Colony-stimulating factor 1 regulates CTP: phosphocholine ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 25, Issue of September 5,pp. 16261-16264,1991 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A .

Communication Colony-stimulating Factor1 Regulates CTP:Phosphocholine Cytidylyltransferase mRNA Levels* (Received for publication, May 22, 1991) Teresa G. Tessnert, Charles 0. Rock& Gabriel B. Kalmar7, RosemaryB. Cornell7, and Suzanne JackowskiSPII From the $Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, the $Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, and the Wepartment of ChernistrylBiochemistry, Simon Fraser University, Burnaby, British Columbia V5A 15’6, Canada

Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle wasinvestigated in the colony-stimulating factor 1 (CSF- 1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested thecells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholinecytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for2 h. The rise inCT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNAabundance as a componentin growth factorstimulated PtdCho synthesis. The rate-controlling (PtdCho)’biosynthesis

enzyme for phosphatidylcholine in mostmammaliansystemsis

* This research was supported by National Institutes of Health Grant GM45737from the National Institute of General Medical Sciences, National Research Service Award T32 CA09346 from the G . T), Cancer Center(CORE) National Cancer Institute(toT. Support Grant CA21765from the National Cancer Institute, the American Lebanese Syrian Associated Charities, and the Natural Science and Engineering Research Council of Canada (to R. B. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed Dept. of Biochemistry, St. Jude Children’s Research Hospital, 322 N. Lauderdale, P. 0. Box 318, Memphis, TN 38105. The abbreviations used are: PtdCho, phosphatidylcholine; CT, CTP:phosphocholine cytidylyltransferase; CSF-1, colony-stimulating factor 1; LCM, L cell-conditioned medium; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; FCS, fetal calf serum; DMEM, Dulbecco’smodifiedEagles’s medium; MOPS, 4-mOrphOlinepropanesulfonic acid kb, kilobase(s); bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; PBS, phosphatebuffered saline.

CTP:phosphocholinecytidylyltransferase (for review, see Refs. 1-3). Regulation of enzyme activity in vivo is correlated with translocation from an inactive, soluble form to an active, membrane-associated form in cell extracts. Membrane association is enhancedby diacylglycerols (1-3), fatty acids(1-3), and PtdCho-deficient membranes(4, 5). C T phosphorylation by CAMP-dependent protein kinase inhibits membrane binding (6,7), and sphingosine acts a competitive as inhibitor with respect to oleic acid (8).In cultured eukaryoticcells, PtdCho is a major membrane constituent and is the precursor to two other abundant membrane phospholipids, phosphatidylethanolamine (9) andsphingomyelin (10). In addition, PtdCho is the precursor to diacylglycerol and phosphatidic acid which may be important in the mitogenic signaling cascade activated by growth factors such as phorbol esters, a-thrombin, bombesin, interleukin-3 (for review, see Ref. ll), and most recently,platelet-derived growth factor (12). Mitogensalso stimulate PtdCho synthesis to compensate for the increased rates of PtdCho degradation(13-15). PtdCho degradation and synthesis are activated incells expressing the productof the ras (16-18) and src (17) oncogenes,suggestinga role for PtdCho cycling in transformation. The recent isolationof a rat CTcDNA clone (19) allowed us to examine whether CSF1regulates C T mRNA abundance. EXPERIMENTAL PROCEDURES

Materials-Sources of supplies were: Whittaker Bioproducts, DMEM; HyClone Laboratories, FCS; Du Pont-New England Nuclear, [methyl-’H]choline chloride (86.7 Ci/mmol), phospho[methyG “Clcholine (50 mCi/mmol), and [”2P]orthophosphate (carrier-free); Amersham Corp., [methyl-3H]choline chloride (75 Ci/mmol) and CDP-[methyl-14C]choline(46 mCi/mmol). Homogeneous human recombinant CSF-1 was kindly provided by Genetics Institute. LCM was the source of CSF-1 for the routine maintenance of BAC1.2F5 cells (20). Plasmids were obtained from either the American Type Culture Collection (murine c-fos and human glyceraldehyde-3-phosphate dehydrogenase) or Dr. John Cleveland, St. Jude Children’s Research Hospital(murine c-myc) or Dr. Laura Sanders, Johns Hopkins University (ornithine decarboxylase). The rat CT cDNA clone was described previously (19). Cell Culture-The BAC1.2F5 cell line is a CSF-1-dependent clone (21,22) thatexhibits many of the properties of macrophages including the production of lysozyme, collagenase, and esterase, and the elaboration of Fc receptors and Ia antigen. BAC1.2F5 cells were routinely maintained in DMEM supplemented with 15% FCS, 25% LCM, and 20 mM HEPES, pH 7.3 (23). Experiments were replicated at least three times (unless otherwise indicated). Phospholipid CompositionofBAC1.2F5 Cells-BAC1.2F5 cells were seeded a t a density of 2 X lo4 cells/60-mm dish and grown for 4 days in medium containing 3 pCi/ml [‘zP]orthophosphate. Adherent cells from duplicate dishes were washed with PBS, harvested by scraping in ice-cold methanol, and thecellular lipids were extracted (24).The total cellular lipids were separated on Silica Gel H plates developed with chloroform/methanol/acetic acid/water (5025:8:3, v/v). Metabolic Labeling Experiments-BAC1.2F5 cultures (60-mm dishes) were growth-arrested by the removal of CSF-1 for 24 h and then were incubated in choline-free DMEM supplemented with 3 p~ [‘Hlcholine (specific activity, 1pCi/mmol), 20 mM HEPES, and 15% dialyzed FCS either with or without 25% dialyzed LCM or 100units/ ml human CSF-1. Cells were harvested by scraping in ice-cold methanol, and the amount of [‘HH]choline incorporated into the phospholipid fraction was determined. The intracellular pool sizes of choline-labeled metabolites were determined by seeding BAC1.2F5 cells at 2 X 104/60-mm dish and growing the cultures for 4 days in choline-free DMEM supplemented with 15% dialyzed FCS, 25% dialyzed LCM, 20 mM HEPES, and 24

16261

CSF-1 Regulation of CT mRNA Levels

16262

[3H]choline chloride (specific activity, 0.125 Ci/mmol). Cells from with its precursor, phosphocholine, indicates that CT cataduplicate disheswere washed twice with PBS, harvested by scraping lyzes the slow step in PtdCho biosynthesis BAC1.2F5 in cells. in ice-cold methanol, and immediately frozen at -20 "C. The cells CSF-1 addition to quiescent BAC1.2F5 cells increased [3H] were extracted(24)and the choline-containingmetabolites were separated on Silica GelG plates developed withmethanol, 0.6% NaCl, choline incorporation into phospholipid, CT mRNA abundance, and CT specific activity in uitro. Approximately 1 h ammonium hydroxide (lO:lO:l, v/v). RNA Isolation and Amlysis-BAC1.2F5 cells were arrested in G1 after CSF-1 addition to G1-arrested BAC1.2F5 cultures, [3H] by culturing for24 h in the absence of CSF-1.At the indicated times choline incorporation into phospholipids accelerated signififollowingthe re-additionof CSF-1,the total cellular RNA was isolated cantly (Fig. 1). PtdCho was the onlylabeledphospholipid by the guanidine thiocyanate lysis procedure followed by CsCl gra- detected in the first2 h, with sphingomyelin reaching 10.1% dient centrifugationto pellet the RNA (25). RNA (10 pg of total RNA) was separated usinga 1.1%agarose, 2.2 of the total [3H]choline-labeled phospholipid at 6 h. These M formaldehydegelrunin20 mM MOPS, 5 mM sodium acetate, 1 data show that the activityof the PtdCho biosynthetic pathmM EDTA, 2.2 M formaldehyde (26). CT mRNA wascapillary-blotted way was stimulated by CSF-1. to nitrocellulose membranes and hybridizedat 42 "C with 5-7.5 X lo6 The levels of C T mRNA were compared with other mRNAs cpm of the 32P-labeled probe usingstandard Northern blot hybridi- known to beregulated by CSF-1 (30, 31). The CT probe zation protocols (26). Probes wereprepared by random primed label- detected a prominent band of 4.8 kb in RNA purified from ing of cDNA fragments(Boehringer Mannheim) isolated from agarose 4.0-kb gelsusing a Geneclean kit.Theprobeswerepreparedfrom the BAC1.2F5 cells (Fig. 2). This band corresponds to the following cDNAs: a 1.2-kb EcoRI fragment of the rat CT; a 3.7-kb C T mRNA species reported previously (19). The smaller CT SstI fragment from murine c-fos; a 0.9-kb XbaI/BglI fragment from 1.9-kb mRNA species (19) waspresent in low to undetectable murine c-myc; a 1.6-kb EcoRI/BarnHI fragment from murine orni- amounts in BAC1.2F5 cells. CSF-1 treatment of quiescent thine decarboxylase; and a 1.06-kbHind111 fragment from murine BAC1.2F5 cells resulted in a transient 4-fold elevation in the glyceraldehyde-3-phosphate dehydrogenase. RNA half-life was cal4.8-kb C T mRNA level (Fig. 2). The CT mRNA pool enlarged culated from the slope of a plot of the log[densiometric intensity] rapidly (within 15 min) andwas reminiscent of the induction versus time. CytidylyltramferaseActivity-Cytidylyltransferase activity was as- kinetics characteristic of immediate-early genes such as csayed essentially as described by Pelech and Vance (27). The cells myc and c-fos (32-35) (Fig. 2). However, the observed changes were washed once, scraped from the dish with ice-cold PBS without i n C T mRNA levels differed from c-myc and c-fos in that CaZ+/Mg2+, collected by centrifugation at 200 X g for 6 min at 4 "C, significant amounts of CT mRNA were detected in the aband resuspendedin200 pl of lysisbuffer(0.25 M sucrose, 1 mM sence of CSF-1 (time0) and CT mRNAincreased only 4-fold EDTA, 10 mM HEPES, pH 7.3,0.2 pg of leupeptin). The mixture was sonicated 4 times using 30-s pulses (cup sonication, Heat Sys- compared with c-myc and c-fos which were induced >50-fold tems-Ultrasonics, Inc., model W-225R cell disrupter, output at 10, (Fig. 2). The rate of change in the levels of C T mRNA was decarboxylase mRNA expression percent duty cycleat 80%).Cell lysates were assayed for CT activity distinctfromornithine in a reaction mixture containing 110 mM bis-Tris, pH 6.5, 2 mM CTP, which requires the prior synthesis of a protein transcriptional (50 mCi/mmol, 0.5 pCi), factor (36, 37). 20 mM MgC12,0.25 mM ph~spho['~C]choline 0.2 mM oleic acid, and 2.5 mM egg PtdCho in a final volume of 40 pl. The elevation of CT mRNApreceded both the increases in Reactions were for 10 min at 37 "C, stopped by freezing at -20 "C, CSF-1-dependent [3H]choline labeling and CTspecific activand the CDP["C]choline formation quantitated by thin-layer chromatography. Cells were separated into cytosol and particulate frac- ity measured in cell lysates. Four hours was selected for the tions by centrifugation of the lysates at 100,000 X g for 1 h at 4 "C C T activity determinations because at this time [3H]choline and the fractionsassayedin the presence of 0.25 mM oleic incorporation had reached its maximum rate (Fig. l), and CT acid:PtdCho (1:l).Protein concentrations were determined by the mRNA content was still elevated (Fig. 2). Comparison of the method of Bradford (28). The specific activity fromeachof three total activity of CT at 0 h (8.1 f 0.69 pmol/min/pg protein, dishes from two separate experimentswas determined at two to three different protein concentrations, summed, and averaged to give the n = 15) and 4 h (12.3 f 1.02 pmol/min/pg, n = 15) after CSFmean (tS.E.) specific activity expressedas pmol/min/pg of protein. 1 addition showed a 50% increase inC T specific activity. CT CT assays both in the presence and absence of lipid activator were was assayed in the presence of oleic acid/PtdCho liposomes linear with respect to protein. pM

RESULTS

CSF-1 is required for both proliferation and survival of bone marrow-derived macrophages (29) and the BAC1.2F5 murine macrophage cell line (21, 23). The transient removal of CSF-1 frommacrophages arrests thecells in early G1, and the readdition of CSF-1 triggers progression through G1 and the remainder of the cell cycle (23). Approximately 33% of the BAC1.2F5 cells were no longer adherent or viable after CSF-1 (LCM) was removed for 24 h. Flow cytofluorometric analysis showed that 98%of the viable, arrested cells were in the G1 phase (notshown). The phospholipid composition of BAC1.2F5 cells was determined by continuous labeling with[3ZP]orthophosphate (see "Experimental Procedures"). The cells contained 37.7% Hours PtdCho, 35.0% phosphatidylethanolamine, 21.4% sphingomyelin, and 5.8% phosphatidylserine/phosphatidylinositol. FIG. 1. [3H]Cholineincorporation into BAC1.2F5 phosphoThe major choline-derived precursor was pho~pho[~H]cholinelipids in the presence or absence of CSF-1. BAC1.2F5 cells were growth-arrestedby removal of CSF-1 for 24 h, then incubated for the (55.5% of the total), while the CDP-[3H]choline pool comindicated times either withor without CSF-1 in media containing 3 prised 7.5% and [3H]choline 10.5% of the soluble pathway PM [3H]choline. The cellswere extracted and the amount of [3H] a product of choline incorporated into the phospholipid fraction was determined intermediates. Glyceroph~spho[~H]choline, PtdCho breakdown, was 26.5% of the total radiolabel in the as described under "Experimental Procedures." Time 0 represents water-soluble pool. The low level of CDP-choline compared the end of the 24-h CSF-1 starvation period.


I

I

I

I

I

I

I

0

1

2

3

4

5

6

Hours Following CSF- 1

CT

1

16263

and the protein synthesis inhibitor cycloheximidedid not prevent the increase in C T mRNA, and CT mRNA levels were neither raised by treatment with cycloheximide alone nor superinduced by CSF-1 plus cycloheximide (Fig. 3). In the absenceof CSF-1, CT mRNAlevels rapidly declined (Fig. 3). The regulation of a typicalimmediateearly gene was determined by analyzing the same blot with a c-myc probe (Fig. 3). Consistent with published findings (32, 34), the cmyc mRNA wasinducedbycycloheximide alone and was superinduced by the combinationof CSF-1 andcycloheximide (Fig. 3). T o determine whether the CT mRNA pool was elevated in the presenceof CSF-1 due to stabilizationof the mRNA, non-synchronous cultures of BAC1.2F5 cells were either incubated with actinomycin D to block de nouo RNA synthesis or CSF-1 wasremoved for 1 h prior to the addition of actinomycin D. A plot of the relative intensity of the CT mRNA signal indicated that the half-life of CT mRNA was approximately 90 min in the absence of the growth factor (Fig. 4).Inthepresence of CSF-1, there was almostno decrease in thelevels of C T mRNA. The effect of CSF-1 on C T mRNA half-life was sufficient to account for the 4-fold increase in C T mRNA abundance (Fig. 2). DISCUSSION

Our data point to growth factor regulation of CT mRNA abundance as a component of the regulatoryprocess that

c-fos

c-myc

-ma--

ODC

“ 0

GAPDH

0-

c~

-

NS 0 .25 .5

CSF-14 CSF- 1

~ 1

2

4

6

Cycloheximide Cycloheximide

rrmom

~

L-

*

. c 01)

C-~YC 0

.25 .50

1

1.5

mmmm

~ -a

~ .25 .50

1

1.5

2 5 .50 1.5 1

Hours Hours FIG. 2. Accumulation of CT mRNA in CSF-1-treated FIG. 3. The effect of cycloheximide on CT mRNA levels. BAC1.2F5 cells. BAC1.2F5 cells were growth-arrested by removal BAC1.2F5 cells were first growth-arrested by removal of CSF-1 for of CSF-1 for 24 h, and at time 0, CSF-1 was added to the cultures. 24 h. At time 0, the cells were treated with either CSF-1 (LCM)(left At the indicated times, total RNA was isolated and fractionated andpanel), 10 pg/mlcycloheximide (middlepanel), or CSF-1(LCM) a nitrocellulose blot was sequentially probedfor a selection of mRNAs containing 10 pg/mlcycloheximide (right panel). Total RNA was as described under “Experimental Procedures” (bottom panel). Ex- isolated at the indicated times, and the amount of C T mRNA (top posure times varied with the probe. NS, non-synchronized; ODC, panel) or c-myc mRNA (bottom panel)was determined by Northern ornithine decarboxylase; GAPDH, glyceraldehyde-3-phosphate de- analysis as described under “Experimental Procedures.” CT autorahydrogenase. The -fold increasein C T mRNA was estimated by diograph wasexposed for48 h, and c-myc autoradiograph was exposed densitometry of the CT autoradiograph(top panel). 10 h.

in order to maximize the CT activity and to overcome the potential modulatoryeffects of phosphorylation (7). However, in the absence of oleic acid/PtdCho in thein vitro assay, we also observed similar increases inC T activity from4.7 & 0.32 pmoles/min/pg ( n= 18)at zero time to6.1 & 0.38 pmol/min/ pg ( n= 17) a t 4 h. Consistent withpreviously published work with tissue homogenates (38, 39), the maximum specific activity in the absence of lipid activator was approximately half of the rate observed in the presence of oleic acid/PtdCho. Neither the percentage of C T activity associated with the membrane fraction nor the specific activity of particulate C T increased between zero time (52%; 22.8 & 2 pmol/min/pg ( n = 9)) and 4 h (53%; 26.4 f 2 pmol/min/pg ( n = 9)). The rapid elevation of CT mRNA suggested that protein synthesis was not required. This property is characteristic of the immediate earlyclass of geneswhose transcriptional activationdonotrequireproteinsynthesisandtheyare superinduced in the presenceof protein synthesis inhibitors (40). Treating BAC1.2F5 cells with a combination of CSF-1

CSF-I Present

CSF- I Absent

“

0 .25 .50 I 1.5

0 .25 .50 I

1.5

Hours Following Actinomycin D Addition FIG.4. Influence of CSF-1onCT mRNA turnover. BAC1.2F5 cultures were treated with 5 &ml actinomycin D in the continuous presence of CSF-1 (left panel)or CSF-1 was removed for 1 h prior to actinomycin D (5 pg/ml) addition and the incubation continued in the absence of CSF-1 (right panel). At the indicated times, total RNAwas isolated and the amountof CT mRNA present was determined by Northern analysis as described under “Experimental Procedures.” The half-lifefor CT mRNAunderthe two conditions was determined by densitometricmeasurement of the decrease in the intensityof the bands.

16264


governs PtdCho synthesis in dividing cells. The elevation in CT mRNA content was accompanied by the stimulation of PtdCho synthesis and an increase in CT activity following the addition of CSF-1 to quiescent cells. These findings imply that the regulation of CT mRNA content may also be associated with the cellular responses to other growth factors and ligands that activate PtdCho synthesis and breakdown. CT mRNA expression differed from ornithine decarboxylase, a gene product required for entry into S phase, in that it appeared within 15 min rather than 3 h after the addition of CSF-1, and prior protein synthesis was not required. On the other hand, the existence of significant levels of CT mRNA in CSF-1-starved cells (Fig. 2), the 4-fold increase in CT mRNA content in stimulated cells (Fig. 2), and the lack of superinduction by cycloheximide (Fig. 3) indicated that CT does not belong to thefamily of immediate early genes typified by c-fos and c-myc(32-35). Rather,the elevation of CT mRNA was attributed to the CSF-1-dependent inhibition of CT mRNA degradation (Fig. 4). Thus, CT mRNA falls into a class of transcripts whose half-lives are altered as part of the pleiotropic cellular response to proliferation factors (for review, see Ref. 41). Previous studies illustrate the importance of post-translational events in the regulation of CT activity, but the possibility remains that modulation of total enzyme content also contributes to increased PtdCho synthesis in some systems. PtdCho formation is enhanced by phorbol ester treatment and is associated with increased CT activity (2). Phosphorylation by CAMP-dependentprotein kinase inhibits CTactivity in uitro (7)and may account for the decreased incorporation of choline into PtdCho by hepatocytes treated with CAMP analogs (42). However, the role of phosphorylation in regulating enzyme activity remains uncertain since CT phosphorylation by either protein kinase C(43) or protein kinase A (7) has not been demonstrated in vivo. Lipid-dependent association of CT with membranes is a major factor in controlling CT activity (for reviews, see Refs. 1-3).Stimulation of PtdCho synthesis uia CT activation results from treatment of cells with phospholipase C to generate diacylglycerols (1, 2), the addition of oleic acid to the medium (1, 2), or the depletion of cellular PtdCho by choline starvation (4,5). However, there are results that cannot be explained by activation/translocation alone. For example, Weinhold et al. (44) report that treatment of HepG2 cellswith oleic acid results inan increase in total CT enzyme activity and immunodetected protein. Other investigators also report that increased PtdCho synthesis correlates with an elevation in membrane-associated CT activity in the absence of a corresponding decrease in cytosolic enzyme content resulting in an increase in total CT activity (2).

14) andinterleukin-2 (45) is attributed to stimulation of choline kinase activity by these mitogens. However, our observation of a temporal correlation between increases in CT mRNA content, enhanced rates of PtdCho synthesis, and higher CT specific activity in vitro raises the possibility that genetic regulation of CT gene expression in growing cells may play a more important role in the overall regulation of the pathway than previously thought. Acknowledgments-We thank Michele Luche for her expert technical assistance and Dr. John Cleveland for his advice. REFERENCES 1. PeAtyh, S. L., and Vance, D. E. (1984) Biochim. Biophys. Acta 7 7 9 , 217ZLDl

2. Tijburg, L. B. M.,Geelen, M. J. H., and van Golde, L. M.G. (1989)Biochim. Blophys. Acta 1 0 0 4 , 1-19 3. Vance, D. E. (1989) in Phosphatidylcholine Metabolism (Vance, D. E., ed) pp. 33-45, CRC Press, Inc., Boca Raton, FL 4. Yao, Z., Jamil, H., and Vance, D. E. (1990) J. Biol. Chem. 265,4326-4331 5. Jamil, H., Yao, Z., and Vance, D..E. (1990) J. Biol. Chem. 265,4332-4339 6. Vance, D.E. (1989) in Phosphatldylchline Metabolism (Vance, D. E., ed) pp. 225-239, CRC Press, Inc., Boca Raton, F L 7. Sanghera, J. S. and Vance, D. E. (1989) J. Bcol. Chem. 264,1215-1223 8. Sohal, P. S., a i d Cornell, R. B. (1990) J. Biol. Chem. 265,11746-11750 9. Voelker, D. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,2669-2673 10. Voelker, D. R., and Kennedy, E. P. (1982).Biochemistry 21,2753-2759 11. Billah, M. M., and Anthes, J. C. (1990) Bwchem. J. 269,281-291 12. Larrodera, P., Cornet, M. E., Diaz-Meco, M. T., Lopez-Barahona, M., DiazLaviada, I., Guddal, P. H., Johansen, T., and Moscat, J. (1990) Cell 6 1 , 1113-1120 13. Warden, C. H., and Friedkin, M. (1984) Biochim. Biophys. Acta 792,270280 14. Warden, C. H., and Friedkin, M. (1985) J. Biol. Chem. 260,6006-6011 15. Pelech, S. L., and Vance, D. E. (1989) Trends Biochern. Sci. 14,28-30 16. Lopez-Barahona, M., Kaplan, P. L., Cornet, M. E., Diaz-Meco, M. T., Larrodera, P., Diaz-Laviada, I., Municio, A. M., and Moscat, J. (1990) J. Biol. Chem. 265,9022-9026 17. Diaz-Lavianda, I., Larrodera, P., Diaz-Meco, M. T., Cornet, M. E., Guddal, P. H., Johansen, T., and Moscat, J. (1990) EMBO J. 9,3907-3912 18. Teegarden, D., Taparowsky, E. J., and Kent, C. (1990) J . Biol. Chem. 2 6 5 , finA9-fiOA7 ""

19. Kalmar. G. B.. Kav. R. J.. Lachance. A,. Aebersold. R.. and Cornell. R. B. (1990) Proc.'Nail.'Acad: Sci. U. S. A. 87,6029-6033 20. Stanley, E. R., and Heard, P. M. (1977) J. BWl. Chem. 252,4305-4312 21. MoTgan:-c., Pollard, J. W., and Stanley, E. R. (1987) J. CelZ. Physiol. 130, 4ZU-4L1

22. Schwarzhaum, S., Halpern, R., and Diamond, B. (1984) J. Immunol. 1 3 2 , 1158-1162 23. Jackowski, S., Rettenmier, C. W., and Rock, C. 0. (1990) J . Biol. Chem. 265,6611-6616 24. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37,911-917 25. Chirgwin, J. M., Przbyla, A. E., MacDonald, R. J., and Ruttle, W. (1979) Bwchemistry 18,5294-5299 26. Manlatis, T., Frltsch, E. F., and.Sambrook, J. (1982) Molecular Cloning; A Laboratory Manual, Cold Sprlng Harbor Laboratory Press, Cold Sprlng Harbor, NY 27. Pelech, S. L., and Vance, D. E. (1982) J. Biol. Chem. 257,14198-14202 28. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 29. Tushinski, R. J., and Stanley, E. R. (1985) J. Cell. Physiol. 122,221-228 30. Muller. R., Curran.. T... Muller, D.. and Guilbert, L. (1985) Nature 3 1 4 , 546-5548 31. Hamilton, J. A., Veis, N., Bordun, A,"., Vairo, G., Gonda, T. J., and Phillips, W. A. (1989) J. Cell. Physiol. 1 4 1 , 618-626 32. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983) Cell 35,603610 33. Blanchard, J-M., Piechaczyk, M., Dani, C., Chambord, J-C., Franchi, A., Pouyssegur, J., and Jeanteur, P. (1985) Nature 317,443-445 p., and Nathans, D. (1987) Proc. Natl. Acad. SCL. U. S . A. 84,118235. Sa;ib;n, E., Luebbers, R., and Kufe, D. (1988) Mol. Cell. Biol. 8,340-346 Katz, A,, and Kahana, C. (1987) Mol. Cell. Biol. 7 , 2641-2643 Kahana, C., and Nathans, D. (1985) Proc. NatL Acad. Sci. U. S. A. 8 1 , 3645-3649 Sleight, R., and Kent, C. (1983) J . BWL Chem. 258 831-835 Roonev. S. A.. Smart, D. A,. Weinhold. P. A., andkeldman, D. A. (1990) Bioc-him. Bwphys. Acta 1044,385-389 Greenberg, M. E., and Ziff, E. B. (1984) Nature 311,433-438 Raghow, R. (1987) Trends Biochem. Sci. 12,358-360 Pelech. S. L.. Pritchard. P. H.. and Vance. D. E. (1981) J. Biol. Chem. 2 5 6 , 8283-8286 Watkins, J. D., and Kent, C. (1990) J. Biol. Chem. 256,2190-2197 Weinhold, P. A., Charles, L., Rounsifer, M. E., and Feldman, D. A. (1991) J. Biol. Chem. 266,6093-6100 Kaplan, O., and Cohen, J. S . (1991) J. Biol. Chem. 266,3688-3694

36. The contributionof increased CT mRNA production to the 37. observed stimulation of PtdCho synthesis remains to be de38. termined. There is not an exact correlation between the 6- 39. fold increase in PtdCho synthesis, the transient4-fold induc- 40. tion of CT mRNA, and the50% increase in total cellular CT 41. activity. Furthermore, there is no detectable change in mem- 42. brane-associated enzyme activity. Other factors must contrib- 43. ute to the stimulation of phospholipid synthesis by growth 44. factors. Increased PtdCho synthesis inresponse to serum (13, 45.