©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Mapping of the N-terminal Regulatory Domain in the Human Raf-1 Protein Kinase (*)

Yu-Hua Chow (1), Kevin Pumiglia (1)(§), Toni H. Jun (1), Paul Dent (2)(¶), Thomas W. Sturgill (2), Richard Jove (1)(**)

From the (1) Department of Microbiology and Immunology and the Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109 and (2) Howard Hughes Medical Institute, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.


INTRODUCTION

The 74-kDa cytoplasmic serine/threonine protein kinase, Raf-1, has a central role in transduction of proliferation and differentiation signals initiated by diverse extracellular factors (1, 2) . Activation of Raf-1, in response to extracellular stimuli transmitted through membrane tyrosine kinases and Ras, initiates activation of a kinase cascade involving the mitogen-activated protein kinases (MAP kinases,() also known as extracellular signal-regulated kinases or ERKs) (3, 4) . The intermediaries between Raf-1 and MAP kinases are the dual specificity tyrosine/serine/threonine kinases, MAP kinase kinases (MKKs or MEKs) (5, 6). Activated MAP kinases are responsible for regulating the functions of a variety of substrates that ultimately control gene expression and DNA synthesis (7, 8) . The findings that multiple isoforms of MKKs and MAP kinases exist suggest complex signaling networks that respond to diverse signals in different cell types (9-12).

Raf-1 is encoded in the c-raf-1 gene (13) , which belongs to a family of protooncogenes that includes two other active members, A-raf-1 (14) and B-raf(15) . A-raf-1 and B-raf genes are expressed specifically in urogenital and brain tissues, respectively, whereas the c-raf-1 gene is ubiquitously expressed in all tissues examined (16) . c-raf-1 was first identified in its tumorigenic form, v-raf, the oncogene of the acutely transforming retrovirus, murine sarcoma virus 3611 (17) . v-raf is expressed as a myristylated Gag-Raf fusion protein in which the N-terminal half of Raf-1 protein is replaced by Gag sequences. Activation of the transforming potential of Raf-1 has also been associated with N-terminal truncation and fusion to other sequences (18, 19, 20) . These observations suggest that the Raf-1 N-terminal sequences contain a negative regulatory domain, deletion of which results in deregulation of the C-terminal kinase domain.

Sequence comparisons among members of the raf family reveal the presence of three blocks of conserved sequences: conserved regions (CR) 1, 2, and 3 (21) . CR1 contains a cysteine-rich ``zinc finger'' motif, related to a similar structure in protein kinase C (22) , that has been shown to coordinate 2 mol of zinc (23) . CR2 consists of a stretch of 20 amino acids rich in serine and threonine residues; these are targets of phosphorylation by Raf-1 autokinase activity as well as by other serine/threonine-specific protein kinases, including protein kinase C (24, 25) . CR3, which spans the C-terminal half of Raf-1, comprises the catalytic domain. Interspersed among these conserved regions are sequences unique to Raf-1. Current models of Raf-1 regulation postulate that the N-terminal regulatory domain represses the transforming potential and kinase activity of the C-terminal catalytic domain (26) .

To fine map functional regions in the N-terminal regulatory domain of Raf-1 protein, we engineered a series of internal deletion mutants that together scan the entire N-terminal half of human Raf-1. These deletions were designed to systematically disrupt conserved as well as unique sequences in the N-terminal half of Raf-1 protein. Wild-type and mutant Raf proteins were expressed in a baculovirus/Sf9 insect cell system to examine effects of these mutations on regulation of Raf-1 kinase activity toward MKK, which is the only known physiological substrate of Raf-1 (27, 28, 29) . Significantly, results show that none of the Raf-1 deletion mutants display greatly increased levels of basal kinase activity. Previous studies demonstrated that Raf-1 protein expressed in insect cells can be synergistically activated by Src and Ras proteins expressed from co-infected recombinant baculoviruses (30) . Analysis of our deletion mutants revealed that, while Ras activates Raf-1 kinase through sequences in CR1 as previously reported (31, 32) , only the C-terminal half of Raf-1 containing CR3 is required for activation by Src. Together, these findings suggest that the function of the Raf-1 N-terminal domain is not merely to repress activity of the C-terminal kinase domain and that regulation of Raf-1 kinase activity is more complex than predicted from current models.


MATERIALS AND METHODS

Mutagenesis of c-raf-1 Gene

Plasmid p627 containing human c-raf-1 cDNA (13) was obtained from the American Type Culture Collection. Dodecameric XhoI linker (sequence CTCGAGCTCGAG) was ligated into restriction enzyme cleavage sites of c-raf-1 according to the linker insertion mutagenesis strategy described previously (33) . Locations of the unique XhoI insertion sites, which are entirely predictable, were identified by detailed restriction mapping. Using selected pairs of XhoI linker insertion mutants, a second generation of mutants was constructed by deleting intervening sequence between two insertion sites.

Recombinant Baculoviruses

A 2.3-kilobase Bsu36I/ApaI fragment of full-length c-raf-1 cDNA from p627 was subcloned into the baculovirus transfer vector, pBlueBac (Invitrogen). BamHI/NsiI fragments of the seven deletion mutants and a 2-kilobase EcoRI/EcoRI fragment encoding the truncation mutant 22W that lacks the N-terminal 305 amino acids (19) were subcloned into a similar baculovirus transfer vector, pBlueBacIII (Invitrogen). The recombinant transfer vectors were then cotransfected with linear wild-type Autographa californica nuclear polyhedrosis virus DNA into Sf9 insect cells. Recombinant baculoviruses containing Raf-coding sequences were screened for Raf protein expression by immunoblot analysis, and positive clones were plaque-purified using standard procedures (34) .

Antibodies

Rabbit polyclonal anti-Raf antibodies were prepared against a peptide corresponding to the extreme C-terminal 12 amino acids (sequence CTLTTSPRLPVF), which was coupled to purified human IgG with m-maleimidobenzoyl-N-hydroxysuccinimide ester (35). Monoclonal anti-Raf antibody was obtained from Transduction Laboratories. Monoclonal anti-Ras antibody, LA-069, was obtained from the NCI Repository (Quality Biotech). The murine hybridoma cell line producing monoclonal anti-Src antibody (monoclonal antibody 327) was kindly provided by J. S. Brugge (ARIAD Pharmaceuticals).

Immune Complex Kinase Assays

Sf9 cells were infected with wild-type baculovirus (A. californica nuclear polyhedrosis virus) or the indicated Raf recombinant viruses at a multiplicity of infection of 5, alone or in combination with c-Src or v-Ras recombinant viruses at multiplicities of infection of 15. Infected cells were harvested at 48-54 h postinfection and frozen at -80 °C until ready to be lysed. Cell pellets were lysed in modified insect lysis buffer (MILY buffer; 20 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM MgCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 1 µM leupeptin, 1 µM antipain, 0.1 µM aprotinin, and 5 mM sodium orthovanadate) (25, 32) for 15 min on ice. Clarified lysates (equivalent to 1 10 cells) were incubated with polyclonal anti-Raf antiserum for 1 h on ice, followed by collection of the immunoprecipitates with protein A-Sepharose (Pharmacia Biotech Inc.). Immune complexes were washed twice with MILY buffer and one time with kinase reaction buffer (25 mM HEPES, pH 7.4, 10 mM MgCl, 5 mM MnCl), and then incubated for 20 min at 25 °C with 200 ng of purified, polyhistidine-tagged MKK1 or MKK2 proteins (chemically inactivated by FSBA treatment) (36) in 30 µl of kinase reaction buffer supplemented with 1 mM dithiothreitol, 15 µM ATP, and 10 µCi [-P]ATP (Amersham Corp.). Reactions were terminated by addition of 4 SDS sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 50% glycerol, 0.002% bromphenol blue) and boiling for 5 min. Reaction products were then electrophoresed through 12% SDS-polyacrylamide gels, transferred electrophoretically onto nitrocellulose membranes, and autoradiographed. These nitrocellulose membranes were analyzed in a Molecular Dynamics PhosphorImager or an AMBIS 4000 radioisotope detector to directly quantify the phosphorylation levels. To confirm that equivalent amounts of Raf proteins were precipitated in the kinase reactions, these same membranes were then probed with anti-Raf antibodies, which were detected using anti-rabbit immunoglobulin followed by enhanced chemiluminescence (ECL; Amersham Corp.).


RESULTS

Generation and Expression of Raf-1 Mutants

To assess the contributions of Raf-1 N-terminal conserved and unique sequences (Fig. 1A) to regulation of its kinase activity, 24 XhoI linker insertion mutations were introduced at predicted enzyme cleavage sites in human c-raf-1 cDNA (Fig. 1B). From these linker insertion mutants, seven internal deletion mutants were derived in which segments between combinations of two linker insertion sites were removed. These in-frame deletions were designed to target blocks of conserved or unique sequences in the N-terminal half of Raf-1, as diagrammed in Fig. 1C. To analyze the kinase activities of these mutants, a baculovirus/insect cell system was used that allows expression of high levels of kinase-active Raf-1 (30) . Full-length Raf-1 and deletion mutants of the expected sizes were expressed at comparable levels from recombinant baculoviruses in infected Sf9 cells (Fig. 2). Raf-1 and deletion mutants d1 through d5 appear as multiple bands on immunoblots, whereas d6 and d7 appear as well defined single bands. Multiple species of Raf-1 have also been detected in mammalian cells and probably result from phosphorylation (21) . For analysis of kinase activity, an in vitro kinase assay was developed using two isoforms of MAP kinase kinase, MKK1 and MKK2, which are physiologically relevant substrates of Raf-1 (27, 28, 29) .


Figure 1: Structures of Raf proteins. Shown in panelA are the linear structures of cellular Raf-1 protein and the Raf sequences retained in v-Raf and the N-terminal truncation mutant, 22W. CRI, CRII, and CRIII, conserved regions 1, 2, and 3, respectively. The cross-hatched box in the v-Raf structure depicts the fused Gag sequence. The cysteine-rich, zinc finger motif is shown as a darkbar in CRI. The 12 amino acids at the extreme C terminus, corresponding to the antigen against which the anti-Raf antiserum was raised, are indicated by wave pattern-filled boxes. PanelB summarizes the positions of the 24 linker insertion mutations, and panelC diagrams the primary structures of the deletion mutants in which the deleted regions are indicated as solidblackboxes. Amino acid positions deleted for each mutant are as follows: Raf-d1 (18-52), Raf-d2 (53-132), Raf-d3 (53-205), Raf-d4 (133-180), Raf-d5 (207-228), Raf-d6 (250-275), and Raf-d7 (276-323).




Figure 2: Expression of Raf proteins in baculovirus-infected insect cells. Sf9 cells were infected with indicated recombinant baculoviruses, and proteins in whole-cell lysates were separated through a 7.5% SDS-polyacrylamide gel and blotted electrophoretically onto nitrocellulose membrane. The membrane was probed with anti-Raf serum and then with anti-rabbit horseradish peroxidase-conjugated secondary antibody, followed by detection with enhanced chemiluminescence (ECL) reagents. Mobilities of all the Raf proteins are consistent with the predicted molecular masses.



Characterization of in Vitro Kinase Activity and Substrate Specificity of Raf-1 Protein

Fig. 3A shows our standard in vitro kinase assay using purified MKK1 protein, irreversibly inactivated with FSBA, which possesses no kinase activity of its own (lane4). Significant levels of phosphorylation on MKK1 were seen in reactions containing anti-Raf immunoprecipitates prepared from lysates of Raf-1 virus-infected Sf9 cells (lane6), and this kinase activity could be competed out when the anti-Raf serum was preincubated with the corresponding synthetic peptide antigen (lane5). These results demonstrate that MKK1 is specifically phosphorylated by Raf-1 in this reaction. A kinetic analysis of Raf-d7 kinase activity on MKK1 shows that the in vitro reaction rate of Raf-d7, which has an elevated basal kinase activity (see below), remains linear for at least 45 min under our standard reaction conditions (Fig. 3B). For all the experiments described below, we used conditions similar to this assay and terminated the reactions at 20 min to ensure that the kinase activities of Raf proteins were analyzed in the linear range. Experiments were also performed using insect cell-expressed Raf340D, a Raf mutant with highly activated kinase (37) , showing that at least 30-fold elevation of the basal kinase activity of Raf-1 can be detected under our in vitro kinase reaction conditions (data not shown).


Figure 3: Characterization of Raf in vitro kinase activity. PanelA, anti-Raf immunoprecipitates (Raf IP) from lysates of Sf9 cells infected with wild-type (A. californica nuclear polyhedrosis virus) baculovirus (lanes 1-3) or with recombinant virus encoding Raf-1 (lanes 5-7) were subjected to an in vitro kinase reaction using purified, chemically inactivated MKK1 as substrate. A kinase reaction containing only MKK1 (lane4) confirms that the substrate lacks intrinsic kinase activity. Phosphorylated MKK1 is indicated on the autoradiograph. Controls for background activities include competition with the synthetic peptide antigen and reactions without added MKK. PanelB, kinetics of Raf-d7 in vitro kinase activity was analyzed. Aliquots of an in vitro kinase reaction mixture containing Raf-d7 immunoprecipitates were removed and the reactions were stopped at the indicated times. Control reactions with MKK1 or Raf-d7 alone were incubated for 45 min. The graph depicts quantification of radioactivity incorporated into MKK1 as detected by a Molecular Dynamics PhosphorImager.



In the same reaction conditions, Raf-1 kinase activities toward purified, FSBA-inactivated preparations of MKK1 and MKK2 were compared (Fig. 4). Raf-1, either at its basal level or activated by co-expressed c-Src or v-Ras proteins, consistently shows a substrate preference toward MKK1 over MKK2. MKK1 was therefore used as exogenous substrate in all of the subsequent in vitro kinase assays.


Figure 4: Substrate specificity of Raf kinases. Lysates of 3 10 Sf9 cells infected with recombinant Raf-1 baculovirus, alone or together with either c-Src or v-Ras baculovirus, were incubated with anti-Raf antiserum followed by collection of the immune complexes with protein A-Sepharose. Beads were washed, resuspended in kinase reaction buffer, and split into triplicates before being subjected to in vitro kinase assays using either 0 or 200 ng of MKK1 or MKK2 as substrate. Following separation by SDS-polyacrylamide gel electrophoresis, the reaction products were transferred to nitrocellulose and then visualized by autoradiography. The nitrocellulose filter was later probed with anti-Raf antibodies to ensure the presence of equal amounts of Raf proteins in the triplicates.



Basal Kinase Activities of Raf Deletion Mutants

Kinase activities of the various Raf mutants were analyzed to evaluate effects of the deletions on Raf-1 basal kinase activity (Fig. 5). In order to normalize the levels of Raf-1 proteins in the in vitro kinase assays, small amounts of immune serum were used so that the anti-Raf antibody would be saturated and immunoprecipitate similar amounts of Raf proteins, as confirmed by Western blotting analysis (Fig. 5B). In several independent experiments, Raf-d7 consistently showed a 2-3-fold higher kinase activity over wild-type Raf-1 (Fig. 5A). In contrast, Raf-d4, lacking the cysteine-rich zinc finger motif, showed only 10-40% the activity of Raf-1. The other deletion mutants revealed no consistently significant differences in kinase activity compared with Raf-1 (Fig. 5B). A kinase-inactive mutant (ATPM) of Raf-1 (25) was used as a negative control to estimate background activity in the in vitro kinase assays. Basal kinase activities of the Raf mutants were relatively unaffected by the presence or absence of 0.5 M NaCl, 0.1% SDS, or 5 mM sodium orthovanadate in the cell lysis buffer, and similar results were obtained using a monoclonal anti-Raf antibody for immunoprecipitation (data not shown). These results demonstrate that deletions that together span the length of the N-terminal regulatory domain do not lead to striking activation of Raf-1 kinase activity in this assay system.


Figure 5: Basal kinase activities of Raf deletion mutants. PanelA shows an autoradiograph of in vitro kinase reaction products from anti-Raf immunoprecipitates using lysates of Sf9 cells infected with viruses encoding wild-type Raf-1, various Raf deletion mutants, or a kinase-negative Raf mutant (ATPM). MKK1 was used as substrate. PanelB, anti-Raf immunoblot using the same nitrocellulose membrane from panelA was performed to confirm equivalent precipitation of Raf proteins in the kinase assays (Raf-d3 migrates close to the immunoglobulin heavy chain band, labeled Ig). PanelC, phosphorylation of MKK1 substrate was quantified by direct counting in an AMBIS 4000 radioisotope detector. The counts representing wild-type Raf-1 kinase activity are arbitrarily defined as 1, and the activities of the mutant Raf proteins are expressed as -fold activity in comparison with Raf-1. Statistically consistent data were obtained from multiple independent experiments, some of which are shown in Figs. 6C and 7C.



Activation of Mutant Raf Proteins by Src or Ras

Earlier studies showed that Raf-1 protein can be activated by co-expressed Src or Ras proteins in the baculovirus/insect cell system (30, 37, 38) . Utilizing a similar system and our panel of Raf-1 mutants, we assessed the regions in the N-terminal half of Raf-1 protein involved in regulation of Raf-1 by these upstream activators. We used constitutively active v-Ras (30) and chicken c-Src (34) , which is active because of lack of the negative-regulating Tyr kinase in insect cells, as the activators. Ras or Src protein was overexpressed with Raf proteins in co-infected Sf9 cells (Figs. 6B and 7B) using multiplicities of infection that ensure favorable activator:Raf ratios.

As shown in Fig. 6, A and C, Raf-d2, Raf-d3, and 22W lost the ability to be activated by Ras, whereas the other deletion mutants and Raf-1 showed 2-4-fold elevated kinase activities in response to Ras. Surprisingly, the basal kinase activity of 22W was consistently lower than that of wild-type Raf-1. The common residues deleted in Raf-d2, Raf-d3, and 22W are amino acids 53-132. Since this region is also required for interaction of Raf-1 with Ras (32, 39, 40) , these results are consistent with the conclusion that the physical interaction of Raf-1 and Ras is essential for Raf-1 activation by Ras.


Figure 6: Activation of mutant Raf proteins by Ras. Sf9 cells were singly infected with various recombinant Raf viruses or doubly infected with the Raf viruses and v-Ras virus as indicated. Kinase activities on MKK1 were analyzed in immune complex assays, and the autoradiograph is shown in panelA. Consistent high levels of Ras protein expressed in the doubly infected cells were confirmed by anti-Ras Western blot of whole-cell lysates shown in panelB. Western blots of whole-cell lysates were also performed to ensure comparable expression levels of the various Raf proteins in singly and doubly infected cells, and equivalent amounts of Raf proteins present in each kinase assay were further confirmed on the nitrocellulose membrane following autoradiography (not shown). PanelC, quantification of MKK1 phosphorylation data in the kinase assays detected with the AMBIS 4000. Kinase activities of the mutant Raf proteins and of various Raf proteins co-expressed with Ras are shown as -fold activity in comparison with wild-type Raf-1 basal activity. Results shown are the means with standard errors of assays done in three independent experiments.



On the other hand, the MKK1 phosphorylating activities of Raf-1, 22W, as well as all seven deletion mutants were activated 5-7-fold by co-expressed Src protein (Fig. 7, A and C). Variable phosphorylation of Raf proteins and immunoglobulin heavy chain, which might partly be due to co-precipitated Src kinase, was observed but did not correspond to the extent of MKK1 phosphorylation. In addition, some of the Raf phosphorylation could be due to autokinase activity enhanced by Src, as previously reported (30) . Raf-1 proteins, but not co-precipitated Src protein, account for the elevated MKK1 kinase activity, as evidenced by the low level of phosphorylation on MKK1 despite substantial immunoglobulin heavy chain phosphorylation in the case of ATPM/Src co-infection. These results demonstrate that the sequences required for activation by Src do not reside in the N-terminal 323 amino acids.


Figure 7: Activation of mutant Raf proteins by Src. Sf9 cells were singly infected with various recombinant Raf viruses or doubly infected with the Raf viruses and c-Src virus as indicated. PanelA, anti-Raf immunoprecipitates were analyzed for in vitro kinase activity on MKK1. Arrowheads denote phosphorylated Raf proteins, and Ig indicates phosphorylated immunoglobulin heavy chain. PanelB, anti-Src immunoblot of whole-cell lysates prepared from doubly infected cells. Comparable expression levels of the various Raf proteins in singly and doubly infected cells and equivalent amounts of Raf proteins present in each kinase assay were also confirmed by Western blotting (not shown). PanelC shows quantification of MKK1 phosphorylation data in the kinase assays analyzed by the AMBIS 4000. Kinase activities of the mutant Raf proteins and of various Raf proteins co-expressed with Src are shown as -fold activity in comparison with Raf-1 basal activity. Results shown are the means with standard errors of assays done in three independent experiments, except for ATPM, Raf-1, and d7, which are from five independent experiments.




DISCUSSION

Genetic analysis of v-Raf and other Raf-1 mutants showed that deletions in the N-terminal half, in some cases accompanied by fusion to unrelated sequences, activate the oncogenic potential of Raf-1 (19, 20) . These findings led to the hypothesis that the N-terminal half of Raf-1 contains a negative regulatory domain, which represses the catalytic domain contained in the C-terminal half of the protein. Direct evidence for specific regions in the N-terminal domain that demonstrably repress Raf-1 protein kinase activity, however, is lacking from previous studies. To explore the mechanism of Raf-1 kinase regulation by the N-terminal domain, we engineered a panel of internal deletion mutants designed to systematically dissect potential regulatory elements in the N-terminal half of Raf-1. Analysis of the Raf-1 deletion mutants revealed that, contrary to expectations from current models, the N-terminal domain does not function to simply repress the C-terminal kinase domain.

Significantly, targeted deletion of various conserved and unique regions in the N-terminal domain of Raf-1 protein did not substantially affect its basal kinase activity, with the notable exceptions of Raf-d4 and Raf-d7. The Raf-d4 mutant has a deletion that removes the zinc finger motif in CR1. This cysteine-rich motif in Raf-1 is of interest because homologous zinc finger structures in protein kinase C have been shown to be essential for phorbol ester binding and kinase activation and have been implicated in translocation of the kinase to the membrane (41, 42). While deletion of the zinc finger in Raf-d4 did result in partially defective kinase activity, a larger deletion in Raf-d3 encompassing the entire CR1 had little effect on the Raf-1 basal kinase activity. One possible explanation for this apparent contradiction is that the zinc finger might have a conformational or functional role only in the context of the intact CR1.

In the case of Raf-d7, deletion of amino acids in the unique sequence between CR2 and CR3 resulted in modestly (2-3-fold) elevated kinase activity. Interestingly, the deleted region of Raf-d7 contains a consensus MAP kinase phosphorylation sequence, Pro-X-Thr*-Pro (amino acids 308-311). MAP kinases have been shown to phosphorylate Raf-1 both in vitro(43) and in vivo in insulin-stimulated cells (44) . Consistent with phosphorylation events occurring in the region deleted in Raf-d7 is the finding that this mutant protein, unlike wild-type Raf-1, migrated as a single species upon electrophoresis through SDS-polyacrylamide gels. It has been suggested that phosphorylation of Raf-1 by MAP kinases may have an important physiological role, such as in an inhibitory feedback loop (8) . While any regulatory effect that might result from phosphorylation of this site remains to be demonstrated, our results on Raf-d7 support the suggestion that a negative regulatory element exists in this region between CR2 and the catalytic domain. Another deletion mutant, Raf-d6, which has the entire CR2 deleted, also appeared as a single species on Western blots. This conserved region is rich in serine and threonine residues that are potential phosphorylation sites, including Ser phosphorylated by protein kinase C (24) and Thr phosphorylated by Raf-1 autokinase activity (25). Because Raf-d6 does not show a significantly different kinase activity compared with wild-type Raf-1, however, no regulatory function of CR2 is required for Raf-1 kinase activity.

Our finding that none of the N-terminal deletion mutants possess greatly elevated kinase activity is surprising in light of the prediction, from earlier genetic studies on activation of Raf-1 oncogenic potential (19, 20) , that at least some of these mutants would have acquired deregulated kinase activity. For example, the 22W Raf-1 mutant, which resembles v-Raf in that it lacks most of the N-terminal regulatory domain, is oncogenic in rodent fibroblasts (19) . Deletion of CR2 also has been associated with activation of the Raf-1 transforming potential (18) . In earlier genetic studies, however, the kinase activities of transforming Raf mutants could not be measured reliably because an appropriate physiological substrate had not been identified yet. In the present study, we find that neither 22W nor Raf-d6, which contains a deletion of CR2, exhibits elevated kinase activity toward MKK. While the in vitro kinase assay that we employ here is highly specific and sensitive to a wide range of Raf-1 kinase activities, we cannot exclude the possibility that this assay is not an accurate measure of in vivo kinase activities. For instance, mutant Raf-1 proteins might be unstable in cell lysates or during incubation in the in vitro kinase assay; however, the fact that all of the deletion mutants examined here can be substantially activated by Src argues against this possibility. Alternatively, the kinase activities of Raf proteins expressed in insect cells might not reflect their activities in mammalian cells due to crucial differences in cellular regulatory molecules. Moreover, it remains possible that oncogenic activation of Raf-1 involves subtle changes in substrate specificity rather than elevated levels of kinase activity.

Earlier studies established that Ras binds with high affinity through its effector domain to the Raf-1 N-terminal domain in a GTP-dependent manner (39, 45, 46, 47, 48) . This physical interaction has been shown to be essential for Raf-1 activation by Ras (31, 32). Studies by Williams et al.(30) showed that co-expressing Raf-1 with either Ras or Src in insect cells led to moderate activation of Raf-1 kinase activity, and that maximal activation required both Src and Ras together. These findings raised the possibility of the presence of both Ras-dependent and Ras-independent mechanisms for Raf activation. Using our panel of mutants, we dissected the N-terminal half of Raf-1 for regions involved in responding to Ras and Src in an attempt to gain further insight into how these activators cooperate. Raf-1 N-terminal deletions that affect amino acids 53-132, which are required for Raf-1 to bind Ras in vitro and in vivo(32, 39, 40) , abolished activation of Raf-1 kinase by Ras. It is notable that no other region outside of CR1 is essential for Raf-1 activation by Ras. On the other hand, our finding that all seven deletion mutants and 22W are activable by Src provides direct evidence that the N-terminal domain is completely dispensable for activation of Raf-1 kinase by Src. Consistent with other recent studies (31, 32) , this finding demonstrates that Src can activate Raf-1 through a Ras-independent mechanism. The Src-response element in Raf-1 might involve the previously described C-terminal tyrosine residues, Tyr and Tyr, which have been identified as the major tyrosine phosphorylation sites of Raf-1 when co-expressed with activated tyrosine kinases in insect cells (37). Additional studies will be required to determine whether phosphorylation on these tyrosine residues of Raf-1 is directly responsible for its activation.

Recent reports have suggested that Ras functions in the activation of Raf-1 by recruiting it to the plasma membrane (49, 50) . In this context, our findings are consistent with a model in which the N-terminal domain has a role in mediating interactions with other molecules that regulate the kinase activity or subcellular localization of Raf-1 rather than simply repressing its kinase activity. Once localized to the membrane, other activation events, such as tyrosine phosphorylation of the C-terminal catalytic domain, may activate Raf-1 kinase activity. In the case of Raf-1 N-terminal mutants that do not bind Ras, overexpression of Src kinase together with these mutants in insect cells may overcome the requirement for Ras. Under normal circumstances in mammalian cells, however, signal transduction from membrane-associated tyrosine kinases may still depend on activated Ras to target Raf-1 to the membrane. This model predicts that Ras and tyrosine kinases normally function in concert for Raf-1 activation, a prediction that is consistent with classical experiments in mammalian cells (51, 52) as well as the finding that maximal activation of Raf-1 kinase in insect cells requires co-expression of both Ras and Src (30) .


FOOTNOTES

*
This research was supported by American Cancer Society Grant BE69D (to T. W. S.) and National Institutes of Health Grant CA55652 (to R. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Parke-Davis/University of Michigan Biotechnology Fellowship.

Fellow of the Juvenile Diabetes Foundation International.

**
To whom correspondence should be addressed: Dept. of Microbiology and Immunology, 6606 Medical Science II, University of Michigan Medical School, Ann Arbor, MI 48109-0620.

The abbreviations used are: MAP kinase, mitogen-activated protein kinase; MKK, MAP kinase kinase; MILY, modified insect lysis buffer; FSBA, 5`-p-fluorosulfonylbenzoyladenosine.


ACKNOWLEDGEMENTS

We thank C.-L. Yu for constructing the baculovirus transfer vector containing wild-type c-raf-1 cDNA, R. Nairn for suggesting the use of human IgG as carrier for peptide antigen, D. Retallack for assistance in preparing the anti-Raf antibody, N. Williams and T. Roberts for v-Ras baculovirus, J. Fabian and D. Morrison for the ATPM-Raf and Raf340D baculoviruses, V. Stanton and G. Cooper for the 22W plasmid DNA, and members of the laboratory for stimulating discussion.


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