THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Bioehemistry and Molecular Biology, Inc

Vol. 269, NO. Issue of March 4, pp. 7030-7035. 1994 Printed in U.S.A.

Mitogen-activated Protein KinaseExtracellular Signal-regulated Protein Kinase Activation by Oncogenes, Serum, and 12-0-Tetradecanoylphorbol-13-acetate Requires Raf and Is Necessary for Transformation*

(Received for publication, June 30, 1993, and in revised form, September 9, 1993)

Jakob Troppmair8, Joseph T. BruderS, Hildita MunozS, Patricia A. Lloyd+, John Kyriakisl, Papia Banerjeel, Joseph Avruchg, and Ulf R. RappH From the $Viral Pathology Section, Laboratory of Viral Carcinogenesis, Frederick, Maryland 21702-1201 and the $Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

The protein kinase cascade Raf”APKK/MEK-MAF’W ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is oblig- atory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNAand a dominant inhibitory mutant of Raf-1 we found that serum and 12-0-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abi/src, Rae, and protein serine/ threonine kinases moslcot) were all “dependent for stimulation of MAPK All of the MAPK stimulating onco- genes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPWERK. Furthermore, the ability to stimulate MAPWERK appears to be critical for transfor- mation by oncogenic Raf-1 as ERK-1 and -2 synergized with u-rufin a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transac- tivation of the oncogene-response element in the poly- oma enhancer. We conclude that this Raf signaling path- way, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.

Raf-1 protein serinelthreonine kinase (PSK)’ has been iden- tified as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the cell mem- brane to changes in the expression of genes involved in the control of cell growth, differentiation, and survival (Rapp, 1991; Rapp et al . , 1993). The position of Raf as an essential cytoplas- mic signal transducer downstream of Ras and membrane-as- sociated tyrosine kinases has been delineated by use of gain of function and dominant negative mutants of Ras and Raf (Smith et al., 1986; Cai et al., 1990; Kolch et al . , 1991; Huleihel et al., 1986; Bruder et al., 1992) as well as cell mutants resistant to transformation by Ras (Noda et al., 1983; Rapp et al., 1988a) and Raf (Kolch et al., 1993a). At the level of Raf we have shown

payment of page charges. This article must therefore be hereby marked * The costs of publication of this article were defrayed in part by the

“aduertisement” in accordance with 18 U.S.C. Section 1734 solely to ~ ~~~ ~

indicate this fact. 1 To whom correspondence should be addressed. ”el.: 301-846-1316;

F a : 301-846-1909. The abbreviations used are: PSK, protein serinekhreonine kinase;

TPA, 12-0-tetradecanoylphorbol-13-acetate.

previously that this enzyme is required for serum-, TPA-, and Ras-induced expression from the Ap-l/ets binding site (Bruder et al., 1992). Furthermore, the expression of activated Raf leads to phoshorylation of c-Jun at serine 63 and 73 in the NH2- terminal transactivation domain and increased transcriptional activity (Smeal et al., 1992). Dominant negative Raf mutants block phosphorylation of c-Jun at these sites in response to Src, Ras, or U V (Smeal et al . , 1992; Radler-Pohl et al., 1993).

Activation of MAPWERK occurs in response to the same set of factors that stimulate Raf-1 and, like Raf activation (Tropp- mair et al . , 1992), is Ras-dependent (Thomas et al., 1992; Wood et al., 1992; Howe et al . , 1992; de Vries-Smits et al., 1992). Phosphorylation and activation of c-Jun has been observed with purified preparations of MAPK (Pulverer et al., 1992). The positioning of Raf and MAF’KiERK in a linear signaling cascade has been suggested recently by the identification of MAPW MEK as substrate for activated Raf-1 (Kyriakis et al., 1992; Dent et al . , 1992; Howe et al . , 1992). These combined data led us to postulate a core signalling unit consisting of Ras-Raf and Raf-regulated kinases such as MAPKKMEK-MAPK, which in- tegrates signals originating from protein tyrosine kinases and protein kinase C at the membrane and connects them to down- stream events including phosphorylation induced changes in the activity of oncogene class transcription factors (Rapp 1991, 1993; Bruder et al . , 1992; Kyriakis et al., 1992).

The aim of this study was to examine whether Raf-1 is an obligatory entry point into the signalling cascade leading to the activation of MAPWERK and whether this enzyme is relevant for Raf-dependent transcription activation and transformation. The experiments presented in this paper show that serum, TPA, and representatives of three classes of oncogenes all de- pend on Raf-l for the activation of MAF’K. In addition, we demonstrate that MAPK function is critical for the activation of transcription and transformation by Raf.

EXPERIMENTAL PROCEDURES Cell Lines, Plasmids, Dansfection, and Chloramphenicol Acetyl-

transferase Assays”NIH3T3, 293 human embryonal kidney cells, and Cos 7 cells were grown in Dulbecco’s modified Eagle’s medium supple- mented with 10% heat-inactivated fetal bovine serum and 100 units/& streptomycin and penicillin. For W m R K kinase assays, 7.5 x lo5 293 cells or 5 x 105 Cos 7 cells were grown in 100-mm tissue culture flasks 48 and 24 h, respectively, prior to transfection. Transfections were car- ried out following a modification of the calcium phosphate coprecipita- tion method (Chen and Okayama, 1987). 5 pg of p44MAF’iERK-1 were cotransfected with the indicated expression plasmids, and adjusted with salmon sperm DNA to a final concentration of 30 pg of DNA/ transfection. Chloramphenicol acetyltransferase assays were carried out as previously described (Bruder et al., 1992). The p44WiERK-1 expression plasmid with an NH2-terminal insertion of a 9-amino acid peptide epitope derived from influenza hemagglutinin (Field et al.,

7030

Raf Control of MAF'KI ERK Activation 7031

A

0

I K w u)

I K w u)

I-

f 2 s + +

B

s 2 + c

m e X

m O K

C k

+ + x x x m m m

o m m m

with 5 pg of p 4 4 W E R K - 1 and 15 pg of RSV-Raf C4 or RSV-C4pml7. 48 h atter transfection cells were starved for 24 h in medium containing FIG. 1. M - 1 is required for ERK-1 activation by serum, TPA, and activated forms of A-, B- and Raf-1. A, 293 cells were cotransfected

0.05% serum prior to stimulation with 206 serum or 100 ng/ml TPA for 15 min. B, 293 cells were cotransfected with 5 pg of p44MAPIERK-1 and 5 pg of activated Raf-1 (RSV-BXB; Bruder et al. (1992)), A-raf (9IV-A-rat Beck et al. (1987)) or B-raf (EH-B-RafL3 C , 5 pg of RSV-BXB DNA were cotransfected with 15 pg of RSV-Raf C4 or RSV-C4pml7. Cells were harvested 72 h after transfection following starvation for 24 h in medium containing 0.05% serum. ERK-1 activity was determined as described under "Experimental Procedures."

1988) has been described previously (Price et al., 1992). MAPIERK Kinase Assay and Protein Analysis-Following incuba-

tion with starvation medium (0.05% serum) for 24 h, plates were rinsed twice with ice-cold phosphate-buffered saline and lysed in 2 ml of RIPA (App et al., 1991). Lysates were precleared by a 15-min centrifugation a t 12,000 x g in an Eppendorf centrifuge and standardized for protein content using the Bio-Rad protein assay. Immunoprecipitation of p44MAPRRKl was camed out with monoclonal antibody 12CA5 (Field et al., 1988) preabsorbed to Protein G-agarose for 3 h at 4 "C. Immuno- precipitates were washed three times in a non-ionic detergent buffer (10 m~ Tris, pH 7.6, 50 mM NaCI, 1% Triton X-100, 30 m~ sodium pyro- phosphate, 1 mM phenylmethylsulfonyl fluoride, 0.1 m~ sodium O-vana- date, 5 m~ benzamidine, 1 m~ EGTA) and one time in Tris-buffered saline supplemented with 1 m~ sodium 0-vanadate and 5 mM benza- midine. Kinase reaction was started by the addition of 30 ml of reaction mixture (30 m~ Hepes, pH 7.4, 10 m~ MgC12. 1 m~ dithiothreitol, 20 q ATP, 20 pCi of [Y-~~PIATP, 5 m~ benzamidine, 2 pg of myelin basic protein) and continued for 30 min at room temperature. Reactions were stopped by the addition of 30 pl of Laemmli buffer and boiling for 5 min. Samples were separated on 12.5% SDS-polyacrylamide electrophoresis gels. Gels were transferred to nitrocellulose and myelin basic protein phosphorylation was visualized by exposure of the blot to x-ray film. Expression of p44MAPRRK-1 was checked by immunoblotting with 12CA5 antibody followed by alkaline phosphatase staining. Immuno- precipitation and Western blot analysis of Raf-1 protein was camed out as previously described (App et al., 1991). RSV-ERK-1 was constructed by inserting the 2-kilobase ClaIISacII fragment from pBSERKl (Boul- ton et al., 1990) into the ClaIISacII site of pKRSPA (Dorn et al., 1990). Expression plasmids for p42MAPKiERK-2 as well as mutants of ERK-2, B3(K52/R52), and C3(Y183/E183) have been described previously (Rob- inson et al., 1993).

Cell ZYansforrnation Assays-I x lo5 NIH3T3 cells were seeded in 100-mm tissue culture plates 1 day prior to transfection. Transfections were carried out by a modified calcium phosphate coprecipitation method (Chen and Okayama, 1987). Focus formation was scored 8 days later.

RESULTS

Raf-1 Is Required for the Activation of p44MAPIERK-1 by Serum Growth Factors and Protein Kinase C-To analyze the effect of various stimulation conditions on the activation of W W E R K we used a transient transfection system. A molecu- lar clone of ERK-1 (the cDNA clone of p44MAPK) NH2-termi- nally fused to the hemagglutinin antigenic region expressed under the control of the cytomegalovirus promoter (Price et al., 1992) was transfected into 293 cells. These cells were chosen because of their high susceptibility to transfection. 72 h later ERK-1 was immunoprecipitated from cell lysates with an epi- tope-specific antibody (Field et al., 1988) and assayed for its ability to phosphorylate myelin basic protein. Expression of the epitope-tagged ERK-1 protein was not effected by cotransfec- tion with any of the activator or inhibitor constructs as judged from immnunoblot analysis (data not shown). Low level myelin

basic protein kinase activity was detected in immunoprecipi- tates from growth factor-deprived cells (Fig. lA) which in- creased after serum stimulation or activation of protein kinase C (Fig. lA ). To test whether Raf-1 is required for the activation of ERK-1 in 293 cells by serum and TPA, a dominant negative mutant of Raf-1 termed C4 (Bruder et al., 1992) was cotrans- fected with ERK-1. The C4 mutant of Raf-1 contains the NH2- terminal regulatory region of Raf-1 and has been shown previ- ously to block Raf-dependent transcription activation from the oncogene responsive element in the polyoma enhancer (Bruder et al., 1992) and a similar mutant, HCR, blocked v-Ha-ras transformation of NIH3T3 cells (Kolch et al., 1991). C4 contains a putative zinc finger structure that is related to a cysteine-rich sequence in protein kinase C and n-chimaerin (Bruder et al., 1992; Ahmed et al., 1990) where it is involved in binding of a regulatory ligand, diacylglycerol, or phorbol ester. Constructs expressing the phorbol ester binding domain of n-chimaerin and protein kinase Cct were found to be functionally distinct from C4 in the transcriptional transactivation assays.' As shown in Fig. lA expression of C4 completely blocked MAPK activation by serum and TPA. Cotransfection with a mutant of C4, C4pm17, which was derived from C4 by a single point mutation in the Raf-1 cysteine finger motif and lacks the domi- nant negative phenotype (Bruder et al., 1992) had no effect. These experiments demonstrate that Raf function is required for serum and TPA induced activation of ERK-1.

The predominant isoform of Raf in 293 cells is Raf-1 (data not shown). To analyze whether two other Raf enzymes, A-Raf and B-Raf, also stimulate ERK-1, gain of function mutants ofA-, B-, and c-Raf were compared in the transient assay system. As previously demonstrated for v-raf in NIH3T3 cells using a dif- ferent approach (Kyriakis et al., 1992) expression of a trunca- tion-activated form of c-raf-l, RSV-BXB (Bruder et al., 1992), in 293 cells results in the activation of ERK-1 (Fig. 1, B and C). This activation was insensitive to the inhibition by C4. The latter finding demonstrates that activation of ERK-1 by acti- vated Raf is not mediated through activation of endogenous Raf-1. To assay A- and B-Raf, transforming versions (9IVA-Raf and EH-B-RaD generated by 5' truncation of wild type cDNA and fusion to gag sequences (Beck et al., 1987)3 were tested. As shown for activated Raf-1 (Fig.U?), cotransfection of 9IVA-Raf and EH-B-Raf with ERK-1 kinase resulted in increased myelin basic protein phosphorylation (Fig. 1B ), thus demonstrating that all three Raf PSKs share the ability to activate ERK-1.

Expression of Dominant Negative Raf Blocks Actiuation of

J. T. Bruder and U. R. Rapp, unpublished data. G. Sithanandam and U. R. Rapp, unpublished data.

7032 Raf Control of MAPKIERK Activation

A

0

e B

s B + 2

0

B

E E s s + +

C

D E

+ + u u

. .

0 0 0 0 0 0 5

+ +

plasmids encoding v-src (Johnson et al., 1985), v-abl, v-Ha-rae (Clanton et al., 1987), RSV-Raf-1 (wild type Raf-1, Bruder et al. (1992)), FIG. 2. Rat-1 is required for ERK-1 activation by oncogenes A-E, 293 cells were cotransfected with 5 pg of p44WERK-1,5 pg Of

v-mos (Bruder et d., 1992). cot (Miyoshi et al., 1991), and 15 pg of RSV-Raf C4 or RSV-Clpml7. Experimental conditions are identical to those described in the legend to Fig. 1.

ERK-1 by Oncogenes-To analyze whether there is redundancy in the signalling pathways leading to ERK-1 activation we extended our studies to include previously identified and po- tential new activators of ERK-l. v-src has been shown to acti- vate MAPK in a Ras-dependent fashion (Howe et al., 1992). Cotransfection of v-src with the dominant negative Raf mutant C4 completely blocked ERK-1 activation, whereas expression of C4pm17 had no effect (Fig. 2A) . Transfection of epitope-tagged ERK-1 with the oncogene expressor constructs did not affect the level at which ERK-1 was expressed as judged from immu- noblotting experiments (data not shown). Identical results were obtained with vabl, another intracellular protein tyro- sine kinase, as shown in Fig. 2B.

Activation of MAPWERK has also been observed after ex- pression of activated p21 Ras in several cell systems (Levers and Marshall, 1992; Pomerance et al., 1992; Shibuya et al., 1992). As shown in Fig. 2C the effect of v-Ha-Ras on the acti- vation of ERK-1 in serum-starved 293 cells was minimal. This low activity of v-Ha-Ras in growth factor-deprived cells is con- sistent with our previous demonstration of the inability of Ras to induce Raf activation under conditions of growth factor re- moval (Reed et al., 1991). However, if v-Ha-ras was cotrans- fected with wild type Raf-1, which by itself only modestly acti- vated ERK-1, strong synergism was observed. This synergistic activation was completely blocked by C4 demonstrating that v-Ha-ras depends on Raf-1 for the activation of MAPK.

We further extended these studies to include the cytosolic PSK oncogenes mos and cot. The cot oncogene was recently isolated by DNA transfection with genomic DNA from a human cell line, TC04, derived from an anaplastic thyroid cancer (Mi- yoshi et d., 1991). Cotransfection of mos or cot with ERK-1

resulted in the activation of ERK-1, which was sensitive to inhibition by the dominant negative Raf-1 mutant C4 (Fig. 2, D and E ) . The Raf dependence of ERK-1 activation as indicated by C4 inhibition predicts that both, Mos and Cot, are activators of Raf-1. This is supported by the observation that Raf-1 pro- tein from serum-starved NIH3T3 cells transformed by either nos or cot is present in the hyperphosphorylated, shifted form that is typical for kinase active Raf-1 (Fig. 31, whereas trans- formation by oncogenes that function downstream of Raf-1, e.g. v-fos, does not result in increased Raf-1 phosphorylation (data not shown).

ERK-1 and 2 Synergizes with Activated Raf-1 in Panscrip- twn Activation and Pansformation and Zs Required for Pans- formation of NZH3T3 Cells by Oncogenic Raf-1-fir demon- strating the essential role of Raf-1 in the activation of ERK-1 by representatives of a broad spectrum of upstream activators we analyzed whether ERK activation was critical for transcrip- tional transactivation as well as transformation by activated Raf-1. To study whether activated Raf-1 and ERK-1 could co- operate in the transcriptional transactivation through Ap-1 sites, RSV-BXB, RSV-ERK-1, and the 5X-TRE-CAT reporter constructs were contransfected into NIH3T3 cells. Chloram- phenicol acetyltransferase activity was dramatically increased in cells cotransfected with Raf-1 BXB and ERK-1 as compared to cells that received either construct alone (Fig. 4).

To determine whether ERK cooperates with activated Raf in transformation, NIH3T3 cells were transfected with expression plasmids for ERK-1 and v-raf (EH-neo, Rapp et al. (1988b)) and assayed for focus yield. As shown in Fig. 5A the number of transformed colonies was dramatically increased in the co- transfection of v-raf and ERK-1 compared to v-raf alone. Iden-

Raf Control of MAPKIERK Activation 7033

e

i

FIG. 3. Hyperphosphorylation of Raf-1 protein in v-mos and cot transformed NIH3T3 cells. Raf-1 protein was immunoprecipitated from the indicated cell lines following 24 h starvation a t 0.05% serum and run out on a 7.5% SDS-polyacrylamide electrophoresis gel and analyzed as described under “Experimental Procedures.” Serum stimu- lation of control NIH3T3 cells was carried out with 20% serum for 20 min.

5x-TRE-cAT

e s

7 1 6-

5 -

4 -

3-

2-

1-

O S

T

.r 5 ug RSV-ERKl 1 ug RSV-BXB 1 ug RSV-EXB

-r v

+ 5ug RSV-ERK1

FIG. 4. Activated Raf-1 and ERK-1 synergize in transcriptional transactivation. NIH3T3 cells were transfected with the 1 pg of the 5xTRE reporter plasmid and expression plasmids for ERK-1 (RSV- ERK-1) and activated Fbf-1 (RSV-BXB) as indicated. The level of chlor- amphenicol acetyltransferase (CAT) expression was determined from cell extracts harvested 48 h post-transfection. The standard deviation of the mean values is indicated by error bars.

tical results were obtained with the epitope-tagged version of ERK-1 used in the transient transfection experiments (data not shown) and with a cytomegalovirus promoter-driven expres- sion plasmid for ERK-2 (~42°K) (Fig. 5B). When kinase inactive versions of ERK-2, constructs B3 and C3 (see “Experi- mental Procedures” for details) were used in the cotransfection assay, the number of v-ruf transformed foci was greatly reduced (Fig. 5B). Cotransfection of B3 and C3 with the neoR marker into NIH3T3 cells did not decrease the yield of antibiotic-resis- tant colonies.

DISCUSSION

In mammalian cells, as in yeast, MAPWERK can apparently be activated through multiple independent pathways (Levin and Errede, 1993; Lange-Carter et ul., 1993). Here we have tested whether a particular set of potential W W E R K acti- vators, oncogene protein kinases, Ras, protein kinase C, and serum, which are all known to affect growth and transforma- tion, would stimulate W W J 3 R K by a single or by multiple mechanisms. One pathway for MAPWERK activation utilizes

A

1600 1 T

r(

I

t

B 1

c1 a &, I c

h

0 r( - Y

4

I c + a c1

c

FIG. 5. Activated Raf and ERK-1 and -2 synergize in the trans- formation of NIH3T3 cells. NIH3T3 cells were cotransfected with expression vector constructs for v-raf (EHneo)(Rapp et al., 198813) and ERK-1 (A) or ERK2 ( B ) as well as kinase inactive versions of ERK2,B3,C3 (B) as described under “Experimental Procedures.” Focus formation was scored 8 days aRer transfection. 1 pg of EHneo DNA yielded 21 foci per plate. Data represent mean values and S.E. from four (A ) and five (B ) experiments, respectively.

Raf-1 PSK for activation of MAPKWMEK (Kyriakis et al., 1992; Dent et al., 1992; Howe et al., 1992). We have used a dominant negative mutant of Raf-1 and examined whether this enzyme is obligatory for activation of a molecular clone of p44MAPKERK-1, in 293 human embryonal kidney cells. The data show that this was the case for all of the above activators. Furthermore, we found that activated forms of the other Raf isozymes, A- and B-Raf, could also function as MAPKK- (MEK-) kinases. Finally, we have addressed the question as to whether MAPK/ERK activation is important for transforma- tion by Raf and by implication other Raf-activating oncogenes. We have observed cooperative transcriptional transactivation of a 5X-TRE reporter construct and transformation of NIH3T3 cells upon cotransfection of activated Raf and ERK-1 or ERK-2, a molecular clone of p42MAPK. Additionally, kinase inactive mutants of ERK-2 inhibit v-ruf transformation as examined in cotransfection experiments with NIH3T3 target cells. We con- clude that MAPK-activating oncogenes, protein kinase C, and serum require Raf for activation of W W E R K , at least in 293 cells, and that MAPWERK is required for transformation of NIH3T3 cells.

In light of the evidence for multiple pathways for the activa- tion of MAPWERK in mammalian cells (Lange-Carter et ul., 1993) our findings of uniform dependence on Raf-1 for MAPW

7034 Raf Control of MAPKIERK Activation

ERK activation by oncogenes and other growth regulators are all the more remarkable. There are several potential explana- tions for this apparent paradox. First, it might be that the cell system that we have chosen is not equipped with the enzymes that are required for alternative routes of MAPWERK activa- tion. Although Lange-Carter et al. (1993), who recently de- scribed the presence of an alternative MEK kinase (MEKK) in mammalian cells, did not specifically test for the presence of this enzyme in 293 cells. They did, however, find MEKK protein in many cell lines including NIH3T3 and Cos 7 and examina- tion of all major organs indicated widespread presence of MEKK-specific transcripts. We have tested some of the onco- genes for Raf-1 dependence of ERK-1 activation in Cos 7 and NIH3T3 cells with results identical to those in 293 cells (data not shown) and therefore consider this explanation as unlikely. Another possibility is that multiple pathways for MAPWERK activation in mammalian cells are functionally distinct, the Raf pathway being reserved for mitogenic signal transduction, similar to the situation in yeast where at least three different protein kinase cascades, each made up by a related set of en- zymes, leads to activation of distinct MAPKK and MAPK iso- forms that mediate different types of signals (Levin and Errede, 1992; Irie et al., 1993; Lee et al., 1993). This is perhaps the most appealing explanation and in line with the general sbservation of a high degree of order in intracellular signal transduction, as exemplified by the hierarchical function of at least four families of PSKs involved in mitogenic signalling (protein kinase C, Raf, MAPKWMEK, and MAPWERK) that had commonly been thought to function in parallel, perhaps redundant pathways. Nevertheless, there are abundant data demonstrating crossover in signalling between different classes of receptors, such as heterotrimeric G-protein uersus Ras- coupled receptors (Kyriakis et al., 1993; Siegel et al., 19931, illustrating either a potential for abnormal crossover events, leading to pathological responses, or the existence of a physi- ologically flexible, complex network. For example, we have ex- amined a series of muscarinic receptors in NIH3T3 and 293 cells for their ability to couple to Raf-1 kinase as measured by shiR inducing hyperpho~phorylation~ and kinase activation (Siegel et aE., 1993). The two types of receptors that can mediate transformation of NIH3T3 cells M1 and M3, but not the trans- formation negative receptors M2 and M4 regulated Raf-1 ki- nases. These findings illustrate that there is no categorical separation between G-protein versus Ras-coupled receptors in terms of their access to different types of M A P - , i.e. MEKK uersus Raf-1. They do not establish, however, that M1 and M3 receptors control Raf-1 in their natural settings as the data were obtained in cells in which they were ectopically ex- pressed. By and large it appears that G-protein-coupled recep- tors trigger responses that are more rapidly terminated than are the responses to Ras-coupled protein tyrosine kinase recep- tors. Ectopic expression of normally tissue-restricted musca- rinic receptors may eliminate aspects of signal termination and thus lead to activation of nonphysiological signal transducers such as Raf. Raf kinase may be especially suited to mediate the long lasting effects of growth factor receptors that are required for complete cell cycle transition. I t will be interesting to test whether protein tyrosine kinase receptors also regulate alter- native MAP- such as MEKK, whether Raf and MEKK differ in their preference for different MAP% that are ex- pected to be present in mammalian cells, and whether these in turn regulate distinct MAPKs/ERKs. The availability of mo- lecular clones of MEKK makes it possible to test some of these possibilities.

A third explanation for the apparent paradox is that the

P. Crespo, N. Xu, U. R. Rapp, and J. S. Gutkind, unpublished data.

oncogenes and other growth regulators we have tested do in fact activate multiple pathways for MAPKK stimulation and that the C4 Raf mutant inhibits all of these. This consideration brings up the question of the mechanism of action of this in- hibitor. Our earlier experiments have indicated that Raf-1 C4 blocks Ras-dependent activation of Raf-1 and that the intact structure of the cysteine-rich conserved region 1 (CR1) was required for this activity (Bruder et al., 1992). These findings led to the speculation that CR1 binds a Ras-controlled Raf- activating ligand which may be protein and/or lipid in nature. Other experiments that demonstrated serum dependence of Ras activation of Raf-1 (Reed et al., 1991) and still others that showed direct activation of Raf-1 by protein kinase C (Kolch et al., 1993b), which also activates Raf enzymes in a Ras depend- ent fashion in vivo led us to propose that Ras functions by bringing cytosolic Raf to Raf kinase kinases in the membrane (Troppmair et al., 1992; Rapp et al., 1993). Recent experiments by us (Zhang et al., 1993) and others (Van Aelst et al., 1993) suggest that in fact Ras directly complexes with Raf and that this interaction requires the intact CR1 (Zn finger) structure of Raf-1. As the binding site on Ras corresponds to the effector loop which is also the site of interaction with GTPase activating protein it is conceivable that C4 blocks more than one Ras effector pathway. Nevertheless, as Raf antisense expression incapacitates Ras for transformation of NIH3T3 cells (Kolch et al., 19911, it appears that if there are other effector kinases for Ras, they do not regulate a redundant set of functions. Based on all of these considerations we tentatively conclude that the growtlddifferentiation regulating activators of MAPWERK, which are all stimulators of Raf-1 kinase activity, do in fact depend on this enzyme for activation of this cascade.

In addition to the above considerations which support an obligatory role for Raf-1 in MAPK activation, there are several other observations that agree well with the data reported here. These include our earlier finding that oncogene activation of Ap-llets-dependent transcription requires Raf-1 (Bruder et al., 1992). Blocking Raf-1 activation eliminated the phosphoryla- tion of c-Jun in the transactivation domain (Smeal et al., 1992), a modification that probably requires MAPK (Pulverer et al., 1991). Experiments with the ets family transcription factor TCF also indicated a role for W K in the activation of this factor (Gille et al., 1992). The effective inhibition of cjun and ets-dependent transcriptional transactivation therefore strongly suggests that the activators that had been tested in that system (protein kinase C, serum, and Ras) (Bruder et al., 1992) do not have alternative routes for activation of MAPK.

While these earlier data bear on our findings with Ras or Ras-dependent inducers of MAPK, the situation is a little dif- ferent for the cytosolic oncogenes Cot and Mos. Both of these oncogene PS& also activate Raf in intact cells but their trans- forming (NIH3T3) (Feig and Cooper, 1988) and differentiation inducing (PCl2)5 activity cannot be blocked with the inhibitory p21 RasAsn-17 mutant. The simplest explanation would be that Mos and Cot can function as Raf kinase kinases, a possi- bility that is currently being examined. The data on the Raf-1 dependence of Mos-mediated MAPK(ERK) activation contrast with our earlier findings of Raf-1 independent transactivation ofAP-Vets-dependent transcription in NIH3T3 cells (Bruder et al., 1992). The basis for this difference is unclear and may include differences in the levels of MAPKWMEK) which has been described as a Mos substrate (Posada et al., 1993; Nebreda and Hunt, 1993) or activation of alternate pathways for tran- scription activation. Two of the four PSKs that make up the membrane to nucleus cascade, the signal initiating Raf kinase kinase, protein kinase C, and the downstream effector kinase

U. R. Rapp, unpublished data.

Raf Control of MAPKI ERK Activation 7035

MAPK(ERK) are known to have multiple substrates (Pulverer et al., 1991; Alvarez et al., 1991; Stokoe et al., 1992; Nemenoff et al., 1993; Lin et al., 1993). In the case of Raf, the only known oncogene kinase in the chain, we also have to consider that MAPKWMEK is only one of several jointly required pathways for transformation. At present there are no genetic epistasis experiments with mammalian cells that would allow ranking of MAPKWMEK as a downstream effector of Raf. Such experi- ments have been carried out in Drosophila looking at a devel- opmental end point and Drosophila MEK was found to func- tionally replace Raf (Tsuda et al., 1993). Our data on cooperative transcriptional transactivation of the oncogene re- sponsive element from the 5X-TRE reporter by Raf and ERK-1 as well as the cooperative transformation of NIH3T3 cells dem- onstrate that ERK-1 and -2 can contribute to the transforming pathway. The transformation blocking experiments with ki- nase negative ERK-2 mutants indicate that MAPWERK activ- ity or another MAPKWMEK substrate is required for Raf transformation at least of this cell type. The mechanism of inhibition by kinase-inactive ERK is probably MAPKWMEK substrate competition. If this enzyme has multiple functionally related or diverse substrates, loss of any one might inhibit transformation. However, the combined cooperative transfor- mation and inhibition data argue for a role of a MAPK in this process.

At least two of the MAPWERK substrates are oncogene class transcription factors, ~ 6 2 ~ ~ ~ (ets family) (Gille et al., 1992) and cJun (Pulverer et al., 1992). We have observed, by use of domi- nant negative mutants of c-Jun that Raf transformation as well as transformation by the other oncogenes tested here for Raf dependence of MAPK activation requires c-Jun andor c-Jun dimerization partners.6 This finding is further evidence for a linear pathway into which most oncogenes funnel and in which MAPWERK is a necessary proximal effector of transformation. In the absence of gain of function mutants of ERK-1 and -2 we cannot yet evaluate whether these enzymes would also be suf- ficient to replace the transforming activity of Raf or whether Raf induces additional activities important for transformation.

Acknowledgments-We thank Drs. Melanie H. Cobb for providing ERK-2 wild type and mutant expression plasmids, George D. Yancopou- 10s for the pBS-ERK-1 plasmid, and Jun Miyoshi for the cot expression plasmid pJJ26.

REFERENCES

Ahmed, S., Kozma, R., Hall, C., Lim, H. H., Smith, P., and Lim, L. (1990) Biochem.

Alvarez, E., Northwood, I. C., Gonzalez, F. A,, Latour, D. A,, Seth, A., Abate, C.,

App, H., Hazan, R., Zilberstein, A,, Ullrich, A., Schlessinger, J., and Rapp, U. R.

Beck, T. W., Huleihel, M., Gunnell, M., Bonner, T. I., and Rapp, U. R. (1987) Nucleic

Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slaughter, C., Moomaw, C., Hsu,

Bmder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes & Deu. 6, 5454-5456 Cai H., Szeberenyi, J., and Cooper, G. M. (1990) Mol. Cell. Biol. 10, 5314-5323

Dent, P., Haser, W., Haystead, T. A,, Vincet, L. A., Roberta, T. M., and Sturgill, T. W. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752

De Vries-Smits, A. M. M., Burgemng, B. M. T., Leevers, S . J., Marschall, C. J., and

J. 272, 767-773

Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266,15277-15285

(1991) Mol. Cell. Biol. 11, 913-919

Acids Res. 15,59&609

J., and Cobb, M. H. (1990) Science 249, 64-67

(1992) Science 257, 1404-1407

Bos, J. (1992) Nature 357, 602404

J. Troppmair and M. Birrer, unpublished data.

Dorn, P., DaSilva, L., Martarano, L., and Derse, D. (1990) J. virol. 64, 1616-1624 Feig, L. A,, and Cooper, G. M. (1988) Mol. Cell. Biol. 8,3235-3243 Field, J., Nikawa, J., B m k , D., MacDonald, B., Rodgers, L., Wilson, I. A,, Lerner,

Gille, H., Sharrocks, A. D., and Shaw, P. E. (1992) Nature 358,414-417 Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P., and Marshall, C. J.

Huleihel, M., Goldsborough, M., Cleveland, J., Gunnell, M., Bonner, T., and Rapp,

Irie, K., Takase, M., Lee, K. S., Levin, D. E., Araki, H., Mastumoto. K., and Oshima,

Johnson, P. J., Coussens, P. M., Danko, A. V., and Shalloway, D. (1985) Mol. Cell.

Kolch, W., Heidecker, G., Lloyd, P., and Rapp, U. R. (1991) Nature 349,426-428 Kolch, W., Heidecker, G., Yanagihara, K., Bassin, R. H., and Rapp, U. R. (1993a)

Oncogene 8,361370 Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, P., Finken-

zeller, G., Marmi?, D., and Rapp, U. R. (1993b) Nature 364,249-252 Kyriakis, J. M., App, H., Zhang, X., Baneiee, P., Brautigan, D. L., Rapp, U. R., and

Avruch, J. (1992) Nature 358,417421 Kyriakis, J. M., Force, T. L., Rapp, U. R., Bonventre, J. V., and Avruch, J. (1993) J.

Biol. Chem. 268,1600%16019 Lange-Carter, C. A., Pleiman, C. M., Gardner, A. M., Blumer, K. J., and Johnson G.

L. (1993) Science 260,315-319 Lee, K. S. , Irie, K., Gotoh, K., Watanabe, Y., Araki, H., Nishida, E., Mataumoto, K.,

and Levin, D. E. (1993) Mol. Cell. Biol. 13,3067-3075 Leevers, S. J., and Marshall, C. J. (1992) EMBO J . 11,569-574 Levin, D. E., and Errede, B. (1993) J. NIH Res. 6,49-52 Lin, L.-L., Wartman, M., Lin, A. Y., Knopf, J. L., Seth, A,, and Davis, R. J. (1993)

Miyoshi, J., Higashi, T., Mukai, H., Ohuchi, T., and Kakunaga, T. (1991) Mol. Cell.

Nebreda, A. R., and Hunt, T. (1993) EMBO J. 12, 1979-1986 Nemenoff, R. A,, Winitz, S . , Quian, N.-X., Van Putten, V., Johnson, G. L., and

Noda, M., Selinger, Z., Scolnick, E., and Bassin R. H. (1983) Proc. Natl. Acad. Sci. Heasley, L. E. (1993) J. Biol. Chem. 268, 1960-1964

Pomerance M., Schweighoffer, F., “que, B., and Pierre, M. (1992) J. Biol. Chem. U. S. A. 80,560%5606

267, 16155-16180 Posada, J., Ahn, N. G., Vande Woude, G . F., and Cooper, J. A. (1993) Mol. Cell. Biol.

Price, D. J., Grove, J. R., Calvo, V., Avruch, J., and Bierer, B. E. (1992) Science 257, 13,254G2553

F’ulverer, B. J., Kyriakis, J. M.,Avruch, J., Nikolauki, E., and Woodgett, J. R. (1991) 973-977

Radler-Pohl, A., Sachsenmaier, C., Gebel, S., Auer, H. P., Bruder, J. T., Rapp, U., Nature 353,670-674

Rapp, U. R. (1991) Oncogene 6,495500 Angel, P., Rahmsdorf, H. J., and Herrlich, P. (1993) EMBO J . 12, 1005-1012

Rapp, U. R., Cleveland, J. T., Morse, H. C., and Rapp, U. R. (1988a) The Oncogene Handbook (Curran, T., Reddy, E. P., and Skalka, A,, eds) pp. 213-253, Elsevier

Rapp, U. R., Heidecker, G., Huleihel, Cleveland, J. L., Choi, W. C., Pawson, T., Ihle, Science Publishers, New York

J. N., and Anderson, W. B. (1988b) Cold Spring Harbor Symp. Quant. Biol. 53, 173-184

Rapp, U. R., Bruder, J. T., and Troppmair, J. (1993) in Critical Reviews in Cancer (Herrlich, P., ed), in press

Reed, J. C.,Yum, S., Cuddy, M. P., Turner, B. C., and Rapp, U. R. (1991) Cell Growth Diver 2, 235-243

Robinson, D. J., Zhen, E., Owaki, H., Vanderbild, C. A., Ebert, D., Geppert, T. D., and Cobb, M. H. (1993) J. Biol. Chem. 268, 50975106

Shibuya, E. K., Polverino, A. J., Chang, E., Wigler, M., and Ruderman, J. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,9831-9835

Siegel, J. N., June, C. H., Yamada, H., Weiss, A., Rapp, U. R., and Samelson, L. E. (1993) J. Immunol. 15,41164127

Smeal, T., Binetmy, G., Mercola, D., Grover-Bardwick, A,, Heidecker, G., Rapp, U. R., and Karin M. (1992) Mol. Cell. Biol. 12, 3507-3513

Smith, M. R., DeGudicibus, S . G., and Stacey, D. W. (1986) Nature 320, 540643 Stokoe, D., Campbell D. G., Nakielny, S., Hidaka, H., Leevers, S. J., Marshall, C.,

Thomas, S. M., DeMarco, M., DArcangelo, G., Halegoua, S., and Bmgge, J. S.

Tsuda, L., Inoue,Y. J., Yoo, M-A., Mizuno, M., Hata, M., Lim,Y-M., Adachi-Yamada,

Troppmair, J., Bruder, J. T., App, H., Cai, H., Liptak, L., Szeberenyi, J., Gooper, G.

Van Aelst, L., Barr, M., Marcus, S., Polverino, A,, and Wigler, M. (1993) Proc. Natl.

Wood, K. W., Sarnecki, C., Roberts, T. M., and Blenis, J. (1992) Cell 68,1041-1050 Zhang, X., Settleman, J., Kyriakis, J. M., Suzuki, E. T., Elledge, S. J., Marshall, M.

R. A,, and Wigler, M. (1988) Mol. Cell. Biol. 8, 2159-2165

(1992) Cell 7, 335-342

U. R. (1986) Mol. Cell. Biol. 6, 2655-2662

Y. (1993) Mol. Cell. Biol. 13, 3076-3083

Biol. 6, 1073-1083

Cell 72, 269-278

Biol. 11, 4088-4096

and Cohen, P. (1992) EMBO J. 11,39853994

(1992) Cell 68, 1031-1040

T., Ryo, H., Masamune, Y., and Nishida, Y. (1993) Cell 72, 407414

M., and Rapp, U. R. (1992) Oncogene 7, 1867-1873

Acad. Sci. U. S. A. 90.62134217

S., Bruder, J., Rapp, U. R., and Avruch, J. (1993) Nature 364, 308313