Growth Factor-stimulated Protein Phosphorylation in Go/G1-arrested Fibroblasts TWO DISTINCT CLASSES OF GROWTH FACTORS WITH POTENTIATING EFFECTS*

(Received for publication, July 1, 1982)

Jean-Claude Chambard$, Arlette Franchi$, Alphonse Le Cam@, and Jacques Pouyssegurt From the $Centre de Biochimie, Centre National de la Recherche Scientifique and 8Faculte de Medecine, Uniuersite de Nice, Parc Valrose, 06034 Nice Cedex, France

Protein phosphorylation of Go/G1-arrested Chinese hamster lung fibroblasts (CC139 line) has been analyzed following stimulation by fetal calf serum (FCS) or by a variety of growth factors. FCS stimulated the phospho- rylation of three major polypeptides separated on so- dium dodecyl sulfate-polyacrylamide gel electrophore- sis: a nuclear protein with a M, of 62,000 daltons, the ribosomal protein S6, and a cytosoluble peptide of 27,- 000 daltons. These phosphorylations occurred rapidly after serum stimulation (1 min for the 27,000-dalton peptide, 5 min for S6 and the 62,000-dalton proteins) and were maximal after 30 min. In nonstimulated cells the 27,000-dalton phosphopeptide exists in two forms with isoelectric points of 5.7 and 6.0; serum increased the amount of the most acidic form.

At low concentrations, the “commitment” growth factors, a-thrombin, eye-derived growth factor (EDGF), platelet-derived growth factor (PDGF), stimulated phosphorylation of the 27,000-dalton peptide. At higher concentrations, these factors alone reinitiated DNA synthesis and, like FCS, stimulated phosphorylation of the three major peptides. In contrast, and suggesting a different mechanism of action, “progression” factors such as insulin (1-10 pg/ml) and multiplication-stimu- lating activity (MSA) are unable to stimulate phospho- rylation of the 27,000-dalton peptide. However, insulin or MSA which are known to potentiate the mitogenic action of a-thrombin, PDGF, EDGF, ... were also found to potentiate phosphorylation of the ribosomal protein S6. These results support the existence of two classes of growth factors and suggest that protein phosphoryl- ation is an early event involved in the control of the cellular Go + GI transition.

Normal cells in culture enter a reversible quiescent state when medium is depleted of serum growth factors (1). Gen- erally, a variety of growth factors are capable of activating the re-entry of these Go/G,-arrested cells into the proliferative state; however, the action of these regulatory peptides at a molecular level remains largely unknown.

Ever increasing evidence indicates that protein phospho-

* This study was supported by grants from the Centre National de la Recherche Scientifique (LP 7300 and ATP 136), the Institut Na- tional de la Sante et de la Recherche Medicale (CRL 80-2016), the Delegation Generale a la Recherche Scientifique et, Technique (81- L0733) and the Fondation pour la Recherche Medicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

rylation is a rapid and general response of cells to growth factors ( 2 4 , yet it is not known whether each growth factor stimulates the phosphorylation of a specific or common set of proteins when added to Go-arrested cells. Another point of interest is the potentiating action observed between growth factors (7,8), in particular between two distinct classes (9-ll), thus suggesting dual modes of action. We asked whether a biochemical basis for their potentiating effect could be defined at the level of protein phosphorylation.

To answer these questions we have used the Chinese ham- ster lung fibroblast line, CC139. This cell line, capable of reversible growth arrest in Gn/GI (12), reinitiates DNA syn- thesis in response to one class of growth factors (11): PDGF,’ FGF, EDGF, and thrombin. A second class of factors that includes insulin and MSA markedly potentiates the mitogenic effect of the first class of growth factors (10, 11, 13). In a preliminary report on the mitogenic response of these cells, we have shown that the potentiation by both classes of factors (e.g. thrombin and insulin) is a very early event since it is observed on the activation of the amiloride-sensitive Na+/H+ exchange system (14). In this report we analyze the early changes in protein phosphorylation of Go/G1-arrested hamster cells stimulated either by FCS or thrombin, PDGF, FGF, EDGF, insulin, or the combination of insulin with other growth factors.

MATERIALS AND METHODS

Chemicals-SDS and Ampholines were purchased from Serva. Phosphoserine and phosphothreonine were from Sigma and phospho- tyrosine was prepared as described (15). Staphylococcus aureus V8 protease was obtained from Miles Laboratories; DNase, grade 1, and RNase were purchased from Boehringer. (.’”P)Orthophosphate (car- rier-free) and [methyl-”Hlthymidine were obtained from the Radi- ochemical Centre Amersham and from the “Commissariat a 1’Energie Atomique” (France), respectively.

Growth Factors-Highly purified human thrombin (>W% electro- phorectically pure, minimum 3000 NIH units/mg) and crystalline bovine insulin (23.6 I.U./mg) were purchased from Sigma; bovine thrombin (1316-1156 units/mg) was from Miles Laboratories. PDGF was prepared in this laboratory according to the method described in Refs. 16 and 17 up to the carboxymethyl Sephadex chromatography step. Bovine pituitary FGF and partially purified EDGF were gifts from Dr. Y. Courtois (Inserm U-118, Paris), and MSA was from Dr. M. Rechler (National Institutes of Health, Bethesda, MD).

’ The abbreviations used are: PDGF, platelet-derived growth fac- tor; FGF, fibroblast growth factor; EDGF, eye-derived growth factor; MSA, multiplication-stimulating activity; FCS, fetal calf serum; DNase, deoxyribonuclease; RNase, ribonuclease; PBS, phosphate- buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; P,, inorganic orthophosphate; DMEM, Dulbecco’s modified eagle’s medium; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth- anesulfonic acid; bistris, 2-[bis(2-hydroxyethyI)amino]-2-(hydroxy- methyl)-1,3-propanediol.

1706

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Cell Culture-Chinese hamster lung fibroblast CCI:19 cells (Amer- ican Type Culture Collection) were generously provided by I'rof. G .

Buttin (Institut Pasteur, Paris) antl vascular smooth muscle cells from bovine aorta (18) were from I h . Y. Courtois: Cultures were maintained in 1)ulhecco's modified Eagle's nlcdiunl (DMEM-Gihco H21) supplemented with 5 5 FCS (Seromed). penicillin (IO() units/ ml), and streptomycin (50 pg/ml). Cells were propagated in tissue culture dishes (Nunc) in a humidified atmosphere of 5? COX at :17 "C.

DNA Sy thes i s A.ssny-Cells were grown in I(i-nlm diameter 24- well plates (Nunc) i n 0.5 nd of IlMEM supplemented with 5'; FCS; confluent cultures were washed twice with serum-free medium and incubated for 30 h without serum i n a mixture ( 1 : I ) of DMEM and Ham's medium (Gihco F12) (19). Quiescent monolayers were then incubated for 24 h in the same medium containing 3 ILM [ 'Hlthvmidine ( 1 pCi/nll) and varying concentrations of either serum or growth factors. Following incorporation. cells were washed with ice-cold I'RS and fixed with I O G trichloroacetic acid containing I O n m thymidine (30 min, 0 "C). Acid-precipitahle fractions were soluhilized in NaOH (0.1 N), and radioactivity was counted by liquid scintillation spectrom- etry. The same protocol o f growth arrest ( 3 0 h serum starvation) and reinitiation (24 h in the presence of growth factors antl :I I'M [.'HI thymidine at 5 pCi/ml) was used t o determine the percentage of labeled nuclei. Cells (in :15-mm diameter dishes) were fixed with trichloroacetic acid and processed for autoradiography as described

I'hchosphotylation I.:.~~~c~,bnc~nts-In all experiments. cells were ar- rested in G,&, by 30 h of serum starvation (12). These conditions reversibly arrest more than 99.55 o f the cell population as judged hy autoradiography. Serum-deprived cells ( i n X - n ~ n ) dialneter tissue culture dishes) were washed twice with Hrpes-huffercd saline without phosphate at 37 "C antl incuhated for 45 nlin at 37 "C i n phosphate- free IlMEM. 20 mM Hepes, pH 7.4. containing 10()-200pCi/n~l of '21',. Different concentrations of serum or growth factors wcre at lde t l and incubation continued for 1.5 min. To stop the reaction. thc mcdium was carefully aspirated and cells were quickly washed four times with I'BS at 0 "C. Cells were immediately Ivsetl i n lOO-20() pI of SIX solution (2'J SIX, 5 n m l',, pH 6.8) and extracts were boiled for 2 min. Before polyacrylamide gel electrophoresis (SIIS-I'AGE), ex- tracts were boiled for an additional 3 min in the presence o f 0 . 1 M dithiothreitol, 1 0 7 glycerol. and O.O()lc; bromphenol blue.

For the two-dimensional I'AGE. cells were lysed in IOO-2OO 111 of lysis buffer (9 M urea, 2% Nonidet 1'-40. 5'; /~-nlc.rc;ll)toethanol. and 27 Ampholines) (21).

Subcellular Frrrctionation-""I'-laheled cells i n Io()-mm diameter dishes were washed three times with I'RS and one time with 2 ml of distilled water at 0 "C. The cells were then incuhated for I O min in 0.5 ml of ice-cold hypotonic solution (10 nlM NaF. I O n m NaH,,I'OI. 1 mM EIlTA, pH 7.4) collected with a ruhher policeman and homog- enized in a Ilounce ( R ) tissue homogenizer on ice. NaF concentration was immediately brought up to 1 0 0 nlM in the cell homogenate. A nuclear and a non-nuclear fraction was ohtained by centrifugation of the lysate for 1 min at 10,OOo X g in an Eppendorf microfuge. The nuclei pellet was resuspended in the same buffer solution ( 1 0 0 n m NaF, containing 0.1'4 (w/v) Nonidet 1'40). centrifuged again for 1 min at 10,OOO X g, and the pellet was soluhilized in 2'; SIlS-5 mM NaHrl'O,, and then boiled for 2 min as described ahove.

The non-nuclear fraction was centrifuged for 15 min at 2O,O()O X g (4 "C). The resulting pellet was solubilized in 2'r SIlS and the supernatant was centrifuged at 150,ooO X g for I h at 4 "C. The high speed pellet, representing the membrane fraction, was solubilized in SDS as above. The soluble proteins contained in the supernatant fraction were precipitated by 20"; trichloroacetic acid. washed twice with ethanol at - 1 0 "C and ethanol-ether ( 1 : l ) at -10 "C, dried, and solubilized in SIX. All SIX-solubilized extracts which contained 1-2 mg of protein per ml were tlwn boiled in the presence of 0.1 M dithiothreitol, I O ? glycerol, and 0.001'r bromphenol blue.

Analysis of Kihosomal Phosl,ho)~,.oteins-Ribosomal proteins from "'l'-labeled cells were extracted and analvzed by two-dimen- sional I'AGE as described (22), except that 150 pg/ml of RNase I and 150 pg/ml of pancreatic DNase were added for 15 min at 0 "C to the cell homogenate before acid extraction (667 acetic acid, 1 0 0 mM MgClr). Ribosomes from rat liver were added as protein carrier.

Polyacylamide Gel I~lectrophore.si.s-One-dimensional SIlS- I'AGE was carried out in slabs according to the method of Laemmli (23) modified by Studier (24). The resolving gel was a linear gradient of 7.5-15"i. acrylamide. The same amount of protein was applied to each lane of a given gel. This amount varied from 40 to 60 pg from an experiment to another.

(2a).

Two-dimensional polyacrylamide gel elect rophoresis was carried out as described (25).

Gels were stained with Coomassie blue. dried under vacuum. and exposed to Kodak X-Onmt S or Fuji IIXL x-ray film with an Ilfortl Hi-plus intensifying screen.

Protein used as molecular weight markers were: phosphorylase b (94.000), bovine serum albumin (68.(XM)). ovalbumin (43.000). rarhonic anhydrase (:IO.(XX)). soybean trypsin inhibitor (20,1(x)). and Iysosymc. (14.800).

Phosphocrmino Acid Ancr/ysis--I'hosphoproteins were recovered from polyacrylamide gels according t o Ref. X . They were then acid hydrolyzed ( 6 N HCI at 110 "C for 2 h) and the phosphonrnino acids

200t

94 - 68

-

42 -

0.5% 6% 22% 707' 3% %LABELLED NUCLEI

FIG. 1. Serum-stimulated protein phosphorylation of CC139 cells. After 30 h of serum starvation, quiescent cells were incubated in DMEM without phosphate, containing IOU pCi/ml o f ,"l', during 45 nlin at 37 "C. Fetal calf serum or I O pglml of insulin (Z.V.'3 were then added and the incubation continued for 15 min. The dishes were quickly rinsed with ice-cold I'BS antl the reaction stopped by addition of 2 5 SDS. Each sample was reduced with 0.1 M dithiothreitol (see under "Materials and Methods") and subjected to electrophoresis in a polyacrylamide discontinuous gel gradient (i.5-It55), one lane con- taining the molecular weight standards. After Coomassie blue staining the gel was dried and exposed for autoradiography. The per cent o f labeled nuclei, determined as described under "Materials and Meth- ods." was measured in a parallel experiment after serunl stimulation of the cells during 24 h in the presence of ["Hlthvmidine.

68-

FIG. 2. T ime course of serum-stimulated protein phospho- rylation. Serum-deprived cells were incubated with 100 pCi/nll of ""I', during 45 min, and 10'r FCS was added ( + I for the time indicated. The reaction was stopped by addition of 2$ S I X and the lysate separated by electrophoresis on a polyacrylamide gel gradient i.5- 15% in SIlS and autoradiographed.

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1708 Growth Factor-stimulated Protein Phosphorylation

separated hy electrophoresis at pH 3.5 on thin layer cellulose plates as tiescrihed (27).

Peptidr Mupping hv L i m i t t d Pr(~/ro/~,~is-One-dimensional pep- tide mapping was carrird out according t o (28) using protease V R from S. rrlrrrus.

"m!l -1 I"

2 h

b62K

*33K

c s c s

FIG. 3 . Serum-st imulated phosphoproteins us u function of the "1'-labeling period. Serum-deprived cells i n :?5-mm diameter culture dishes were incubated with 75 yCi/ml of.ILI', at 37 "C for the times indicated. For each time of laheltng. cells were stimulated with 10'; FCS during the last 15 minutes. (', control. 5'. stimulated.

94

68

42

30

20-

c. MW 40-3

cb 0

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62-

1'" 1"

- MWJO-~

FIG. 4. Subcellular localization of serum-stimulated phos- phoproteins. Quiescent cells in 1oO-mm diameter culture dishes were incubated with ZOO pCi/ml o f ."I>, ftrr 'L h at 37 "C; one-half was stimulated with 10'; FCS during the last 15 min. The cells were fractionated as described under "Materials and Methods" and thP diffcrenr fractions analyzed IJ.V t4rc.t IO~J~IU,CSIS UII S1)S-IwIywryl- amide gel gradient (i.5-15fi) and autoradiographed. C. control, S. stimulatrd. W H . whole homogenate: NL:C. nuclei; MA. 15 min 20,OOO x fi pellet: M H , ($0 min IOO.O(X) x g pellet: SOI,. 60 min 100.000 x g supernatant.

B C FIG. 5. Two-dimensional electrophoresis of "'P-labeled extracts enr iched in r ibosomal proteins . Hi-

hosomal proteins were extracted from ,"P-laheled CC139 cells with acetic acid-MgCI,! as described under "Materials and Methods." Electrophoresis in the first dimension was performed at pH 5.5 with 8 M urea, 105; P-mercaptoeth- anol, 0.01 M histris-acetic acid in a 4% polyacrylamide gel. Voltage was constant and maintained at 150 V during 6 h. The second dimension in S I X was performed in a slah gel with 1 I ? acrylamide. A, autoradiography of a gel with extracts from quiescent cells; B , autoradiography of a gel with extracts from a cell stimulated for 30 min with 10'7 FCS. Arrows indicate positions of 27,000- and 33.000-dalton proteins. C, Coomassie hlue-stained gel with the position of S6 according to Ref. 22.

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Growth Factor-stimulated Protein Phosphorylation 1709

B l 30

C

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FIG. 6. Electrophoretic analysis of the 27,000-dalton phosphoprotein. A, resolution of the 27,oOO-dalton phosphoprotein by two-dimensional polyacrylamide gel electrophoresis. "1'-labeled soluble fraction from quiescent cells (left) or cells stimulated for 15 min with 10'; FCS (r ig/t / ) were subjected to a two-dimensional electrophoresis according to Ref. 21. The first dimension is an isoelectrofo~using (IF,'F) in a pH gradient formed by Ampholines in the pH range 5-7. The pH gradient was measured with a microelectrode on 5-mm slices of the gel following 30 min of incubation in 200 pl of 8 M urea. The second dimension is an SIH-PAGE with 11$ of acrylamide. R. limited proteolysis of the two forms of the 27,.O(X)-dalton phosphoprotein. Serum-deprived cells in 6O-mm diameter culture dishes were intensively laheled during I h with 1 mCi of ,"I', and stimulated during the last 15 min with 10'; FCS. The soluble fraction was separated as described for the subcellular fractionation (supernatant, 150,000 X g for 1 h) and subjected to a two-dimensional polyacrylanlide gel electrophoresis t o separate the 27,000-dalton protein into two spots as in Fig. 6. Each spot was excised and subjected to a limited proteolysis by the protease V8 from S. aurew in an SI%-polyacrylamide gel as described by Cleveland et nl. (28). The gel was 15'; acrylamide; each sample received 200 ng of protease. I,rrnc, 1. autoradiogram showing the phosphopeptides produced from the pI 5.7 form of the 27,000-dalton protein. Lune 2. autoradiogram showing the phosphopeptides produced from the pI 6.0 form of the 25,000-dalton protein. C, phosphoamino acid analysis. The "'1'-labeled 27,000-dalton protein was extracted from a polvacrylanlide gel as tlescrihetl by Hunter and Sefton (27) anti subjected to acid hydrolysis in 6 N HCI at 110 "C for 2 h. Amino acids were produced and standards were separated on a cellulose plate by high voltage electrophoresis at pH 3.5 in acetic acid-pyridine-H?O (505945). The autoradiogram of the plate is shown with the position of standards revealed by ninhydrin staining (hatched bars). 1. phosphoamino acids from the 27,000-dalton phosllhoprotein. 2, phos1)hoamino acids standards: phosphoserine ( p S E H ) . phosphothreonine ( p T H H ) , and phosphot;rosine ( p T Y H ) . .

msmm Serum-stimulated Protein Phosphoplation in CC1.79

Cells-Addition of FCS to serum-deprived CC139 cells leads to DNA replication after a lag period of 8-10 h (12). Exposure of ~'v'F"labeled GtI-arrested cells to FCS for 15 min resulted in marked alterations in the degree of phosphorylation of several polypeptides with M,- = 62,000, 33,000, and 27,000 (Fig. 1). A 4- to 6-fold stimulation is generally observed for these 3 phosphopeptides; however, this is an underestimation due to the incomplete resolution of these proteins in one dimension (see below). In addition to these main changes, consistently observed throughout all experiments, some other minor al- tered phosphorylations were detected (increased phosphoryl- ation of proteins with M , = i0,000, 45,000, and 22,000 and decreased phosphorylation of proteins with M , = 57,000 and 25,000). Raising the serum concentration from 0 to 109; re- sulted in an increase in the percentage of cells traversing the S phase (0.5 to io% of labeled nuclei) which paralleled the increase in ".P incorporation into the three major peptides of M , = 27,000, 33,000, and 62,000 daltons (Fig. 1).

Time Course of Serum-stimulated Protein Phosphoryls- tion-Gll-arrested cells prelabeled with :"Pi for 45 min were stimulated from 1 to 30 min with FCS. Fig. 2 shows that serum-stimulated protein phosphorylation is a rapid event; stimulation of the 27,000-dalton polypeptide was observed after 1 min of serum addition. Phosphorylation of the 33,000- and 62,000-dalton peptides was delayed and appeared simul-

taneously after 5 min. In all cases maximum effect of serum was seen after 30 min. Note that the 33,000-dalton protein migrates slower as its phosphorylation increases.

The effect of serum was independent of the specific activity of the ATP pool since a similar pattern of phosphoproteins was obtained in cells prelabeled for 2, 10, or 20 h with "'P, (Fig. 3 ) .

Subcellular Localization and Partial Identification of the Serum-stimulated Phosphoproteins-Control and serum- stimulated "'P-labeled cells were homogenized and separated into soluble, nuclear, and membrane fractions. Fig. 4 shows that the 27,000-dalton polypeptide is mainly cytosoluble. It is not found in nuclei, and the small amount present in the membrane fraction could represent a contamination. The 62,000-dalton polypeptide is highly enriched in the nuclear fraction. As far as the 33,000-dalton polypeptide is concerned, because of its localization in the microsomal and membrane fraction and its peculiar migration behavior in SDS-polyacryl- amide, it was assumed to he the ribosomal protein S6 (29,30). Ribosomal proteins were acid-extracted (22) and separated on two-dimensional PAGE (Fig. 5); the 33,000-dalton protein is very basic and was found to comigrate with the ribosomal protein S6 of rat polysomes introduced as carrier and visual- ized by Coomassie blue staining (Fig. 5 0 .

As shown by two-dimensional gel analysis, the 27,000-dalton polypeptide exists as two variants (Fig. 6). Assuming an isoe- lectric point of 5.4 for actin (31), the two 27,000-dalton phos-

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1710 Growth Factor-stimulated Protein Phosphorylation

" " o r . . . " . .

4

4 3 3 k

4 2 7 k

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433K

4. 427K

462K

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427 K

52% 40% 81- l% Labeled Nuclei

FIG. 7. Effect of growth factors (thrombin, PDGF, EDGF) on the phosphorylation of CC139 proteins. The procedure is the same as in Fig. 1. Cells are arrested in G / G , , labeled with ,"l', for 45 min, and stimulated with various concentrations o f growth factors during 15 min. A , H . and C show the autoradiogram o f extracts separated on SIN-PACE. A . dose effect of a-human thrombin ( I unit/nll = I O nM) on protein phosphorylation with the corresponding percentage of labeled nuclei. R, effect o f platelet-derived growth factor extract. I'IXF X I O (50 pg/ ml) corresponds to the maximally mitogenic concentration (40-255'r laheled nuclei). C. effect o f eye-derived growth factor extract. EDGF X 1 0 (10 pg/ml) is a concentration which gives 15-20$ of labeled nuclei.

phorylated peptides have isoelectric points of 5.7 and 6.0, respectively. Both forms are phosphorylated in serum-de- prived cells, but serum enhances only the more acidic form (Fig. 6A) . The fact that the two phosphopeptides correspond to the same protein with a different phosphate content is indicated by the similarity between the peptide maps shown on Fig. 6R. Analysis of the phosphoamino acids present in the 27,000-dalton phosphoprotein stimulated by serum revealed equal amounts of phosphoserine and phosphothreonine (Fig. 6C).

Effects of Various Growth Factors on Protein Phosphoryl- ation in CCl.?9 Cells-PDGF, FGF, EDGF, and thrombin are all capable o f reinitiating DNA synthesis in G,,/G,-arrested CC139 cells. A second class of polypeptides, including insulin a t high concentrations (1-10 pg/ml) or MSA (0.1-1 pg/ml), although not mitogenic alone, markedly potentiates the growth-promoting effect of PDGF, FGF, EDGF, and throm- bin. In addition, these growth factors can replace serum and ensure continuous exponential growth of CC139 cells as effi- ciently as 10% FCS (13). A minimal and very potent combi-

nation of purified growth factors. allowing exponential growth over six days, was found to be human thrombin (0.1 unit/ml) and insulin, although thrombin alone at higher concentrations (1 unit/ml) is also able to trigger many rounds of cell division without cell damage.' It was, therefore, of interest to analyze the thrombin-stimulated protein phosphorylation of GI,-ar- rested cells. Fig. 7A shows that thrombin alone stimulates the phosphorylation of a n identical set of proteins as found in serum-stimulated cells: ribosomal protein S6, the nuclear 62,000-dalton and the 27,000-dalton peptides. As with serum, increasing thrombin concentrations lead to an increase in the percentage of cells traversing the S phase. This increase in labeled nuclei parallels the increase of ."P incorporated into the growth factor-stimulated peptides.

The other partially purified growth factors, including I'DGF and EDGF, stimulate the phosphorylation of 27,000-, 62,000-, and 33,000-dalton proteins at concentrations which

E. Van Obberghen-Schilling and J . I'ouyssPgur. manuscript in .~ ~" - ~ .~ ~ ~

preparation.

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Growth Factor-stimulated Protein Phosphorylation 1711

initiate DNA synthesis o f a substantial number o f cells (15 to 35% of laheled nuclei) (Fig. 7, R and C). Lower concentrations of these factors which reinitiate less than lO',Y of cells stimulate only the phosphorylation of the 27.000-dalton peptide. This property is shared by PDGF, FGF, EDGF, and thrombin. Epidermal growth factor alone, a poor mitogen for CC139 cells (7-10'4) of labeled nuclei in the range of 10 to 100 ng/ml), also stimulated the phosphorylation of the 27,000-dalton peptide and very slightly that of S6 and of the 62,000-dalton protein (data not shown).

In contrast, the second class of growth factors, insulin and MSA, which alone have almost no effect on the reinitiation of

4 3 3 K

4 2 7 K

FIG. 8. Effect of insulin on the phosphorylation of CC139 proteins. '"I'-labeled G,)-arrested cells (as in Fig. 1 ) were stimulated for 15 min with two concentrations of insulin: A. insulin, 1 pg/nd; H, insulin, 1 0 pg/ml; C, no addition: U. IO'; FCS.

DNA synthesis ( 4 3 4 of labeled nuclei) stimulate only the phosphorylation of the ribosomal protein S6 and the 62,000- dalton peptide (Fig. 8). T h e 27,000-dalton peptide is absolutely insensitive to their action. Rut the most important aspect of the action of these factors is that they potentiate the growth- promoting activity of low concentrations of PDGF. EDGF, and thrombin. Interestingly, we also observed that insulin markedly potentiates the thrombin-stimulated protein phos- phorylation (Fig. YA). This potentiating effect, which is most pronounced for S6 and the 62,000-dalton protein, is observed with thrombin concentrations ranging from IO-.' to 10-' units/ ml and is not restricted to thrombin, since it was also observed with EDGF (Fig. 9R). Furthermore, it is not a peculiarity of these Chinese hamster lung fibroblasts. As illustrated in Fig. YC, insulin markedly potentiated specific phosphorylation stimulated by either EDGF or thrombin in G,,/GI-arrested vascular smooth muscle cells.

All the effects reported here with 1 to 10 pg/ml of insulin, potentiation of DNA synthesis and potentiation of ribosomal protein S6 and 62,000-dalton peptide, were also observed with the insulin-like growth factor, MSA at a concentration of 0.1 to 1 pg/ml (data not shown).

Finally, we found that addition of A-RrcAMP (0.5 m i d to quiescent CC139 cells has no apparent effect on the pattern of phosphorylation. Therefore, the growth factor-induced phos- phorylation of the 27,000-, 62,000-dalton and S6 proteins is mediated through a CAMP-independent phosphorylating mechanism.

DISCUSSION

Growth of CC139 cells in litre, like that of secondary cultures of fibroblasts, is highly serum-dependent. This serum requirement reflects the dependence of normal cells for serum growth factors. Indeed, a chemically defined medium contain- ing a set of purified growth factors can support growth of

C

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FIG. 9. Potentiating effect of insulin on thrombin or EDGF-stimulated protein phosphorylation. '"1'- labeled G,,-arrested cells (as in Fig. I ) were stimulated for 15 min with growth factors added separately or together. A, synergistic effect of thrombin and insulin on CC139 cells. The arrows indicate when insulin ( INS , 10 pg/ml) and n-human thrombin (TM are added together. B, synergistic effect of EIIGF and insulin on CC139 cells. EDGF was used at 1 pg/ml and insulin at 1 0 pg/ml. C, synergistic effect of either thrombin or EIIGF with insulin on vascular smooth muscle cells. Confluent vascular smooth muscle cells were arrested and labeled with " P , as described for CCll9 cells (legend to Fig. 1). (+) or (-) indicate whether growth factors were present or absent during the 15 min of stimulation. I N S (insulin at I O pg/ml); EIIGF ( I pg/ml); TH (thrombin at 10 ' units/ml).

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CC139 cells as efficiently as FCS (13). When the culture medium is depleted of growth factors, growth stops reversibly in Go/GI (1, 12). The addition of exogenous growth factors or the acquisition by the cell of a mutation leading to growth factor “relaxation” are two means of preventing growth arrest. We have shown that a mutation leading to growth factor independence is a prerequisite for tumoral growth of CC139 cells (20, 32).

In the present communication we report that phosphoryl- ation of at least 3 peptides, ribosomal protein S6, the nuclear 62,000-dalton, and the cytoplasmic 27,000-dalton peptides, constitutes an early biochemical response of arrested cells to serum. We believe that these three major changes play an important role in the molecular events controlling the revers- ible Go/GI arrest point for the following reasons: 1) the in- creased phosphorylation of the three peptides is observed not only in response to serum but also in response to each growth factor that is capable of reinitiating DNA synthesis (thrombin, FGF, PDGF, and EDGF). 2) The extent of phosphorylation of the three peptides parallels the percentage of labeled nuclei. 3) CC139-derived clones capable of “autonomous” growth after in vitro or in vivo selection (20, 32) display a “constitutive” phosphorylation level of these three peptides.”

To approach the molecular events controlling cell growth it appears essential to understand the functional nature of these growth factor-stimulated proteins. Although the ability of CAMP, insulin, growth factors, or sperm to stimulate S6 phosphorylation has now been largely documented in various cell systems (29, 30,33-35), the functional role of phosphoryl- ated S6 is unknown. What is clear from this study, which confwms the report of Thomas et al. (30) with quiescent 3T3 cells, is that S6 phosphorylation and protein synthesis can be activated by insulin independently of reinitiation of DNA synthesis. Moreover, Go-arrested CC139 cells incubated in the presence of insulin for 15 to 24 h remain in the Go/G1-arrested state, in spite of S6 phosphorylation and protein synthesis activation. Indeed, addition of thrombin (1 unit/ml) to these cells stimulates DNA synthesis only after a characteristic lag of 8-10 h (12).” Similarly, insulin fails to modify the lag following stimulation by growth factors of quiescent Balb or Swiss 3T3 cells (36, 37). These observations indicate that the phosphorylation of S6 is not sufficient to allow Go-arrested cells to progress into G1. So far, no role can be attributed to the 62,000- and 27,000-dalton phosphoproteins. However, the 27,000-dalton phosphoprotein possesses interesting features. Its stimulation is very rapid and it is elicited by all the growth factors known to reinitiate DNA synthesis of CC139 cells (thrombin, FGF, EDGF, ... ). In addition, the 27,000-dalton protein is also a target of thrombin-stimulated phosphoryla- tion in human platelets. Indeed, we found that the similar phosphopeptide (Mr = 27,000) stimulated in platelets and in fibroblasts generates identical phosphopeptides when it is subjected to limited proteoly~is.~ This finding suggests that thrombin elicits a common postreceptor biochemical event in these two cell types (38).

On the contrary, we reported that the second class of growth factors, i.e. insulin and MSA, has no stimulatory effect on the 27,000-dalton phosphopeptide. This result supports the con- cept of two classes of growth factors delivering distinct trans- membrane signals (10, 11). Along this line, the increased phosphorylation of S6 by both classes of factors as well as their synergistic action vn this phosphorylation may indicate the existence of two distinct pathways leading to ribosomal

J.-C. Chambard, A. Franchi and J. Pouyssegur, manuscript in

J. C. Chambard, and J . Pouyssegur, in preparation. preparation.

protein S6 phosphorylation. The inhibition of a phosphatase activity has been implicated for the phosphorylation of S6 in sea urchin eggs activated by sperm (33). Such a mechanism was also recently postulated for insulin action in hepatocytes (39). In addition, we have reported that the regulation of intracellular pH via the amiloride-sensitive Na’/H’ exchange system constitutes a limiting step for growth factor-stimulated S6 phosphorylation (14, 40). Future work should clarify the nature of these different intracellular messengers leading to alterations in protein phosphorylation and to cell prolifera- tion.

Acknowledgments-We thank E. Van Obberghen-Schilling and G. Oillaux for their help in preparing the manuscript.

Note Added in Proof-Recent reports by Thomas et al. (41) and Nilsen-Hamilton et al. (42) with 3T3 cells have shown a synergistic stimulation of S6 ribosomal protein phosphorylation with either PGF?,, and EGF or EGF and insulin.

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J C Chambard, A Franchi, A Le Cam and J PouysségurTwo distinct classes of growth factors with potentiating effects.

Growth factor-stimulated protein phosphorylation in G0/G1-arrested fibroblasts.

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