase activity (data not shown). Therefore, PtdIns(3)P 4-kinase seems to be distinct from any PtdIns kinase which has been described previously. PtdIns 3-kinase and PtdIns(3)P 4-kinase pathways might be responding to different agonists and they might be activated by different mechanisms. PtdIns 3-kinase appears to be activated by a tyrosine kinase-dependent phosphorylation (47). The mechanism of control of PtdIns(3)P 4-kinase is unknown; however, the ability of phorbol esters and thrombin to stimulate PtdIns(3,4)P2 production in platelets raises the possibility that protein kinase C may regulate this activity (16-18). Our present data showing that human platelet hothe activity that phosphorylates mogenate contains PtdIns(3)P to PtdIns(3,4)P2 give further support to this possibility. In conclusion, we have demonstrated a novel pathway for production of PtdIns(3,4)Pz from PtdIns(3)P in human platelets, human erythroleukemia cells and human red cells. This pathway may operate in conjunction with the PtdIns 3-kinase pathway to produce 3-inositol phospholipids in stimulated cells. Most unstimulated cells contain a significant quantity of PtdIns(3)P; thus the appearance of PtdIns(3,4)P2 in response to a hormone or growth factor could result from activation of PtdIns(3)P 4-kinase without the operation of PtdIns 3-kinase. The relative contribution of these two enzymes to the production of PtdIns(3,4)Pz and PtdIns(3,4,5)P:, in response to activation of receptors with intrinsic proteintyrosine kinase activity versus those that interact with G proteins, requires further investigation. REFERENCES M., Dowries, C. P., Keeler, M., Keller, T., and Cantley, L. C. (1988) Nature 332.644-646 Auger, K. R., Serunian, L. A., S&off, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175 Carpenter, C., Duckworth, B. C., Auger, K. R., Cohen, B., Schaffbausen, B. S., and Cantley, L. C. (1990) J. Biol. &em. 265, 19704-19711 Talmage, D. A., Freund, R., Young, A. T., Dahl, J., Dawe, C. J., and Benjamin, T. L. (1989) Cell 59,55-65 Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L. C., and Roberts, T. M. (1987) Ceil 50,1021-1029 Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. (1. S. A. 87, 1411-1415 Fukui, Y., and Hanafusa, H. (1989) Mol. Cell. Biol. 9, 1651-1658 Whitman, M., Kaplan, D. R., Schaftbausen, B., Cantley, L. C., and Roberts, T. M. (1985) Nature 315,239-242 Coughlin, S. R., Escobedo, J. A., and Williams, L. T. (1989) Science 243,

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