THE JOURNAL OF BIOLOGICAL CHEMISTRY (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 17, Issue of June 15, pp. 10941-10945,1991 Printed in lJ S. A.

Epidermal Growth Factor Stimulates Phosphorylation of RAF- 1 Independently of Receptor Autophosphorylation and Internalization*

(Received for publication, December 7, 1990)

Manuela BaccariniS, Gordon N. Gills, and E. Richard Stanleyll From the Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, New York, New York 10461 and the 6Dewartment of Medicine. Diuision of Endocrinology and Metabolism, School of Medicine, University of California, La Jolla, Cajifokia 92093 ’

Phosphorylation of the RAF- 1 protooncogene prod- uct and activation of its associated serinelthreonine kinase are common features of the response of cells to peptide growth factors. We have used wild-type and mutant epidermal growth factor (EGF) receptors to investigate mechanisms of RAF- 1 phosphorylation. I n uiuo EGF treatment rapidly stimulated phosphoryla- tion of RAF-1 exclusively on serine residues. Stimu- lation of RAF-1 phosphorylation occurred at 37 “C but not at 4 “C and persisted after dissociation of EGF from its receptor. EGF-induced RAF- 1 serine phosphoryla- tion required the intrinsic tyrosine kinase activity of the EGF receptor but was independent of EGF receptor self-phosphorylation and of ligand-induced receptor internalization. Down-regulation of protein kinase C did not affect the EGF-induced increase in RAF-1 phosphorylation. These data suggest that the activated tyrosine kinase activity of the EGF receptor enhances serine phosphorylation of RAF-1 via an intermediary molecule(s).

Cytosolic protein serine/threonine kinases have been im- plicated as intermediates in a phosphorylation cascade in- duced by the binding of growth factors to tyrosine kinase receptors. Microtubule-associatedprotein-2 kinase, S6 kinase, casein kinase 11, and protein kinase C are among the best documented examples (1-7). Recently, EGF’ treatment of murine fibroblasts has been shown to stimulate seven distinct cytosolic serine/threonine kinase activities by a mechanism involving phosphoryl transfer (8, 9). Observations in several growth factor systems, e.g. EGF (IO), PDGF (ll), CSF-1 (la), and insulin (13, 14), indicate that ligand-induced stimulation of tyrosine kinase receptors leads to phosphorylation of the RAF-1 protooncogene product and activation of its associated serine/threonine kinase. Dephosphorylation of immunoiso-

* This work was supported by National Institutes of Health Grants CA 26504 and CA 32551 (to E. R. S.)and DDK AM13149 (to G. N. G.), by Albert Einstein Core Cancer Grant P30-CA 1330, and by the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of fellowships from the European Molecular Biology Organization (EMBO) and the Cancer Research Institute, New York.

7 To whom correspondence should he addressed: Dept. of Devel- opmental Biology and Cancer, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.

’ The abbreviations used are: EGF, epidermal growth factor; PDGF, platelet-derived gr0wt.h factor; CSF-1, colony-stimulating fac- tor; wt, wild type; PMA, phorbol 12-myristate 13-acetate; HEPES, 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium do- decyl sulfate; PAGE, polyacrylamide gel electrophoresis.

lated RAF-1 from PDGF- and CSF-1-stimulated cells leads to inactivation of the RAF-1-associated kinase (11, 12).

RAF-1 is present in most tissues and cell lines (15). Trun- cation of the amino-terminal domain of RAF-1 increases its kinase activity in uitro, and microinjection of the amino- terminally truncated RAF-1 protein into serum-starved 3T3 fibroblasts induces proliferation of these cells, indicating a role for this kinase in mediating cell proliferation (15-17).

The mechanism by which the phosphorylation and activa- tion of the RAF-1 protein are regulated in vivo is unknown. RAF-1 is reported to be one of the proteins phosphorylated on tyrosine residues in response to PDGF in uiuo (11). The PDGF receptor is able to phosphorylate RAF-1 on tyrosine residues in an in uitro kinase assay, and PDGF receptor- mediated RAF-1 tyrosine phosphorylation and activation have been shown in a baculovirus system on co-expression of the PDGF receptor and RAF-1 (18). However, only serine phosphorylation of RAF-1 has been reproducibly detected after in vivo activation of CSF-1 and insulin receptor tyrosine kinases (12-14). Moreover, we were unable to detect PDGF- stimulated tyrosine phosphorylation of authentic RAF-1 in 3T3 cells overexpressing RAF-1 (la). It is thus uncertain whether RAF-1 is activated as a direct consequence of tyrosine phosphorylation by ligand-activated growth factor receptors or whether RAF-1 is activated subsequent to an intermediate event in growth factor action.

To study further the mechanism of RAF-1 regulation we have investigated the ability of ligand-activated wild-type and mutant EGF receptors to phosphorylate RAF-1 in uiuo. We show that EGF rapidly stimulates in vivo phosphorylation of RAF-1 exclusively on serine residues. Although tyrosine ki- nase is activated at both 37 and 4 “C, RAF-1 phosphorylation occurs only at the higher temperature. EGF receptor tyrosine kinase activity, but neither receptor autophosphorylat,ion nor induced receptor internalization, is essential for RAF-1 phos- phorylation. Down-regulation of protein kinase C does not abrogate EGF-induced phosphorylation of RAF-1. These re- sults indicate that signal transduction processes initiated by the EGF receptor tyrosine kinase activate RAF-1 via cellular intermediates.

MATERIALS AND METHODS

Construction and Expression of Mutant EGF Receptors-Construc- tion of the mutant EGF receptors has been described previously (19). Carboxyl-terminal truncations were made either in the context of the wild-type (wt) Lys”’ human EGF receptor cDNA (20) or in the context of the Met’2’ EGF receptor cDNA as described by Chen et al. (21). DNA transfection in mouse B82L cells, lacking endogenous EGF receptor mRNA or protein (22), was performed using calcium phos- phate precipitation (23), and clonal cell lines were selected and amplified using methotrexate. Mutant EGF receptors expressed in the cell lines are identified by their carboxyl-terminal amino acid

10941

10942 EGF-stimulated RAF-1 Phosphorylation residue based on the cDNA sequence (24) and on the presence of lysine or methionine at position 721, i.c. Lys:"/carhoxyl 973, Met:", Met:"/carboxyl 10'22.

Cell Culture and Labeling-Permanent clonal transfectants ex- pressing t he various human EGF receptor constructions were cultured in Dulhecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1 V M methotrexate. Confluent cultures were grown in medium containing 0.055 fetal calf serum and 1 FM methotrexate for 16 h to up-regulate receptors. In selected experiments protein kinase C down-modulation was performed by treating cultures with 5 pM phorhol 12"myristate 13-acetate (PMA, Sigma) in dimethyl sulfoxide (14 mM final concentration) or with dimethyl sulfoxide alone for 20 11 (4 ,25) . Cells were subsequently incuhat,ed for 90 min in phosphate- free Dulhecco's modified Eagle's medium supplemented with 25 mM HEPES buffer (GIBCO) prior to labeling by incubation for 3 h in phosphate-free medium containing 1 mCi/ml carrier-free ('"Plortho- phosphate (9,000 Ci/mmol, Amersham Corp. and Du Pont-New Eng- land Nuclear). Laheling media contained 10% of the usual concentra- tion of NaHCO:% (2.6 mM; isotonicity of the media was maintained by adjusting the NaCl concentration to 23.6 mM) and 25 mM HEPES buffer (26). For growth factor stimulation, cells were equilibrated at the desired temperature and incubated for different periods of time with 100 nM purified mouse submaxillary gland EGF (Collaborative Research, Bedford, MA). Incubations were terminated by aspirating the medium and washing five times with ice-cold phosphate-buffered saline. When indicated, cells were suhjected to a mild acid wash (three times in a buffer containing 50 mM glycine, 100 mM NaCI, pH 4.0, at 37 "C) to remove cell surface-bound EGF prior to incuhation in the presence or absence of EGF.

Cell Solubilization and Immunoprecipitation-Cells were solubi- lized by the addition of 1 ml of ice-cold lysis buffer (20 mM Tris-HCI, 137 mM NaCI, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM I)henylmethylsulfonyI fluoride, and 1 mM sodium vanadate, pH 8.0). Insoluble material was removed hy centrifugation (15,000 rpm, 30 min, 4 "C) and the protein concentrations of the clarified lysates determined (27). Volumes of lysates containing equal amounts of protein were cleared by incuhation with bovine serum alhumin- Sepharose heads for 60 min at 4 "C prior to incubation for 3 h a t 4 "C with different antibodies. Rabbit polyclonal antisera raised against a carboxyl-terminal peptide of RAF-1 (SP63, CTLTTSPRLPVF, 28) or against a :30-kDa carboxyl-terminal fragment of v-raf produced in Ikhv-ichia coli (29) were used to immunoprecipitate RAF-1 mole- cules. EGF receptors were immunoprecipitated with the mouse anti- human EGF receptor monoclonal antibody 528 (167 ng/ml lysate), specific for the ligand binding domain (30). Immune complexes were incuhated for 45 min a t 4 "C with protein A-Sepharose beads and washed once with lysis buffer, twice with 0.5 M LiCI, 0.1 M Tris-HC1, pH 7.4, and once with 10 mM Tris-HCI, pH 7.4. Protein was eluted from the heads by boiling in Laemmli sample buffer containing 2- mercaptoethanol (31), and the eluates were analyzed by 7.5% SDS- PAGE. The gels were stained, dried, and subjected to autoradiography a t -70 "C usingpreflashed Kodak XAR-5 film. Alternatively, proteins were electrophoretically transferred onto Immobilon polyvinylidene difluoride paper (Millipore Co., Bedford, MA), the blots probed with anti-RAF-1 (anti-SP63, 1:1000; anti-30-kDa, 1500) antisera, and the antigen-antibody complexes visualized (12).

Phosphoamino Acid Anal~~sis-'"P-Laheled proteins were separated by SDS-PAGE, and individual hands were extracted from the gels and suhjected to tryptic digestion. The digests were hydrolyzed in 6 N HCI at 110 "C for 1 h, and the hydrolysates were separated by two- dimensional electrophoresis on cellulose-coated TLC plates (32). Phosphoamino acids were visualized by autoradiography a t -70 "C on preflashed Kodak XAR-5 film.

Detection of Phosphot~~rosine-containing Proteins-Cells were treated without or with 100 nM EGF for the indicated times. Medium was removed, and the reaction was terminated by the addition of hot Laemmli sample buffer. Cell extracts were heated a t 100 "C for 2 min in a capped tube, and proteins were separated on 7.5% SDS-PAGE and transferred to Immobilon. Membranes were blocked with 2.5% bovine serum albumin, 0.1% NaN:,, 10 mM Tris-HCI, pH 7.5, 0.9% NaCI, 0.05% Tween 20 for 2 h a t room temperature followed by incubation for 2 h with ",.'I-labeled mouse monoclonal anti-phospho- tyrosine antihody PY-20 (20) and a rabbit antipeptide antihody, N13, directed against the amino terminus of the human EGF receptor. After washing with blocking buffer, dried membranes were suhjected to autoradiography to quantitate phosphotyrosine. EGF receptors were detected using a goat anti-rahbit/alkaline phosphatase system.

RESULTS

EGF-stimulated Phosphorylation of RAF-1 Is Temperature Dependent-The addition of EGF to B82L cells expressing holo or carboxyl 973 human EGF receptors rapidly increased tyrosine phosphorylation of cellular substrates (Fig. 1). The major tyrosine-phosphorylated substrate in cells expressing holo EGF receptors is the receptor itself which contains five identified phosphorylation sites in its carboxyl terminus (33- 35). Self-phosphorylation is also evident by the decreased migration of receptors as defined by immunostaining. As reported previously (35), truncation to residue 973 removes all EGF receptor self-phosphorylation sites but enhances phosphorylation of cellular substrates. Activation of in uiuo tyrosine kinase activity of both holo and carboxyl 973 EGF receptors also occurs a t 4 "C with kinetics that are similar to those observed a t 37 "C (Fig. 1).

To determine whether RAF-1 was phosphorylated in re- sponse to activation of the EGF receptor, RAF-1 was immu- noisolated from control and EGF-treated cells. Fig. 2 shows that RAF-1 is a phosphoprotein. At 37 "C phosphorylation of RAF-1 was rapidly increased when cells were treated with EGF, and this enhanced phosphorylation persisted for at least 45 min. Enhanced phosphorylation was seen both by :'2P incorporated into RAF-1 (panel A ) and decreased electropho- retic mobility characteristic of phosphorylated proteins (panel B ) . In contrast to tyrosine phosphorylation of several cellular proteins, neither the phosphorylation of RAF-1 (panel C) nor its electrophoretic mobility (panel D ) was altered by EGF treatment a t 4 "C. EGF-induced RAF-1 phosphorylation was thus strongly temperature dependent.

RAF-1 Phosphorylation Requires the Intrinsic Tyrosine Ki- nase Actiuity of the EGF Receptor but Is Independent of Receptor Internalization-In cells stimulated with EGF at 4 "C, the receptor and an array of other cytosolic proteins become phosphorylated on tyrosine, but clustering and inter-

I 37% , 4%

A. ' B. + c

9 , "+- P

E [r CL

C.

U LL E

D.

c

0 1 15 30 60 0 2 30 60120

TIME (min)

FIG. 1. EGF-stimulated tyrosine phosphorylation in vivo at 37 "C and 4 "C. B82L cells expressing holo or carboxyl 973 EGF receptors were treated without or with 100 nM EGF for the indicated times at 37 "C or 4 "C. Total cell proteins were extracted with hot Laemmli sample buffer, and EGF receptors and tyrosine phosphoryl- ated proteins were measured by Western blotting. Immunostained EGF receptors are shown above each autoradiogram, which were developed with "..'I-laheled monoclonal PY-20 anti-phosphotyrosine antibody.

EGF-stimulated RAF-1 Phosphorylation 10943

A

E G F - + + + + - + + + TIME 0 1.5' 5' 15' 45' 0 30' lh 2h 04 -

44 -

EG F

+ s * T

Y Y C

D

44 - - a- TEMPERATURE 37°C 4'C

FIG. 2. Changes in RAF-I phosphorylation and electropho- retic mobility after EGF stimulation of B82L cells expressing the holo EGF receptor. [:"P]Orthophosphate-labeled cells were stimulated with 100 nM EGF at 37 "C (A and H ) or a t 4 "C (C and 11) for different times prior to solubilization in 1% Triton X-100 containing Na:tVO,. RAF-1 molecules were immunoprecipitated with the anti-RAF-1 (SP63) rabbit polyclonal antiserum and the immune precipitates subjected to 7.5% SDS-PAGE, anti-RAF-1 immunoblot- ting and autoradiography. A and C, autoradiograms of the blots (exposure times: A, 1 h; C, 2.5 h). B and D, RAF-1 immunoblots. Density of the RAF-1 bands in A and C relative to the density of the zero time bands (= 1.00) were: A, 1.5 min, 2.07; 5 min, 2.49; 15 min, 2.88; 45 min, 2.72; C, 30 min, 0.97; 1 h, 1.14; 2 h, 1.02.

EGF - + - + - + - + Mr

( X 10-3) 110 -

84 -

66 - "UU

EGFR Wt K721/ c ' 973 M721 M72l/c' 1022

FIG. 3. Effect of EGF treatment on RAF-1 phosphorylation in B82L cell lines expressing mutant EGF receptors. ['"PI Orthophosphate-labeled cells were incubated in the absence (-) or presence (+) of 100 nM EGF for 15 min a t 37 "C prior to solubilization in 1% Triton X-100 containing Na,lVO,,. RAF-1 molecules were immunoprecipitated with the anti-RAF-1 (SP63) rabbit polyclonal antiserum and the immune precipitates subjected to 7.5% SDS- PAGE, anti-RAF-1 immunoblotting, and autoradiography. Exposure time was 2 h.

nalization of the receptor are blocked (26, 36, 37). To deter- mine whether the temperature dependence of RAF-1 phos- phorylation could be explained by a lack of receptor internal- ization at the nonpermissive temperature, we used a series of B82L cell lines expressing holo and mutant EGF receptors which exhibit varying properties. Truncation to residue 973 eliminates all of the self-phosphorylation sites and results in a kinase-active EGF receptor that is defective in ligand- induced Ca'+ uptake, internalization, down-regulation, and degradation (19,38). Fig. 3 shows that EGF stimulated RAF- 1 phosphorylation equally well in cells expressing holo and carboxyl 973 EGF receptors. This result suggests that the blocking of occupancy-induced receptor internalization at 4 "C is not responsible for the failure to increase RAF-1 phosphorylation at this temperature. Although a concentra- tion of 100 nM was used in this and all subsequent experi- ments, RAF-1 phosphorylation was as effectively stimulated at EGF concentrations as low as 1 nM (data not shown).

- S * T Y

Y

EGFR Wt K72'/C'973

FIG. 4. Phosphoaminoacid analysis of RAF-1 from EGF- stimulated or control cells. RAF-1 immunoprecipitates were pre- pared from 2 X 10" '"P-laheled R82L cells expressing either wt or Lys7"/carboxy1973 EGF receptors that had been incubated for 5 min at 37 "C in the absence (-) or presence (+) of 100 nM EGF and subjected to 7.5% SDS-PAGE. The RAF-1 bands were excised and processed for phosphoamino acid analysis. 2,000 (-) or 2,800 Ceren- kov cpm (+) were subjected to two-dimensional electrophoresis in each panel. Autoradiography exposure time was 2 days. The positions of the ninhydrin-stained reference phosphoamino acids are indicated. S, phosphoserine; T, phosphothreonine; Y , phosphotyrosine. Densi- tometric analyses of the phosphotyrosine spots revealed a 1.8-fold increase in intensity with stimulation for wt cells and a 1.4-fold increase in intensity for Lys:"/carboxyl973 cells.

Although RAF-1 phosphorylation is not increased at 4 "C under conditions in which activation of EGF receptor protein tyrosine kinase occurs, the tyrosine kinase activity of the EGF receptor is essential. The addition of EGF to cells expressing kinase-inactive Met"' EGF receptors did not affect RAF-1 phosphorylation (Fig. 3). Phosphorylation of RAF-1 was also examined using cells expressing kinase-inactive Met'"' car- boxyl 1022 EGF receptors which undergo ligand-induced in- ternalization and down-regulation2 (19). Fig. 3 shows that EGF failed to increase RAF-1 phosphorylation in these cells, indicating that in the absence of tyrosine kinase activity ligand-induced internalization had no effect on RAF-1.

EGF-stimulated Phosphorylation of RAF-1 Occurs Exclu- sively on Serine Residues-The temperature dependence of stimulation of RAF-1 phosphorylation suggested that a proc- ess additional to activation of the tyrosine kinase activity of the EGF receptor was required. Because the tyrosine kinase activity of the EGF receptor was essential for RAF-1 phos- phorylation, we investigated whether RAF-1 was a direct substrate for the EGF receptor tyrosine kinase by determining the phosphoamino acid content of RAF-1 from control and EGF-treated cells. Fig. 4 shows that in untreated cells RAF- 1 is phosphorylated exclusively on serine residues. As expected from the results of the densitometric analyses presented in the legend to Fig. 2, the extent of serine phosphorylation was increased by EGF activation of holo (1.8-fold) and carboxyl 973 (1.4-fold) receptors (5 min), but phosphotyrosine was not detected in RAF-1. Similar results were obtained when cells were stimulated with EGF for 1.5 min (data not shown). App et al. (10) also failed to demonstrate tyrosine phosphorylation of RAF-1 in antiphosphotyrosine immunoblots of the holo receptor from EGF-stimulated cells. These findings indicate that RAF-1 is not directly phosphorylated by the EGF recep- tor kinase and suggest that enhanced serine phosphorylation of RAF-1 requires an intermediate step that is temperature dependent.

RAF-1 Phosphorylation Persists after EGF Withdrawal- To investigate the coupling between EGF receptor activation

' Wiley, H. S., Herbst, J. J., Walsh, B. J., Lauffenberger, D. A., Rosenfeld, M. G., and Gill, G. N. (1991) J. Riol. Chern. 266, 11083- 11094.

10944 EGF-stimulated RAF-1 Phosphorylation

and RAF-1 phosphorylation, we determined whether the EGF-mediated stimulation of RAF-1 phosphorylation could be reversed by EGF withdrawal. Cells expressing either the wt or the Lys"'lcarboxy1 973 EGF receptors were incubated with EGF for 3 min a t 37 "C prior to stripping bound EGF from the cell surface using mild acid conditions (39, 40). Cells were subsequently incubated without or with EGF for an additional 5 or 15 min a t 37 "C in medium containing ["PI orthophosphate. Identical results were obtained using both receptors. The acid wash procedure, which took 5 min to perform, did not inhibit either the EGF-induced RAF-1 phos- phorylation or the associated decrease in electrophoretic mo- bility (compare lanes 1-3 in Fig. 5A); similarly, these two parameters were unaltered by subsequent incubation of the acid-washed cells with (lanes 4 and 5) or without (lane 6) EGF. During this second incubation in labeling medium, additional :'?P incorporation was reproducibly observed (lane 3 versus lanes 4-6 in panel A ). Examination of the correspond- ing EGF receptor immunoprecipitates from cells expressing the wt EGF receptor (Fig. 5B) indicated that EGF stimulated an increase in receptor phosphorylation. However, in contrast to RAF-1, the wt EGF receptor was dephosphorylated after dissociation of EGF by the acid wash procedure (lane 6). Readdition of EGF after the acid wash maintained the phos- phorylated state of the EGF receptor (cf. lanes 3-5, Fig. 5B) but did not alter the phosphorylation of RAF-1 (lanes 4-6, Fig. 5A). These data indicate that RAF-1 phosphorylation is not dependent on the persistence of the phosphorylated state of the EGF receptor, in agreement with the persistence of RAF-1 phosphorylation after down-regulation of >70% of the EGF or CSF-1 receptors during the normal response to growth factor (12, 20, 37). Consistent with the absence of a tight coupling between the phosphorylation states of RAF-1 and

E G F - + + + + +

+ + + + + + -

Acid Wash EGF

X 10-3) Mr

116 97

200

- 116 - 97 B

- 66

1 2 3 4 5 6

FIG. 5. Failure of growth factor removal to revert the EGF- induced effects on RAF-1 phosphorylation. [:"PP]Orthophos- phate-labeled B82L cells expressing either the Lys"'/carboxyl 973 (panel A ) or the wt (panel R ) EGF receptor were incubated in the absence (-) or presence (+) of 100 nM EGF for 3 min a t 37 "C prior to either solubilization in 1% Triton X-I00 (lanes I and 2) or removal of receptor bound growth factor by acid stripping (lanes 3-6) followed by either solubilization (lane 3 ) or reincubation with (lane 4, 5 min; lane fi, 15 min) or without the growth factor (lane 6, 15 min). RAF-1 (panel A ) or EGF receptor (panel R ) immunoprecipitates were sub- jected to 7.5% SDS-PAGE and autoradiography. Exposure time was 7 h.

the EGF receptor, we were unable, in co-precipitation exper- iments, to demonstrate association of RAF-1 with the wild- type EGF receptor in any of the mutants studied (data not shown).

Down-modulation of Protein Kinase C Does Not Affect EGF- stimulated RAF-1 Phosphorylation-These data suggest the existence of "third party" molecule(s), distinct from but reg- ulated by growth factor receptors, which deliver a stable intracellular signal for RAF-1 phosphorylation. Protein ki- nase C is activated by EGF stimulation and as a consequence translocates to the cell membrane with kinetics that are similar to those of RAF-1 phosphorylation (4, 41). Further- more, activation of protein kinase C by acute PMA treatment causes RAF-1 phosphorylation in fibroblasts (11). The pos- sibility that EGF-induced RAF-1 phosphorylation is mediated through a protein kinase C-dependent mechanism was tested by using chronic PMA treatment to deplete the intracellular protein kinase C pool (4, 25). EGF-induced RAF-1 phos- phorylation was not affected by pretreatment with PMA (data not shown), suggesting that the increased serine phosphoryl- ation of RAF-1 is not mediated via protein kinase C.

DISCUSSION

The present results demonstrate that EGF stimulates phos- phorylation of RAF-1 on serine residues exclusively and that RAF-1 phosphorylation occurs only when cells are stimulated at physiological temperatures (37 "C). Stimulation a t 4 "C allowed EGF-stimulated protein tyrosine phosphorylation but prevented receptor internalization. However, analysis of cells expressing internalization-defective EGF receptors indicates that receptor internalization is not necessary for ligand-in- duced RAF-1 phosphorylation. Although self-phosphorylation of the EGF receptor was also not required, receptor kinase activity was necessary. Removal of bound ligand, shown pre- viously to cause dephosphorylation of both the EGF receptor and its candidate substrate phospholipase C (42), did not affect the growth factor-induced changes in RAF-1 phos- phorylation and electrophoretic mobility, suggesting the ex- istence of an intermediate molecule(s), distinct from but regulated by the EGF receptor, which delivers a stable intra- cellular signal for RAF-1 phosphorylation. Interestingly, chronic PMA pretreatment of cells did not abrogate the effect of EGF on RAF-1 phosphorylation, suggesting that protein kinase C is not involved in this pathway. Furthermore, the failure of carboxyl 973 EGF receptor to increase cytosolic [Ca'+] (19) indicates that the postulated intermediates are not regulated via changes in [Ca'+].

RAF-1 as well as a number of other proteins involved in signal transduction (i.e. phosphoinositol trisphosphate ki- nase, phospholipase C, GTPase-activating protein, and GTPase-activating protein-associated proteins) have been hy- pothesized to be part of a signal transduction complex asso- ciated with growth factor receptors (43-49). The studies that support this hypothesis rely mainly on the ability of antibod- ies to a component of the complex to co-precipitate one or more of the other proteins involved. The presence of these proteins in the immunoprecipitates is revealed subsequently by Western blot analysis. Using this technique controversial results have been obtained in different laboratories (11, 12, 49, 50). In the case of RAF-1, studies conducted in our laboratory failed to demonstrate ligand-induced association of this molecule with either the CSF-1 receptor, the human PDGF receptor, or the murine PDGF receptor (12). In the present study we also failed to detect RAF-1 in immunopre- cipitates of the various mutant EGF receptors after reaction of cell lysates with monoclonal antibody 528 directed against

EGF-stimulated RAF-1 Phosphorylation 10945

the extracellular domain of the EGF receptor (data not shown). We therefore conclude that, consistent with our pre- vious data obtained in the CSF-1 system, EGF-mediated phosphorylation of RAF-1 occurs in the absence of complex formation with the receptor.

These data are consistent with an hypothesis in which growth factor receptors regulate a molecule(s) capable of sustaining a stable intracellular signal for RAF-1 phosphoryl- ation. Candidate molecules could conceivably include serine kinases responsible for RAF-1 phosphorylation or phospha- tase inhibitors that counteract RAF-1 dephosphorylation. A recent report (36) has shown that although EGF-induced phosphorylation of several proteins can be demonstrated at 4 "C as well as at 37 "C, tyrosine phosphorylation of three protein species (p38, p78, and p120) was temperature-depend- ent and, like RAF-1 phosphorylation, could not be demon- strated at 4 "C. Since a functional EGF receptor kinase is required for RAF-1 phosphorylation, it will be of interest to study the possible interactions between RAF-1 and the pro- teins rapidly phosphorylated on tyrosine after EGF stimula- tion, in particular those whose phosphorylation is tempera- ture dependent.

Acknowledgments-The anti-RAF-l antisera were a kind gift of Dr. Ulf R. Rapp, NCI, Frederick, MD. We wish to express our appreciation to Drs. Thomas Decker, Yeung Yee-Guide, Orin Chish- d m , and Wei Li for critically reviewing this manuscript, to Gordon M. Walton for analysis of tyrosine phosphorylation, and to Dr. M. G. Rosenfeld for advice and discussion.

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