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Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms. Darlene Barnard1,2, Bruce Diaz1,2, David Clawson1,2 and Mark ...
Oncogene (1998) 17, 1539 ± 1547  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms Darlene Barnard1,2, Bruce Diaz1,2, David Clawson1,2 and Mark Marshall1,2 1

Department of Medicine, Division of Hematology and Oncology, 2Department of Biochemistry and Molecular Biology, and Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut St., Indianapolis, Indiana 46202, USA

We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12myristate 13-acetate (PMA) to activate Raf-1. Speci®cally, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491 ± 508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCa phosphorylation sites failed to signi®cantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no e€ect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of di€erent stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a signi®cant mechanism of Raf-1 kinase regulation in COS-7 cells. Keywords: Oncogene; Ras; Raf; PKC; Src; EGF

Introduction Three mammalian Raf genes, raf-1, B-raf and A-raf, have been identi®ed as viral and cellular oncogenes capable of inducing a ras-like transformed phenotype in cell culture. The Raf proteins all share homologous structural segments designated Conserved Region (CR) 1, 2 and 3. CR1 contains a Ras binding domain (RBD) and a PKC-like zinc binding repeat (Heidecker et al., 1992). CR2 contains multiple phosphorylated residues shown to have regulatory function and serves as a binding site for the 14.3.3 sca€olding protein. The CR3 domain constitutes the actual kinase domain of Raf and also has at least one site of 14.3.3 attachment (Muslin et al., 1996). CR1 and CR2 are both located within the amino-terminal 300 amino acids of Raf-1

Correspondence: M Marshall Received 26 November 1997; revised 21 April 1998; accepted 21 April 1998

and have been shown to constitute a negative regulatory domain. A 14.3.3 dimer may bridge CR2 to a separate binding site in CR3, maintaining an inactive conformation (Luo et al., 1995). Removal of CR1 and CR2 or removal of the 14.3.3 binding site in CR2 (serine 259) results in the oncogenic activation of Raf-1 (Heidecker et al., 1990; Morrison, 1996). Normally localized in the cytosol in an inactive form, Raf-1 associates with Ras at the plasma membrane following growth factor-induced Ras activation (Traverse et al., 1993). Once at the membrane, Raf-1 becomes catalytically activated through a complex and still partially unde®ned mechanism. These activation steps may include a conformational change which would relieve the inhibition imposed by the Raf-1 amino-terminus, multiple phosphorylations of CR2 and CR3, and the presence of accessory proteins such as hsp90 and 14.3.3 (Heidecker et al., 1992; Morrison and Cutler, 1997). It has been demonstrated that activation of Raf by Ras requires multiple contacts with both the RBD and the zincbinding region (Brtva et al., 1995; Chuang et al., 1994). While the RBD provides a high anity site for RasGTP, the zinc binding region interacts with Rasfarnesyl and is critical for the activation of the protein kinase (Chuang et al., 1994; Hu et al., 1995; Luo et al., 1997). Association with Ras dislodges 14.3.3 from CR2, possibly opening up the molecule for activation by further modi®cation (Clark et al., 1997; Rommel et al., 1996). Raf activity clearly can be regulated by phosphorylation. A number of regulatory phosphorylation sites have been identi®ed on Raf-1 which can be characterized as either constitutive or inducible. The primary constitutive in vivo phosphorylations have been identi®ed on serines 43, 259 and 621 (Morrison, 1996). Phosphorylation of serine 43 and serine 259 cause down regulation of Raf-1 in response to protein kinase A (Morrison and Cutler, 1997). Phosphoserine 621 may also constitute a carboxy-terminal binding site for 14.3.3 (Muslin et al., 1996). In addition to this, phosphorylation of serine 621 is also a prerequisite for the inducible phosphorylation of serine 624 (Ferrier et al., 1997). Substitution of either of these serines with alanine inactivates Raf-1. Additional inducible phosphorylations of Raf-1 have been reported on threonine 269, serines 338 and 339, tyrosines 340 and 341, serine 497 and serine 499. Threonine 269 is phosphorylated by a ceramide-activated protein kinase, also known as Ksr (Sundaram and Han, 1995; Zhang et al., 1997). Phosphorylation of this residue has been shown to enhance the activation of Raf-1 by tyrosine kinases. Src-family tyrosine kinases have been shown to strongly activate the kinase and transforming activity

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of Raf-1 through the phosphorylation of tyrosines 340 and 341 (Fabian et al., 1993). While Src alone appears sucient to stimulate Raf-1 activity, the presence of oncogenic Ras increases Raf-1 activation (Marais et al., 1995). The diculty in detecting phosphotyrosine on Raf-1 has provoked some debate as to the signi®cance of this modi®cation in physiological systems. However, most recent studies agree that tyrosine phosphorylation is important for the Rasdependent regulation of Raf-1 (Dent et al., 1995a; Jelinek et al., 1996). Phosphorylation of serine at residues 338 and 339 in Raf-1 has recently been shown to be Ras-inducible and necessary for Raf-1 activation by both oncogenic Ras and vSrc (Diaz et al., 1997). However, unlike tyrosines 340/341, phosphorylation of these serines is not of itself activating but a prerequisite for activation. The functional relationship between the phosphorylated serines and tyrosines in this unusual region is not known, although genetic and biochemical analysis suggests separate functions (Diaz et al., 1997). The protein kinase responsible for phosphorylation of serine 338/339 has not yet been identi®ed. PKCa, PKCZ and PKCe have been reported as being Raf activating kinases (Cai et al., 1997; Schonwasser et al., 1998). PKCa has been reported to activate Raf-1 via phosphorylation of serine 499 in the activation loop, although this mechanism has been disputed (Dent et al., 1995b; MacDonald et al., 1993; Schonwasser et al., 1998). PKC-dependent phosphorylation of serine 497 has also been detected in hematopoetic cells in response to IL-3 (Carroll and May, 1994). Both serine 497 and serine 499 are positioned in the Raf-1 activation loop, a site of regulatory phosphorylation conserved among many protein kinases. A potential physiological role for PKCa in the Raf pathway is indicated by the ability of Raf-1 and PKCa to transform ®broblasts when transfected together (Kolch et al., 1993). Furthermore, treatment of cells with phorbol esters results in the rapid activation of Raf-1 (Cai et al., 1997). Phorbol ester-dependent activation of Raf-1 has been reported as being both Ras-dependent and Ras-independent (Arai and Escobedo, 1996; Burgering and Bos, 1995; Cai et al., 1993, 1997; Ueda et al., 1996; Zou et al., 1996). One hypothesis is that certain PKC isoforms function as Raf kinase kinases, which together with Ras-GTP, promotes activation of Raf-1 (Burgering and Bos, 1995; Schonwasser et al., 1998). Additional support for an activation mechanism requiring discrete phosphorylation of the Raf-1 activation loop is provided by the observation that oligomerization of Raf-1 results in catalytic activation (Farrar et al., 1996; Luo et al., 1996). As with receptor tyrosine kinases, oligomerization of Raf-1 protein might result in the transphosphorylation of the activation loop of the catalytic domain, resulting in a catalytically active enzyme. Attempting to make sense of all the regulatory mechanisms proposed for Raf-1 is a dicult endeavor. Many key studies of Raf-1 regulation have been performed in non-mammalian systems. Some of the confusion concerning the role of Raf-1 tyrosine phosphorylation for example may have resulted from the comparison of heterologous Raf regulation in insect, amphibian and mammalian cells. Typically, extremely high catalytic activities have been

equated with physiological mechanisms of regulation. However, two recent studies reported that only low levels of Raf activity are mitogenic in primary cells, while high levels of Raf activity are antiproliferative (Sewing et al., 1997; Woods et al., 1997). Many of the older studies concerning the role of PKC as a Raf regulator were not able to use a physiological Raf substrate (Mek) in their kinase assays, leaving the question unanswered as to whether or not conventional PKCs can directly stimulate Raf-1 activity towards physiological substrates. Other studies which concluded that phorbol-ester stimulation of Raf-1 was independent of Ras, relied upon the use of the dominant-negative RasN17 allele, which is frequently unreliable. Finally, the role of inducible phosphorylation in Raf regulation has recently been questioned altogether. Stokoe and McCormick reported that protein kinases were not required at all for Rasloaded plasma membranes to stimulate immunoprecipitated Raf-1 (Stokoe and McCormick, 1997). In light of these seemingly contradictory data, we have used the battery of Raf-1 mutants available to us to probe speci®c in vivo requirements for Raf regulation by oncogenes, growth factors and phorbol esters in a single, well de®ned mammalian cell culture system. We report that each of these stimuli have a common requirement for Ras association and the phosphorylation of both serines 338/339 and tyrosines 340/341. We found no evidence for activation loop phosphorylation during activation by Ras or Src, although regulation by phorbol esters may involve modi®cation of the loop. In addition, we observed that phorbol ester directly promoted Ras-GTP accumulation in COS-7 cells.

Figure 1 Stimulation of Raf-1 activity in COS-7 cells by oncogenes, EGF and phorbol ester. COS-7 cells were transfected with pcEXV-Raf-1 by electroporation, either alone or together with pcEXV-Ras[V12] or pvSrc. Oncogene cotransfected cultures were harvested after 3 days for analysis. Raf-1 transfected cultures were stimulated with EGF or PMA for 5 min following 18 h serum-starvation. Myc-tagged Raf-1 was immuneprecipitated and tested for Raf activity using a coupled MEK-ERK-MBP assay. All results were normalized for Raf-1 protein and standardized to Raf-1 stimulated by co-expression with Ras[V12] which is expressed here as 100%. These results were obtained from 3 ± 5 separate experiments performed in duplicate

Regulation of the Raf-1 protein kinase D Barnard et al

Results Oncogene, EGF and phorbol ester regulation of Raf-1 We wished to address disagreements in the literature concerning the various mechanisms of Raf-1 activation utilized by di€erent stimuli. Figure 1 shows the comparative abilities of oncogenic Ras[V12], vSrc, EGF and phorbol ester to activate heterologously expressed Raf-1 in COS-7 cells. Raf-1 mutants defective for Ras binding (Raf-1[A84A86A87]), phosphorylation of serines 338/339 (Raf-1[A338A339]) and tyrosines 340/341 (Raf-1[F340F341]) were assayed for responsiveness to these stimuli (Figure 2). As expected, inactivation of the Ras binding domain signi®cantly reduced activation by Ras[V12] (Figure 2a). Similarly, the presence of serine at residues 338 and 339 was

con®rmed as essential for catalytic activation. The loss of tyrosines at 340 and 341 also signi®cantly impaired the Ras-response, but to a lesser degree than the alanine 338/339 mutant. Similar results were observed for Raf-1 coexpressed with vSrc, except that the phenylalanine 340/341 mutant was more impaired than the alanine 338/339 mutant (Figure 2b). The general responsiveness of each mutant Raf-1 protein to EGF treatment was similar to that observed with Ras[V12] and vSrc, but with some variation in the extent of the defect (Figure 2c). The loss of the Ras binding site in Raf-1 clearly prevented signi®cant activation by EGF (490% reduction in inducible activity). However, alanine substitution of serines 338/ 339 and tyrosines 340/341 resulted in no more than a 70% decrease in inducible activity. It can be noted in Figure 2 that the basal activity (unstimulated) of the

Figure 2 Regulatory requirements for Raf-1 activation by oncogenes, EGF and phorbol ester. COS-7 cells were transfected with pcEXV-Raf-1, pcEXV-Raf-1[A84A86A87] designated RBD (Barnard et al., 1995), pcEXV-Raf-1[A338A339] and pcEXV-Raf1[F340F341] by electroporation, either alone or together with pcEXV-Ras[V12] or pvSrc. Oncogene cotransfected cultures were harvested after 3 days for analysis. Singly transfected cultures were stimulated with EGF or PMA for 5 min following 18 h serumstarvation. Myc-tagged Raf-1 was immuneprecipitated and tested for Raf activity using a coupled MEK-ERK-MBP assay. All results were normalized for Raf-1 protein and standardized to stimulated Raf-1, which is expressed as 100%. These results were obtained from 3 ± 5 separate experiments performed in duplicate. (a) Ras[V12] cotransfected with pcEXV-Raf-1, (b) vSrc cotransfected with pcEXV-Raf-1, (c) EGF stimulated pcEXV-RAf-1 transfected cells, (d) PMA stimulated pcEXV-Raf-1 transfected cells. Light bars are unstimulated activities, dark bars are stimulated activities

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alanine 338/339 mutant is less than wild type Raf-1 (2.6 vs 3.5). The signi®cance of the basal activities is dicult to determine due to reduced accuracy of the assay when measuring low levels of kinase activity. COS-7 cells transfected with each Raf-1 mutant were also treated with the phorbol ester, PMA (Figure 2d). PMA-induction of Raf-1 activity was observed to require both an intact Ras binding domain and the serines at 338/339. As with Ras[V12]-activated Raf-1, PMA activation was observed to be only partially dependent upon the presence of tyrosine at 340 and 341. The requirement of an intact RBD for phorbol ester stimulation indicated that Raf-1 associates with Ras-GTP following treatment of the cell with phorbol esters. To determine if phorbol ester-treatment results in activation of Ras by GTP binding, the ratio of RasGTP to Ras-GDP was directly measured in COS cells prior to and following a brief treatment with PMA (Figure 3). The basal fraction of Ras-GTP in serumdeprived COS-7 cells was measured at 1.8% and 5.1%. Following a ®ve minute treatment with PMA, the fraction of Ras-GTP increased substantially to approximately 13.5%. EGF was found to be a superior activator of endogenous Ras, resulting in 28% Ras-GTP. This level of GTP loading of Ras was similar to the 30% fraction observed in cells transiently expressing Ras[V12]. These results demonstrate that phorbol esters activate Ras in COS-7 cells by promoting the accumulation of Ras-GTP, consistent with our observation that Raf-1 must be able to associate with Ras in order to be stimulated by PMA. The close physical and functional association of the serine 338/339 and tyrosine 340/341 phosphorylation sites raises the obvious question as to whether or not the phosphorylation of one site is dependent upon phosphorylation of the adjacent residues. Some data

addressing this question have been previously published. We further examined this possibility using the constitutive, isoprenylated Raf-CX mutant as a probe to compare the biological and catalytic importance of serine 338/339 phosphorylation with tyrosine 340/341 phosphorylation (Diaz et al., 1997). The A338A339 and F340F341 substitutions were separately introduced into Raf-CX. Each mutant was compared for the ability to induce focus formation in Rat1 ®broblasts as well as for Mek kinase activity in COS-7 cells (Table 1). While the Raf-CX[A338A339] mutant was completely defective in both focus formation and enzymatic activity, the Raf-CX[F340F341] mutant was fully half as potent as the `wild type' Raf-CX protein in both assays. These results suggest that the phosphorylation of serine 338/339, but not tyrosine 340/341, is essential for Raf-CX activity. Considered together, these data indicate that the phosphorylation and function of serine 338/339 does not require phosphotyrosine at positions 340 or 341. The role of the activation loop in Raf-1 regulation A common mechanism for protein kinase regulation is the phosphorylation of one or more residues in the activation loop which is part of the catalytic site of many protein kinases. Phosphorylation of this loop can occur through autophosphorylation (PKA), transphosphorylation (IRK) or by other regulatory protein kinases (Mek, Erk). Alignment of the conserved catalytic domain sequences of vertebrate and invertebrate Raf with the known structures of Erk2, Cdk2, IRK and PKA (Figure 4) indicated that the Raf protein kinases also possess a region analogous to an activation loop connecting subdomains VII and VIII. We tested the hypothesis that phosphorylation of the Raf-1 activation loop is required for catalytic activation by diverse stimuli. Within the putative Raf-1 loop, are six conserved serine and threonine residues which could potentially serve as regulatory phosphorylation sites.

Table 1 Role of serine 338/339 and tyrosine 340/341 in the function of isoprenylated Raf-CX Construct Vector Raf-CX Raf-CX[A338A339]c Raf-CX[F340F341] a

Figure 3 Phorbol esters induce Ras GTP loading. Subcon¯uent COS-7 cell cultures were serum-deprived for 18 h, phosphatedeprived for 2 h and 32P-orthophosphate labeled for 3 h. DMSO, PMA and EGF treated cultures were harvested after 5 min along with Ras[V12]-transfected and mock cultures. Total Ras was immuneprecipitated under conditions inhibitory towards p120 RasGAP and the bound guanine nucleotides identi®ed and quanti®ed by TLC and phosphoimager analysis. One Ras[V12] transfected culture was mock precipitated with a non-immune antibody. The percentage of GTP to GDP was calculated following adjustments of the raw counts to re¯ect the stoichiometry of phosphate in GDP and GTP

Focus formation (% of Raf-CX)a

Raf activity (Fold increase)b

0 100 0 56+14

1.0+0.1 12.0+0.5 1.0+0.1 6.9+1.5

Mutated genes for Raf-CX cloned into the pBabepuro vector were transfected into Rat-1 cells by calcium phosphate precipitation. Transfections were plated without drug selection and foci were counted following 2 weeks of incubation. Focus number was standardized to the number of puromycin resistant colonies obtained from plating one-tenth of the transfection under puromycin selection. Experiments were standardized to the `wild-type' Raf-CX gene. Typically, 10 mg of pBabepuro-Raf-CX would result in the formation of 700 foci. The values shown are the means and standard errors of the means of four experiments. bMutated genes for Raf-CX cloned into the pcEXV-3 vector were transfected into COS-7 cells by electroporation. After 3 days, the transfected cells were harvested and soluble extracts were tested for Raf activity using a coupled MEKERK-MBP assay (Diaz et al., 1997). Activity is expressed as the fold increase over endogenous Raf activity following normalization to protein expression levels. These results are derived from three separate assays performed in duplicate. cData for this mutant was previously published (Diaz et al., 1997)

Regulation of the Raf-1 protein kinase D Barnard et al

Two of these residues are serine 497 and 499 which have been previously implicated in the activation of Raf-1 by PKCa. Each of these six residues in the Raf-1 activation loop were substituted with alanine and assayed for constitutive, Ras[V12], vSrc and PMA-inducible activity in COS-7 cells. As shown in Figure 5, all of the Raf-1 loop mutants remained inducible, although speci®c mutants responded somewhat di€erently to each stimulus. For example, the Raf-1[A491] mutant was fully inducible by vSrc but only 40% inducible by either Ras[V12] or PMA. The Raf-1[A494] and Raf-1[A508] mutants were signi®cantly less responsive to PMA than to either oncogene. Signi®cantly, the loss of either serine 497 or 499 failed to signi®cantly decrease Raf-1 activation by PMA. In fact, Raf-1[A499] was reprodu-

cibly found to be stimulated by PMA nearly twofold better than wild type Raf-1. To control for the possible redundant phosphorylation of either serine 497 or serine 499 by PKC, a Raf-1[A497A499] double mutant was also tested for inducibility. This mutant was activatable by all stimuli including PMA, demonstrating a lack of regulatory function for these two residues in COS-7 cells. To investigate the possibility of multiple sites of phosphorylation other than 497 and 499, a single mutant was constructed in which every serine and threonine in the activation loop was substituted with alanine (multiloop mutant). This mutant was moderately responsive to vSrc, less responsive to Ras[V12] and poorly induced by PMA suggesting a role for the activation loop in response to phorbol esters in CPS-7 cells.

Figure 4 Potential phosphorylation sites within the predicted activation loop of Raf-1. The amino acid sequence of conserved subdomains VII and VIII of Raf-1, B-Raf, A-Raf, D. melanogaster Raf (Dm-Raf) and C. elegans Raf were aligned with the corresponding subdomains of Erk2, Cdk2, insulin receptor kinase (IRK) and the cAMP-dependent protein kinase (cAPK). Through this alignment, the boundaries of the activation loop in the Raf kinases was predicted and all serine and threonine residues within the putative loop marked with bold print. The location of the loop phosphorylations on Erk2, Cdk2, IRK and cAPK are shown in outlined print and the location of each site is emphasized by the underlying black bars

Figure 5 Activation loop mutants of Raf-1 remain activatable by Ras[V12] and vSrc. COS-7 cells were transfected with pcEXVRaf-1, pcEXV-Raf-1[A491], pcEXV-Raf-1[A494], pcEXV-Raf-1[A497], pcEXV-Raf-1[A499], pcEXV-Raf-1[A506], pcEXV-Raf1[A508], pcEXV-Raf-1[A497A499] and pcEXV-Raf-1[multi-loop] by electroporation, either alone or together with pcEXV-Ras[V12] or pvSrc. Each culture was harvested after 3 days for analysis. Singly transfected cultures were stimulated with PMA for 5 min following 18 h serum-starvation. Myc-tagged Raf-1 proteins were immuneprecipitated and tested for Raf activity using a coupled MEK-ERK-MBP assay. All results were normalized for Raf-1 protein and standardized to stimulated wild type Raf-1, which is expressed here as 100%. These results were obtained from 2 ± 5 separate experiments performed in duplicate. Light bars are unstimulated, black bars are Ras[V12] stimulated, stippled bars are vSrc stimulated and grey bars are PMA stimulated

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Figure 6 Regulatory steps in the regulation of the Raf-1 protein kinase in COS-7 cells. Ras, Src, EGF and phorbol esters induce a set of common regulatory interactions and phosphorylations required to activate Raf-1. Raf-1 must associate with Ras-GTP at the plasma membrane, presumably to induce an activatable conformation in the protein kinase. Complex phosphorylation of serine 338/339 and tyrosine 340/341 then occurs, resulting in the catalytic activation of the enzyme. Phosphorylation of the activation loop does not appear important for regulation by Ras or Src in this model system, although it may occur in response to phorbol esters. Arrows indicate the required components for Raf-1 regulation by each stimulus rather than direct interactions between each protein. PMA e€ects are presumed to be directly mediated through conventional PKC's. PSK designates protein serine kinase

Discussion We have determined that a variety of diverse stimuli regulate the Raf-1 protein kinase through a common mechanism in COS-7 cells. Previous studies have demonstrated a multitude of regulatory steps for Raf1, but with disagreement on the general role of each step in the variety of model systems examined. Previous studies have demonstrated that association with Ras is necessary for Raf-1 activation by oncogenic Ras[V12] and EGF-family growth factors. However, activation of Raf-1 by vSrc and phorbol esters has been argued to be both dependent and independent of Ras. In this study we show that Raf-1 can only be activated by Ras[V12], vSrc, EGF and phorbol esters when complexed with Ras-GTP. Interaction with Ras at the plasma membrane probably has multiple e€ects on Raf-1. First of all, Ras association is believed to drive the conformational change necessary to reverse the inhibitory e€ects of the amino terminus of Raf-1 (Hu et al., 1995; Luo et al., 1997). Second, docking with Ras brings Raf-1 to the plasma membrane which is the primary site of action for vSrc, the EGF receptor and phorbol esters (Traverse et al., 1993). Stimulation of Raf-1 kinase activity by tyrosine phosphorylation has been demonstrated in both vertebrate and invertebrate cells. Tyrosine phosphatase treatment of Raf-1 inactivates the protein kinase consistent with these prior reports. While the actual

tyrosine kinase directly responsible for phosphorylation of Raf-1 has not been identi®ed, coexpression with oncogenic Src results in the phosphorylation of tyrosines 340 and 341. The importance of tyrosine 340/341 phosphorylation for activation of Raf-1 by Ras or phorbol esters has not yet been de®nitively demonstrated. In fact a recent study using an in vitro activation system for Raf-1 suggested that tyrosine phosphorylation was not required for activation by oncogenic Ras (Stokoe and McCormick, 1997). Our in vivo results suggest the opposite conclusion. We ®nd a clear requirement for the presence of tyrosine at residues 340 and 341 in order for Ras to fully activate Raf-1. We interpret this result as evidence for a requirement for phosphorylation of these residues. It should be noted that in the case of Ras[V12], EGF and PMA stimulation, as well as Raf-CX, the loss of tyrosine 340/341 resulted in only a 50 ± 70% reduction in inducible activity. Since tyrosine 340/341 phosphorylation is not an absolute prerequisite for Raf-1 activation, except by vSrc, less sensitive assay conditions might readily overlook the e€ects of tyrosine phosphorylation on Raf-1 regulation. Therefore, our data supports previous observations that tyrosine phosphorylation of Raf-1 on 340 and 341 is a part of normal regulation by diverse stimuli. However, the contribution of phosphotyrosine to Raf-1 regulation in mitotically dividing cells appears to be additive with other mechanisms and not an all-or-nothing form of regulation. Adjacent to tyrosines 340/341 in Raf-1 is a conserved serine at position 338. It has recently been shown that this serine and possibly serine 339 as well are phosphorylated in cells expressing Ras[V12]. Raf-1 mutants lacking these serines are not activated in response to either oncogenic Ras[V12] or vSrc. It had not been previously shown whether or not this modi®cation was necessary for Raf-1 regulation by growth factors or phorbol esters. Our results indicate that the phosphorylation of serine 338 and possibly 339 is a clear requirement for full Raf-1 activation by oncogenes, growth factors and phorbol esters. Since the loss of either serines 338/339 or tyrosines 340/341 appears to have similar e€ects on Raf-1 regulation, it might be supposed that the phosphorylation of either set of residues might be redundant in function. Alternatively, the presence of a phosphoamino acid at either site might direct the phosphorylation of the adjacent site. We have previously demonstrated that mutations altering either phosphorylation site have di€ering e€ects (Diaz et al., 1997). Raf-1 mutants with acidic amino acid substitutions of the serines at 338/ 339 remain Ras[V12] and vSrc inducible, but are not directly activating as are acidic substitutions of the tyrosines at 340/341. One interpretation of these results is that phosphoserine at 338 or 339 enhances the recognition of tyrosine 340/341 by tyrosine kinases. While this may be correct, we have previously shown that alanine substitution of serine 338 inactivates the constitutive Raf-1[D340D341] mutant, suggesting that the serine and tyrosine phosphorylations can be functionally distinguished. It is also possible that the presence of phosphotyrosine at 340/341 enhances the serine phosphorylation of 338/339. In this study we further demonstrate a separate regulatory function for these two adjacent phosphorylation sites using the

Regulation of the Raf-1 protein kinase D Barnard et al

isoprenylated Raf-CX mutant. Loss of the tyrosine 340/341 site had only minor e€ects on the catalytic and transforming function of Raf-CX whereas loss of the serine 338/339 site signi®cantly impaired both. This result indicates that the phosphorylation of serine 338/ 339 remains at least partially independent of the phosphorylation of tyrosine 340/341. We hypothesize that the phosphorylation of serine 338/339 is an obligatory but intermediate step in Raf-1 activation, while the level of phosphorylation of tyrosines 340/341 determines the intensity of Raf-1 activation. It is important to attempt to put this regulatory information into a physiological context. Nearly every Raf reconstitution system (including our own) depends upon the overexpression of Raf, Ras and Src. Without overexpression, it is dicult to measure activity and the di€erences between mutant forms of the kinase. In cell based systems which provide a biological read out, such as ®broblast transformation, much less Raf activity is required to see an e€ect. For example, expression of the free catalytic domain of Raf-1 in ®broblasts is transforming and independent of tyrosine 340/341 (Morrison, 1996). However, the basal activity of the free catalytic domain of Raf-1 expressed in COS7 cells is approximately 10-fold less than similar amounts of Ras[V12] stimulated full-length Raf-1 (data not shown). Sewing and others have recently reported that di€erent levels of Raf activity can have signi®cantly di€erent e€ects on primary cells (Sewing et al., 1997; Woods et al., 1997). Low levels of Raf activation have a strong proliferative e€ect while high levels of Raf activity induce cellular senescence. Perhaps regulation of Raf-1 signal strength is accomplished by a balance of phosphorylations on the multiple serine and tyrosine residues located at positions 338 ± 341. A-Raf could be similarly modulated based upon the conservation of this sequence. BRaf however, is independent of tyrosine phosphorylation but does have a serine at a position analogous to S338. Clearly the multiple serine and tyrosine phosphorylation of Raf-1 residues 338 ± 341 represents an unusual regulatory mechanism. Homology modeling of the Raf-1 catalytic domain with the insulin receptor kinase catalytic domain locates the 338 ± 341 phosphorylation patch away from the catalytic cleft towards the rear of the `top' lobe of the kinase domain. This suggests that phosphorylated serine 338/339 and tyrosine 340/341 may not directly regulate the catalytic site but rather interact with other proteins or with other sites within Raf-1 itself. The conventional protein kinase C enzymes have been proposed to function distal to Ras as Raf kinase kinases, capable of activating Raf-1 directly by phosphorylating the activation loop on serine 497 or serine 499. Furthermore, we suggested in an earlier study that Raf-1 oligomers might transactivate through phosphorylation of resides within the activation loop of the kinase (Luo et al., 1996). To evaluate the potential regulatory role of activation loop phosphorylation, we generated Raf-1 mutants in which every potentially phosphorylated residue within the putative activation loop was replaced with an alanine. In every case, oncogenic Ras, Src and PMA were able to activate each mutant protein to nearly 30% of wild type activity, representing a ten-fold increase over basal activity. If one of these residues was an essential site of

phosphorylation, a near complete loss of inducible activity would have been expected. It should be noted that in general, a number of these mutants (A491, A494, A508) were less responsive to PMA than to Ras[V12] or vSrc. Evaluation of the multi-loop substitution mutant supported these results. Although the introduction of multiple amino acid substitutions within the catalytic site of Raf-1 would be expected to have structurally adverse consequences, this mutant was reasonably inducible by Ras[V12] (®vefold) and vSrc (ninefold) but poorly induced by PMA (5twofold). Activation of the alanine 497 and alanine 499 double mutant by PMA demonstrated that these particular residues are not activating sites of PKCa phosphorylation in COS-7 cells. Surprisingly, replacement of Raf-1 S499 with alanine resulted in a protein which was super inducible by PMA. This is consistent with a recently published report suggesting that PKCa phosphorylation of S499 may decrease activity of Raf1 (Schonwasser et al., 1998). These results suggest that induction of Raf-1 activity by either Ras or Src does not require phosphorylation of the activation loop. However, phorbol ester does appear to utilize a mechanism of Raf-1 regulation which is in part mediated through changes in the activation loop. Although we have failed to detect a positive role for phosphorylation of S497 or 499 in COS-7 cells, regulatory phosphorylation of these residues might be more critical in other cell types. We also observed that phorbol ester-induced Raf activation requires co-localization of Raf-1 with RasGTP at the plasma membrane and that the accumulation of Ras-GTP is induced by PMA. This e€ect could be a result of either increased guanine nucleotide exchange or a decrease in RasGAP activity, an e€ect previously documented in T-cells (Downward et al., 1990). Several reports have detailed the use of the RasN17 dominant negative mutant to demonstrate the Ras-independence of phorbol ester-dependent Raf regulation (Arai and Escobedo, 1996; Zou et al., 1996). However, RasN17 would have little e€ect if phorbol esters were increasing Ras-GTP by reducing RasGAP-stimulation of the Ras GTPase. Induction of a Ras-dependent pathway by phorbol esters could increase the activity of Raf protein phosphorylated by PKC. GTP loading of Ras stimulated by phorbol ester may in fact be the reason we were unable to observe clear evidence for activating phosphorylation of the Raf-1 activation loop by PKC. In summary, the regulation of the Raf-1 protein kinase by di€erent growth stimuli may be simpler than the complex image presented by a thorough review of the literature. Raf-1 is tightly regulated by multiple protein interactions and phosphorylation events which control the functions of the regulatory amino terminal regions of Raf-1 and of the carboxy-terminal catalytic domain. Our results suggest that one primary route to Raf-1 activation exists independent of what the stimulus might be. These common regulatory events include plasma membrane association with Ras-GTP and serine/tyrosine phosphorylation of the 338 ± 341 regulatory site (Figure 6). These events are a basic part of the complex regulatory pathway controlling the activity of the Raf kinases which may include other as yet unknown components. Phosphorylation of the catalytic activation loop does not appear to be a

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Regulation of the Raf-1 protein kinase D Barnard et al

1546

signi®cant mechanism for Raf-1 activation by Ras or Src in our COS-7 cell system but may play a role in phorbol ester regulation through conventional PKCs.

Materials and methods Cell culture COS-7 cells were maintained in DMEM medium supplemented with 10% calf serum in a 10% CO2 atmosphere. These cells were transfected by electroporation using a Biorad Gene Pulser (Chu et al., 1987). Cells to be stimulated with 10 ng/ml epidermal growth factor (EGF) or 50 nM phorbol 12-myristate 13-acetate (PMA) were ®rst grown overnight in low serum-containing medium (DMEM with 0.5% calf serum). Cells were harvested on ice after 5 min of stimulation. Rat1 ®broblasts were maintained in DMEM medium supplemented with 5% fetal calf serum and 5% calf serum. The focus forming ability of Raf-1 constructs was assayed by transfecting Rat1 cells with 10 mg of the desired DNA using a calcium phosphate transfection kit (GIBCO ± BRL). Cells were split 2 days after transfection, with 1/10th volume onto a plate containing medium supplemented with puromycin (2.5 mg/ml) and the remainder onto a 150 mm plate without drug selection. Foci were counted by staining the plates 3 weeks post-transfection. Puromycin-resistant colonies were also counted to ensure equivalent transfection eciencies for each DNA. Immunoblotting The expression levels of Raf proteins in transfected COS-7 were determined by lysing cells in bu€er A (20 mM Tris-Cl, pH 8.0, 2 mM EDTA, 50 mM b-glycerophosphate, 1 mM Na3VO4, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM PMSF, 1 mg/ml pepstatin A, 1 mg/ml leupeptin and 2.2 mg/ ml aprotinin) (Kyriakis et al., 1992). The lysate was cleared by centrifugation at 10 000 g for 10 min at 48C. Protein concentrations were determined by a BCA assay (Pierce Corp.). Equal amounts of lysate were analysed by SDS ± PAGE and transfer to PVDF membranes. Blots were probed with the 9E10 anti-myc antibody (Evan et al., 1985), (a gift from Robert Deschenes) and scanned with an Epson 1200C scanner with Bioimage IQ software to allow estimation of relative expression levels. DNA manipulation and mutagenesis Full-length Raf-1 was tagged at the amino terminus with a myc epitope (EQKLISEEDL) as previously described (Kolodziej and Young, 1991). All mutations were made using the Altered Sites Mutagenesis kit (Promega Corp.) and were con®rmed by DNA sequencing with Thermosequenase (Amersham). Full-length Raf-1 constructs were subcloned into the mammalian expression vector pcEXV-3 (Miller and Germain, 1986).

EGTA) to which 10 ml of GST-Mek (0.5 mg) and GST-Erk (1.1 mg) and 5 ml of Mg2+/ATP (50 mM MgCl2 and 0.8 mM ATP) were added to initiate the reaction. After 20 min at 308C, 10 ml of the supernatant were diluted into 40 ml of dilution bu€er containing 1 mM Na3VO4 and 1 mM DTT. Ten ml of this mixture was then assayed in duplicate by the addition of 38 ml of 0.4 mg/ml myelin basic protein in dilution bu€er with 1 mM Na3VO4, 1 mM DTT and 2 ml of 37.5 mM MgCl2 and 1.25 mM ATP containing 5 mCi [32P]ATP. After 10 min at 308C, 40 ml of the reaction mixture was spotted onto P81 ®lters, washed six times in 75 mM H3PO4, once in acetone, dried and quantitated by scintillation counting. For analysis by Western blotting (9E10 antibody, generously provided by Robert Deschenes), an equal volume of SDS ± PAGE sample bu€er was added to the remainder of the immunoprecipitates followed by SDS ± PAGE. The measured fold induction of Raf-1 was then normalized to the amount of Raf-1 protein in the reaction. The assay remained linear within the activity range of the positive controls used. Immunoblot analysis indicated that less than 1.0% of endogenous Mek non-speci®cally associated with the Sepharose beads during immunoprecipitation. This was not increased when myc-tagged Raf-1 was speci®cally precipitated. Guanine nucleotide loading of Ras COS-7 cells were serum-deprived overnight (18.5 h) in DMEM plus 0.5% dialyzed calf serum. Cells were depleted of phosphate by incubation for 3 h in phosphate-free media containing 0.5% dialyzed calf serum for 3 h. Cells were then metabolically labeled with 32Porthophosphate (126 mCi/ml) for approximately 5 h prior to addition of 0.8 ml/ml of DMSO, 10 ng/ml EGF or 50 nM PMA. After 5 min, the cells were placed on ice and lysed by the addition of ice cold lysis bu€er containing 25 mM Tris at pH 7.5, 150 mM NaCl, 16 mM MgCl2, 1% NP-40, 1 mM PMSF, 11 mg/ml aprotinin, 50 mg/ml leupeptin and 5 mg/ml Ras antibody (Y13-259, Oncogene Science) or 5 mg/ml Rat IgG as a negative control. Cells were scraped, aspirated through a 25G needle and centrifuged for 10 min at 14 000 g at 48C. After adjusting the concentration of NaCl to 0.5 M, additional Y13-259 antibody was added and the lysates mixed by rotation at 48C for 2 h. Forty ml of washed, 50% Gamma Bind G Plus beads were added to each sample and rotated for an additional 90 min followed by ®ve washes with lysis bu€er lacking antibody. Elution was performed in 30 ml of elution bu€er (2 mM EDTA, 0.2% SDS and 2 mM DTT) at 1008C for 3 min followed by centrifugation at 14 000 g for 5 min. Supernatants were stored at 7808C. Samples (15 ml) were spotted along with GDP/GTP standards onto polyethyleneimine cellulose plates coated with ¯uorescent indicator and chromatographically separated in 1.0 M KH2PO4, pH 3.5 bu€er (Vaillancourt et al., 1994). Labelled nucleotides were visualized and quantitated by phosphoimager analysis.

Raf kinase assays Total cell lysates were prepared by lysis in bu€er A. Cells were scraped, aspirated through a 25G needle and centrifuged at 10 000 g for 10 min at 48C. Equivalent amounts of protein were immunoprecipitated with 9E10 antibody prebound to GammaBind Plus Sepharose (Pharmacia) for 3 h at 48C. Complexes were collected by brief centrifugation. After three washes with lysis bu€er, the complexes were resuspended in 10 ml of dilution bu€er (50 mM Tris at pH 7.5, 5 mM MgCl2, 75 mM NaCl, 5 mM

Acknowledgements We would like to thank Susan MacDonald and Adele Filson for assistance with the Raf kinase protocol and Joseph Avruch and Wenyan Miao for helpful discussions. This study was supported by the American Cancer Society and a research grant from Eli Lilly Co.

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References Arai H and Escobedo JA. (1996). Mol. Pharmacol., 50, 522 ± 528. Barnard D, Diaz B, Hettich L, Chuang E, Zhang X, Avruch J and Marshall M. (1995). Oncogene, 10, 1283 ± 1290. Brtva TR, Drugan JK, Ghosh S, Terrell RS, Campbell-Burk S, Bell RM and Der CJ. (1995). J. Biol. Chem., 270, 9809 ± 9812. Burgering BM and Bos JL. (1995). Trends Biochem. Sci., 20, 18 ± 22. Cai H, Erhardt P, Troppmair J, Diaz-Meco MT, Sithanandam G, Rapp UR, Moscat J and Cooper GM. (1993). Mol. Cell. Biol., 13, 7645 ± 7651. Cai H, Smola U, Wixler V, Eisenmann-Tappe I, Diaz-Meco MT, Moscat J, Rapp U and Cooper GM. (1997). Mol. Cell. Biol., 17, 732 ± 741. Carroll MP and May WS. (1994). J. Biol. Chem., 269, 1249 ± 1256. Chu G, Hayakawa H and Berg P. (1987). Nucl. Acids Res., 15, 1311 ± 1326. Chuang E, Barnard D, Hettich L, Zhang X, Avruch J and Marshall M. (1994). Mol. Cell. Biol., 14, 5318 ± 5325. Clark GJ, Drugan JK, Rossman KL, Carpenter JW, RogersGraham K, Fu H, Der CJ and Campbell SL. (1997). J. Biol. Chem., 272, 20990 ± 20993. Dent P, Jelinek T, Morrison DK, Weber MJ and Sturgill TW. (1995a). Science, 268, 1902 ± 1906. Dent P, Reardon DB, Morrison DK and Sturgill TW. (1995b). Mol. Cell. Biol., 15, 4125 ± 4135. Diaz B, Barnard D, Filson A, Macdonald S, King A and Marshall M. (1997). Mol. Cell. Biol., 17, 4509 ± 4516. Downward J, Graves JD, Warne PH, Rayter S and Cantrell DA. (1990). Nature, 346, 719 ± 723. Evan GI, Lewis GK, Ramsay G and Bishop JM. (1985). Mol. Cell. Biol., 5, 3610 ± 3616. Fabian JR, Daar IO and Morrison DK. (1993). Mol. Cell. Biol., 13, 7170 ± 7179. Farrar MA, Alberola-Ila J and Perlmutter RM. (1996). Nature, 383, 178 ± 181. Ferrier AF, Lee M, Anderson WB, Benvenuto G, Morrison DK, Lowy DR and DeClue JE. (1997). J. Biol. Chem., 272, 2136 ± 2142. Heidecker G, Huleihel M, Cleveland JL, Kolch W, Beck TW, Lloyd P, Pawson T and Rapp UR. (1990). Mol. Cell. Biol., 10, 2503 ± 2512. Heidecker G, Kolch W, Morrison DK and Rapp UR. (1992). Adv. Cancer Res., 58, 53 ± 73. Hu C, Kariya K, Tamada M, Akasaka K, Shirouzu M, Yokoyama S and Kataoka T. (1995). J. Biol. Chem., 270, 30274 ± 30277. Jelinek T, Dent P, Sturgill TW and Weber MJ (1996). Mol. Cell. Biol., 16, 1027 ± 1034.

Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, Finkenzeller G, Marme D and Rapp UR. (1993). Nature, 364, 249 ± 252. Kolodziej PA and Young RA. (1991). Meth. Enzymol., 194, 508 ± 519. Kyriakis JM, App H, Zhang X-F, Banerjee P, Brautigan DL, Rapp UR and Avruch J. (1992). Nature, 358, 417 ± 421. Luo Z, Diaz B, Marshall MS and Avruch J. (1997). Mol. Cell. Biol., 17, 46 ± 53. Luo Z, Tzivion G, Belshaw PJ, Marshall MS and Avruch MS. (1996). Nature, 383, 181 ± 185. Luo ZJ, Zhang XF, Rapp U and Avruch J. (1995). J. Biol. Chem., 270, 23681 ± 23687. MacDonald SG, Crews CM, Wu L, Driller J, Clark R, Erikson RL and McCormick F. (1983). Mol. Cell. Biol., 13, 6615 ± 6620. Marais R, Light Y, Paterson HF and Marshall CJ. (1995). EMBO J., 14, 3136 ± 3145. Miller J and Germain RN. (1986). J. Exp. Med., 164, 1478 ± 1489. Morrison DK. (1996). Mol. Reprod. Devel., 42, 507 ± 514. Morrison DK and Cutler Jr RE. (1997). Curr. Opin. Cell. Biol., 9, 174 ± 179. Muslin AJ, Tanner JW, Allen PM and Shaw AS. (1996). Cell, 84, 889 ± 897. Rommel C, Radziwill G, Lovric J, Noeldeke J, Heinicke T, Jones D, Aitken A and Moelling K. (1996). Oncogene, 12, 609 ± 619. Schonwasser DC, Marais RM, Marshall CJ and Parker PJ. (1998). Mol. Cell. Biol., 18, 790 ± 798. Sewing A, Wiseman B, Lloyd AC and Land H. (1997). Mol. Cell. Biol., 17, 5588 ± 5597. Stokoe D and McCormick F. (1997). EMBO J., 16, 2384 ± 2396. Sundaram M and Han M. (1995). Cell, 83, 889 ± 901. Traverse S, Cohen P, Paterson H, Marshall C, Rapp U and Grand RJ. (1993). Oncogene, 8, 3175 ± 3181. Ueda Y, Hirai S, Osada S, Suzuki A, Mizuno K and Ohno S. (1996). J. Biol. Chem., 271, 23512 ± 23519. Vaillancourt RR, Gardner AM and Johnson GL. (1994). Mol. Cell. Biol., 14, 6522 ± 6530. Woods D, Parry D, Cherwinski H, Bosch E, Lees E and McMahon M. (1997). Mol. Cell. Biol., 17, 5598 ± 5611. Zhang Y, Yao B, Delikat S, Bayoumy S, Lin XH, Basu S, McGinley M, Chan-Hui PY, Lichenstein H and Kolesnick R. (1997). Cell, 89, 63 ± 72. Zou Y, Komuro I, Yamazaki T, Aikawa R, Kudoh S, Shiojima I, Hiroi Y, Mizuno T and Yazaki Y. (1996). J. Biol. Chem., 271, 33592 ± 33597.