Disruption of the 14-3-3 Binding Site within the B-Raf Kinase Domain Uncouples Catalytic Activity from PC12 Cell Differentiation*

A number of Raf-associated proteins have recently been identified, including members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3-3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulates Raf activity have not been fully established. We report that mutational disruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf biological activity. Expression of the isolated B-Raf catalytic domain (B-Rafcat) induces PC12 cell differentiation in the absence of nerve growth factor. By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was severely compromised for the induction of PC12 cell differentiation. Interestingly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro catalytic activity. InXenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activation of endogenous mitogen-activated protein kinase kinase and mitogen-activated protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant inhibited nerve growth factor-stimulated PC12 cell differentiation. We conclude that 14-3-3 interaction with the catalytic domain is not required for kinase activity per sebut is essential to couple B-Raf catalytic activity to downstream effector activation.

Members of the Raf family of serine/threonine protein kinases have been shown to be key mediators of growth factor signaling in diverse biological systems. A complex multistep process of Raf-1 activation by growth factors has begun to emerge. The activity of Raf proteins appears to be primarily dependent upon relief of the inhibitory interaction between the N-terminal and C-terminal domains (1)(2)(3)(4)(5). The relief of negative regulation appears to involve both phosphorylation and altered interactions with associated proteins. Initially, growth factor stimulated activation of the Ras protein induces a translocation of Raf-1 from the cytoplasm to the plasma membrane (6 -10). A series of subsequent activation events take place at the membrane (11,12), which may involve Raf-1 phosphorylation, association with additional proteins, and complex formation with downstream effectors MEK 1 and MAP kinase.
Over the last several years, a number of Raf-1-associated proteins have been identified, including members of the 14-3-3 family of proteins (13)(14)(15)(16)(17). The 14-3-3 proteins have been shown to bind to phosphoserine or phosphothreonine residues within the context of specific amino acid sequence motifs defined as RXX(S/T)XP (where X represents any amino acid and S/T represent either a serine or threonine residue) (18). It has been proposed that in an analogous manner to Src homology domain 2-containing proteins, the 14-3-3 family of proteins, by virtue of their ability to dimerize, may function as adapter proteins that link different intracellular signaling molecules in higher order complexes (18,19).
Mutational analysis of the Raf-1 14-3-3 binding sites has provided some insight into the effect of 14-3-3 binding upon Raf-1. Ablation of the Raf-1 N-terminal regulatory domain 14-3-3 binding site consensus sequence results in a constitutively active enzyme, implying an inhibitory role for 14-3-3 binding in the regulation of Raf-1 activation (25). However, 14-3-3 association with the N-terminal domain has also been shown to facilitate Ras-dependent Raf-1 activation (27), suggesting that 14-3-3 interaction maintains Raf-1 in an inactive, but activable, conformation. Consistent with this interpretation, 14-3-3 protein is displaced from the N-terminal domain of Raf-1 in response to Ras binding (29). Mutation of the 14-3-3 binding site within the Raf-1 C-terminal catalytic domain (18), by contrast, renders the mutant Raf-1 protein catalytically inactive (30). This finding suggests that interaction of 14-3-3 with the C-terminal catalytic domain is required for Raf-1 kinase activity.
Very little is known about the role of 14-3-3 in the regulation of the B-Raf activity. 14-3-3 proteins have been demonstrated to interact with B-Raf in vivo (31), and in a manner similar to Raf-1, these interactions appear to occur through separate Nand C-terminal interaction sites (32). A consensus 14-3-3 interaction motif is found centered around serine 728 (Ser-728) in the C-terminal catalytic domain (equivalent to Ser-621 in Raf-1). In this study, we have determined the effect of mutational perturbation of 14-3-3 interaction with the B-Raf catalytic domain. Our findings suggest that 14-3-3 binding to the catalytic domain is not necessary for B-Raf kinase activity per se but rather facilitates B-Raf biological activity through the coupling of B-Raf to downstream effector complexes.
Protein Expression in Xenopus Oocytes-Defolliculated Xenopus oocytes were microinjected with in vitro transcribed RNA (5-10 ng of RNA per embryo) encoding glutathione S-transferase (GST) fusion proteins as indicated. The extent of oocyte maturation (in the presence or absence of 2 g/ml progesterone (Sigma)) was monitored morphologically by the appearance of a white spot on the animal hemisphere, which is indicative of germinal vesicle breakdown. Pools of 20 injected oocytes were lysed in Nonidet P-40 lysis buffer (10 mM Tris, pH 7.5, 137 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM NaPP i , 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 0.2 units of aprotinin (Sigma)/ml, 0.1 mM NaVO 4 , and 25 M leupeptin) (34). For cyclin B protein injections, human ⌬87 cyclin B1 was expressed and purified from sf9 cells as described previously (36), and 50 -100 pg of cyclin protein was injected per oocyte. Subcellular fractionation of membrane and cytosolic components was performed as described previously (37).
PC12 Cell Culture, Transfection, and Differentiation-PC12 cells were grown in Dulbecco's modified Eagle's medium with 10% equine serum and 5% fetal calf serum. For transfections PC12 cells were plated on 35 mm Biocoat dishes (Becton Dickinson) at 30% confluency and transfected by the LipofectAMINE method (Life Technologies, Inc.). Transfected cells were cultured in low serum medium (2% equine serum ϩ 1% fetal bovine serum in Dulbecco's modified Eagle's medium) in order to maximize their differentiation response (38). NGF (Roche Molecular Biochemicals; final concentration, 100 ng/ml) was added in low serum medium. Transfected cells were visualized by expression of the co-transfected ␤-galactosidase gene and blue X-gal staining (Life Technologies, Inc.). NGF-and B-Rafcat-stimulated differentiation was scored by counting hypertrophic cells with one or more growth cone tipped neurites greater than 2 cell bodies in length. To prepare protein lysates, transfected cells were washed with cold phosphate-buffered saline and lysed in Nonidet P-40 lysis buffer. Cell debris was removed by centrifugation (12,000 ϫ g for 5 min at 4°C), and the resulting supernatant was assayed for total protein by Bradford assay (Pierce).
Glutathione-Sepharose Affinity Purification, Immunoprecipitation, Kinase Assays, and Western Blotting-GST fusion proteins were partially purified from PC12 and Xenopus cell lysates as described previously (33). The GST fusion proteins were then assayed for Raf activity in 20 mM Tris, pH 7.5, 10 mM MnCl 2 , 10 mM MgCl 2 , 25 mM ␤ glycerophosphate, 1 mM dithiothreitol, 50 M ATP, 10 Ci of [␥-32 P]ATP, and 100 ng of kinase-negative MEK for 10 min at 30°C (35,39). Radiolabeled phosphate incorporation into kinase-negative MEK was determined by PhosphorImager analysis (33). Western blotting to determine GST fusion protein levels was performed with a GST antibody (Santa Cruz Biotechnology). The activation status of MEK and MAP kinase was visualized using antibodies specific for the diphosphorylated, activated forms of each enzyme (New England Biolabs). Bound antibody was visualized by ECL (Amersham Pharmacia Biotech) and exposure to hyperfilm (Amersham Pharmacia Biotech). For 14-3-3 association experiments, the partially purified GST fusion proteins were washed twice in Nonidet P-40 lysis buffer containing 1 M NaCl, and associated 14-3-3 protein was detected after SDS-polyacrylamide gel electrophoresis and Western transfer with a 14-3-3␤ antibody (Santa Cruz Biotechnology). To visualize vRas after subcellular fractionation, Western blotting was performed with a Ras monoclonal antibody (Transduction Laboratories).

RESULTS
The amino acids residues encoding the Raf-1 C-terminal catalytic domain 14-3-3 recognition motif (RSASEP) are conserved in B-Raf and are centered around serine 728. We utilized the yeast two-hybrid protein association assay to determine whether mutation of serine 728 to an alanine residue (S728A) disrupted 14-3-3 protein interaction with the catalytic domain of B-Raf. The cDNAs encoding the C-terminal catalytic domain (B-Rafcat) and a mutant encoding the serine to alanine change (B-Rafcat S728A) were subcloned in-frame with the GAL4 DNA binding domain of the yeast two-hybrid pAS1 vector. A series of yeast transformations was performed with the GAL4 transactivation domain fusion pGAD vector encoding 14-3-3␤ or lamin (an unrelated, control protein). The S728A mutation disrupted association of 14-3-3␤ protein with the B-Raf catalytic domain as assessed by GAL4-dependent ␤-galactosidase expression (data not shown). No association of B-Rafcat or B-Rafcat S728A with lamin was detected.
To determine the signaling properties of the mutant B-Rafcat S728A protein in vivo, we utilized the Xenopus oocyte maturation model system. Because every oocyte expresses injected RNA transcripts, it is possible to analyze the effect of exogenous proteins on endogenous effector pathways. The isolated B-Raf catalytic domain and the S728A derivative were subcloned into the pXen vector (35) to engineer an in-frame N-terminal GST epitope tag, and in vitro transcribed RNA was prepared and injected into immature Xenopus oocytes. The GST fusion proteins were partially purified, and any associated 14-3-3 protein was analyzed by Western blotting with a 14-3-3␤-specific antiserum. As can be seen in Fig. 1, the S728A mutation abrogated 14-3-3 interaction with the catalytic domain of B-Raf, confirming the results of the yeast two-hybrid analysis. We next determined whether expression of GST B-Rafcat or GST B-Rafcat S728A could induce Xenopus oocyte maturation. Whereas GST B-Rafcat effectively induces maturation in Ͼ90% of the injected oocytes, the B-Rafcat S728A mutant protein failed to induce maturation after 20 h of culture ( Fig. 2A). The kinase activity of B-Raf was necessary for the induction of oocyte maturation because B-Rafcat K482M (a kinase deficient mutant) failed to induce maturation at any time point examined (data not shown). An analysis of the activation status of endogenous MEK and MAP kinase in oocytes expressing the GST-tagged proteins revealed that in contrast to GST B-Rafcat, the mutant GST B-Rafcat S728A had a significantly reduced ability to activate MEK and MAP kinase (Fig. 2B). GST B-Rafcat S728A induced MEK activation was barely detectable, although a modest activation of MAP kinase (as compared with progesterone or GST B-Rafcat) was reproducibly observed. This GST B-Rafcat S728A induced MAP kinase activation probably reflects an amplification of the weak MEK activation signal. No significant differences in subcellular localization were detected (Fig. 2C, left panel), and both GST B-Rafcat and GST B-Rafcat S728A proteins were found pre-dominantly in the cytosolic compartment. As a control for fractionation, ectopic vRas (Ras V12 ) was predominantly localized to the membrane fraction (Fig. 2C, right panel).
We next determined whether the failure to induce oocyte maturation was due to the S728A mutation rendering the B-Rafcat S728A protein catalytically inactive. Suprisingly, the GST B-Rafcat S728A mutant protein possessed 45.3% (Ϯ 7.1 S.E.; n ϭ 3) of the specific in vitro kinase activity of GST B-Rafcat (Fig. 3, upper panel) when the relative catalytic activities are normalized for GST fusion protein expression (Fig. 3, lower panel). Thus, despite possessing significant in vitro catalytic activity the GST B-Rafcat S728A protein failed to effectively activate downstream effectors in vivo (Fig. 2). Because equivalent levels of GST B-Rafcat and GST B-Rafcat S728A were expressed (Fig. 3, lower panel), the differential ability to exert a biological effect cannot be attributable to a decreased stability of the mutant protein. When the analogous 14-3-3 interaction site mutation was made in the Raf-1 enzyme (changing serine 621 to alanine) the Rafcat S621A protein was essentially inactive (7% Rafcat activity) consistent with an earlier report (40). In these experiments, the GST Rafcat and GST Rafcat S621A proteins were expressed to significantly higher levels than the GST B-Rafcat and GST B-Rafcat S728A proteins (Fig. 3, lower panel). These results are consistent with earlier findings (41) in which the isolated Rafcat, despite being oncogenic, has a lower specific activity than the isolated B-Rafcat.
Because the catalytic domain of B-Raf S728A appears to possess in vitro catalytic activity but fails to activate MEK in vivo or to elicit a biological response, we wished to determine whether the S728A mutation in the context of the full-length protein would exert a dominant inhibitory influence on progesterone-stimulated oocyte maturation. As can be seen in , and the extent of oocyte maturation was determined morphologically (by the appearance of a white spot on the animal hemisphere, which is indicative of germinal vesicle breakdown (GVBD)) after 20 h of culture. The histogram represents the combined data from three independent experiments, and the numbers above each column indicate the number of mature oocytes over the total number characterized. No maturation was observed in uninjected oocytes (control). B, protein lysates were prepared from pools of oocytes analyzed in A, and the activation status of MEK and MAP kinase was determined by Western blotting with antiserum specific for the activated (phosphorylated) forms of each enzyme (arrowheads). For negative and positive controls, protein lysates were prepared from immature (Imm) and progesterone matured (Prog) oocytes, respectively. Equivalent levels of GST B-Rafcat and GST B-Rafcat S728A were expressed. A representative Western blot is shown. C, hypotonic protein lysates were prepared from pools of oocytes expressing GST B-Rafcat, GST B-Rafcat S728A, or oncogenic Ras V12 (vRas) (14) and fractionated into membrane (M) and cytosolic (C) fractions. Following SDS-polyacrylamide gel electrophoresis, the B-Rafcat and B-Rafcat S728A proteins were detected with a GST antibody (left panel), and vRas was detected with a Ras monoclonal antibody (right panel). The expressed vRas protein migrates as a doublet. A representative experiment is shown. (Fig. 4B). To demonstrate that the expression of GST B-Rafcat S728A is not simply cytotoxic to oocytes, we stimulated maturation with cyclin B protein (42). Cyclin B protein induced maturation in either the absence (83% maturation, 60 oocytes scored) or presence of co-expressed GST B-Rafcat S728A (80% maturation, 60 oocytes scored). When protein lysates were prepared from these oocytes, cyclin B-mediated MAP kinase activation was fully induced in the GST B-Rafcat S728A expressing oocytes, although we did observed some attenuation of MEK activation (Fig. 4B). These findings demonstrate that expression of GST B-Raf S728A specifically attenuates progesterone-induced oocyte maturation.
To examine the effect of the B-Raf S728A mutation in a cellular context in which B-Raf is normally expressed, PC12 cells were transiently co-transfected with GST-tagged forms of B-Rafcat or B-Rafcat S728A and a ␤-galactosidase reporter construct. The transfected cells were scored for differentiation in response to expression of GST B-Rafcat or GST B-Raf S728A. Differentiation was scored as the number of blue staining (transfected) cells with neurite extensions of greater than 2 cell diameters. As can be seen in Fig. 5, expression of GST B-Rafcat induced PC12 cell differentiation. By contrast, expression of the 14-3-3 binding mutant GST B-Rafcat S728A protein had dramatically reduced ability to induce PC12 differentiation (4.7% differentiation Ϯ 1.9 S.E.; n ϭ 3). The expressed GST B-Rafcat and GST B-Rafcat S728A proteins were partially purified from PC12 cell lysate using glutathione-Sepharose. GST Western blotting confirmed that equivalent levels of each protein were expressed in these experiments (data not shown), suggesting that the differential effects on PC12 cell differentiation were due to the differential activities of B-Rafcat and B-Rafcat S728A.
To directly analyze the relative activity of the B-Rafcat S728A 14-3-3 binding site mutant, the GST B-Rafcat and GST B-Rafcat S728A proteins were partially purified from transfected PC12 cells, and their activity was measured in an in vitro kinase assay using kinase-negative MEK as an exogenous substrate. Similar to our findings with B-Raf proteins expressed in Xenopus oocytes, we found that the mutant GST B-Rafcat S728A protein did retain significant catalytic activity compared with GST B-Rafcat (38.4% activity Ϯ 3.5 S.E.; n ϭ 3) (Fig. 6A, compare lanes 2 and 3). It has been demonstrated that quantitative differences in Raf-induced signal strength can result in qualitatively different cellular responses (43,44). We therefore wished to determine whether the failure of GST B-Rafcat S728A to induce PC12 differentiation was attributable to the lower activity of the mutant protein. To address this issue, we performed a series of transient transfections in which we decreased the amount of GST B-Rafcat expression (by decreasing the amount of transfected plasmid DNA from 0.5 to 0.01 g) relative to a constant amount of GST B-Rafcat S728A

FIG. 3. Disruption of the C-terminal 14-3-3 interaction site has a differential effect on Raf-1 and B-Raf in vitro catalytic activity.
Immature Xenopus oocytes were microinjected with RNA encoding the indicated proteins. Protein lysates were prepared, the GST fusion proteins were partially purified, and their catalytic activity was determined using kinase-negative MEK as a substrate. The relative levels of MEK kinase activity are indicated relative to GST Rafcat (for Rafcat S621A) and B-Rafcat (for B-Rafcat S728A). Phosphorylated MEK protein was visualized and quantitated by PhosphorImager analysis after SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose membrane (arrowhead, upper panel). The levels of each GST fusion protein were determined by Western blot analysis of the same filter using GST antiserum (arrowheads, lower panel). The GST B-Rafcat proteins are slightly larger than the GST Rafcat proteins. A representative experiment is shown.

FIG. 4. B-Raf S728A expression antagonizes MEK activation in response to extracellular stimuli.
A, immature Xenopus oocytes were injected with RNA encoding GST B-Raf, GST B-Raf S728A, or a control RNA (GST alone), cultured for 12 h, and then stimulated with progesterone. The extent of oocyte maturation was assessed morphologically as described in the legend to Fig. 2. The histogram represents the combined data from three independent experiments, and the numbers above each column indicate the number of mature oocytes over the total number of injected oocytes characterized. B, protein lysates were prepared from immature (Imm) or progesterone-treated oocytes that had been injected with GST B-Raf or GST B-Raf S728A. Protein lysates were also prepared from cyclin B protein-injected oocytes or GST B-Raf S728A/cyclin B-co-injected oocytes. The activation status of MEK and MAP kinase in these lysates were determined by Western blot analysis using activation-specific antisera (arrowheads). Equivalent levels of GST B-Raf and GST B-Raf S728A were expressed as determined by Western blotting with GST antiserum (arrowhead, bottom panel). The injection of GST RNA (control) led to levels of GST expression comparable to those of the GST B-Raf and GST B-Raf S728A proteins (data not shown). A faint nonspecific anti-GST reactive band migrates slightly above the GST B-Raf and GST B-Raf S728A proteins and was observed in uninjected (Imm) and control RNA-injected oocytes. GVBD, germinal vesicle breakdown. plasmid DNA (0.5 g). When the levels of GST B-Rafcat activity were equivalent to or less than the activity of GST B-Rafcat S728A (Fig. 6A, compare lane 2 and lanes 4 -6), significant PC12 cell differentiation was induced (Fig. 6B). Under these same conditions, GST B-Rafcat S728A induced PC12 cell differentiation in only 2% of the transfected cells. We next tested whether the reduced specific activity of the GST B-Rafcat S728A mutant protein, rather than reduced total catalytic activity, was responsible for the dramatically reduced ability to induce PC12 cell differentiation. PC12 cells were transiently transfected with the isolated Raf-1 catalytic domain (GST Rafcat) or GST B-Rafcat S728A in order to compare their ability to induce differentiation. In these experiments, the GST Rafcat protein had a lower specific activity than GST B-Rafcat S728A (see also Fig. 3) when the in vitro catalytic activities are normalized for the amount of each GST fusion protein expressed (Fig. 6A, compare lanes 1 and 2). Rafcat induced significant PC12 cell differentiation (66% of the transfected cells were differentiated) under these conditions (Fig. 6B), indicating that the reduced ability of the GST B-Rafcat S728A protein to induce a biological response is not directly correlated with overall catalytic activity or with the reduced specific activity of the GST B-Rafcat S728A mutant protein.
To examine the effect of expression of the full-length B-Raf S728A protein on PC12 cell differentiation, PC12 cells were transiently co-transfected with a ␤-galactosidase reporter and either B-Raf or B-Raf S728A and subsequently stimulated with NGF. As can be seen in Fig. 7, expression of B-Raf slightly potentiated NGF-stimulated differentiation. By contrast, expression of the B-Raf S728A mutant attenuated NGF-stimulated differentiation of PC12 cells when compared with SR␣ vector-transfected control cells (53.1% inhibition of differentiation Ϯ 3.9 S.E.; n ϭ 3). DISCUSSION In this study, we have characterized the effects of disrupting 14-3-3 association with the C-terminal catalytic domain of B-Raf.
The B-Raf catalytic domain has one consensus 14-3-3 interaction motif centered around serine 728 (Ser-728), which is analogous to the C-terminal 14-3-3 site centered around Ser-621 in the Raf-1 isoform. We demonstrate that mutation of this serine to alanine (S728A) disrupts the association of 14-3-3 with the C-terminal catalytic domain of B-Raf (B-Rafcat S728A). Unlike the analogous Raf-1 mutant protein (Rafcat S621A), which does not efficiently phosphorylate MEK substrate, the B-Rafcat S728A protein possesses significant in vitro kinase activity (approximately 40 -50% of the specific activity of B-Rafcat). However, despite measurable in vitro catalytic activity, the biological activity of the B-Rafcat S728A protein was severely compromised, and B-Rafcat S728A had a dramatically reduced ability to induce PC12 cell differentiation.
There are several possible explanations for the reduced efficacy of the B-Rafcat S728A protein to induce PC12 cell differentiation. These include subthreshold signal strength, altered conformation of the catalytic domain, altered subcellular localization, and an impaired ability to form functional signaling complexes in vivo. It has been reported that changes in Raf signal strength can have profound biological consequences (43-  6. B-Rafcat S728A expressed in PC12 cells retains catalytic activity. Duplicate plates of PC12 cells were transiently transfected with a ␤-galactosidase reporter plasmid and the indicated amount of each indicated plasmid (g of DNA) and cultured for 3 days. A, one set of transfections was lysed, and the GST fusion proteins were partially purified and assayed for their in vitro catalytic activity using kinase-negative MEK as a substrate (arrowhead, upper panel). The relative levels of MEK kinase activity are indicated relative to GST B-Rafcat. No phosphorylation of MEK was detected in lysates prepared from SR␣ vector-transfected cells. The relative expression levels of the GST fusion proteins were determined by Western blot analysis using GST antiserum. The relative positions of the GST fusion proteins are indicated (arrowhead, lower panel). B, the second set of transfected cells was fixed and stained, and the extent of PC12 cell differentiation was assessed, as described for Fig. 5. The transfection conditions are indicated by the numbers below the histogram, which match the lane numbers and transfection conditions in A. A representative experiment is shown. 45). Because the B-Rafcat S728A mutant protein has a lower specific activity compared with B-Rafcat, it is possible that the compromised biological activity is a direct consequence of reduced signal strength. However, when the levels of B-Rafcat activity were titrated down to equal that of B-Rafcat S728A, significant PC12 cell differentiation still occurred (Fig. 6), suggesting that the loss of ability to induce PC12 cell differentiation is not be solely attributable to the reduced signal strength of B-Rafcat S728A. Moreover, when the level of B-Rafcat activity was less than 25% of the activity of B-Rafcat S728A, B-Rafcat still induced significant differentiation of PC12 cells (Fig. 6). Likewise, expression of Rafcat induced PC12 cell differentiation, despite having less overall in vitro kinase activity and a lower specific activity than the B-Rafcat S728A protein.
We conclude from these experiments that neither the reduction in total B-Raf catalytic activity nor the reduction in B-Rafcat specific activity can fully account for the compromised biological activity of the B-Rafcat S728A mutant protein. Interestingly, the role of 14-3-3 binding appears to be one of facilitation of B-Raf activity in vivo rather than obligatory, because we did observe some differentiation of PC12 cells when we transfected in a large amount of GST B-Rafcat S728A plasmid DNA (2 g), although the extent of differentiation was much less than that observed with an equivalent amount of the GST B-Rafcat plasmid DNA (data not shown). These data demonstrate that the GST B-Rafcat S728A is not catalytically inert in vivo. Consistent with these findings, B-Rafcat S728A stimulated a low level of activation of endogenous MEK and MAP kinase (Fig. 2B) and eventually induced some Xenopus oocyte maturation, although the extent and rate of maturation was much reduced compared with that induced by B-Rafcat. Our results from both Xenopus oocytes and PC12 cells suggest that the abrogation of 14-3-3 binding caused by the S728A mutation attenuates B-Rafcat biological activity disproportionally to the reduced specific ac-tivity of the B-Rafcat S728A enzyme. It is thus apparent that the catalytic potential of B-Rafcat S728A, as measured in vitro, does not does not reflect the biological activity of the mutant protein in vivo. The S728A mutation does not significantly alter the subcellular localization of the B-Rafcat protein, and both B-Rafcat and B-Rafcat S728A are found predominantly in the cytosol (Fig. 2C).
It has been suggested previously that the Raf-1 enzyme is hypersensitive to mutation of residues surrounding the C-terminal domain 14-3-3 binding site, Ser-621 (40). The approximately 50 -60% decrease in in vitro catalytic activity that we observed in the B-Rafcat S728A mutant may be due to a similar sensitivity. However, unlike Rafcat S621A, B-Rafcat S728A still retains significant in vitro kinase activity, suggesting that the conformation of the catalytic domain is not grossly perturbed. Moreover, our data suggest that the integrity of Ser-728 and interaction with 14-3-3 are not essential for catalytic activity per se, but rather contribute to B-Raf effector activation in vivo.
Our data support a model in which 14-3-3 binding to the C-terminal catalytic domain of B-Raf contributes to functional downstream signaling in vivo and is consistent with a genetically defined role for 14-3-3⑀ downstream of Drosophila Raf signaling (24). The inability of the B-Rafcat S728A protein to effectively activate downstream effectors (Fig. 2B) suggests that 14-3-3 interaction with the C-terminal kinase domain may participate in the coupling of B-Raf activity to MEK activation in vivo. The requirement for 14-3-3 to mediate B-Raf effector coupling is independent of 14-3-3 binding to the N-terminal regulatory domain, because this domain has been deleted in the B-Rafcat protein. The role of 14-3-3 in mediating B-Raf effector signaling could be to stabilize a productive B-Raf-MEK interaction. Given that B-Rafcat S728A can phosphorylate MEK in vitro, it would be anticipated to phosphorylate the endogenous MEK to approximately 40 -50% of the extent of B-Rafcat in vivo if it were present in a B-Rafcat S728A-MEK complex. However, B-RaFcat S728A did not induce significant MEK phosphorylation in vivo (Fig. 2B). Like the B-Raf S728A protein, mutant MEK proteins have been characterized that have in vitro catalytic activity but that fail to efficiently activate downstream effectors in vivo (46,47). It had been postulated that the discrepancy between in vivo and in vitro activity of these MEK proteins may be explained by the necessity for an additional associated protein, found in vivo, which interacts with the wild type but not the mutant MEK proteins to facilitate complex formation with MAP kinase. Subsequent work has identified the MP-1 adapter protein, which couples MEK and MAP kinase and facilitates their activation by B-Raf (48).
The applicability of our B-Raf findings for understanding the role of 14-3-3 interaction with the Raf-1 catalytic domain remains to be determined. A mutational substitution of alanine for serine (S621A) at the Raf-1 C-terminal 14-3-3 interaction site ablates 14-3-3 binding to the C-terminal domain of Raf-1 (18) and renders the mutant Raf-1 protein catalytically inactive (30). These findings suggest that phosphorylation of Ser-621 and interaction with 14-3-3 are essential for Raf-1 kinase activity. The requirement for 14-3-3 binding for Raf-1 catalytic activity has been further addressed through the use of synthetic phosphopeptides or detergent extraction to strip associated 14-3-3 away from in vivo activated Raf-1. These studies have yielded conflicting findings. When 14-3-3 was removed by detergent treatment, Raf-1 activity was comparable to that observed when 14-3-3 was present, suggesting that 14-3-3 association was not required for Raf-1 activity (25). In other studies, removal of 14-3-3 with detergent (49) or a competing phosphopeptide (50) resulted in a loss of Raf-1 catalytic activity. These conflicting results may be explained in part by the different in vitro assay conditions employed. In the first study, in which removal of 14-3-3 had no effect on Raf-1 catalytic activity, Raf-1 activity was measured directly using a kinasenegative MEK substrate. In the studies in which 14-3-3 appeared to be necessary for Raf-1 activity, Raf-1 activity was measured in an indirect coupled assay that required that Raf-1 activate purified MEK, which in turn then phosphorylated kinase-negative MAP kinase. It is possible that 14-3-3-depleted Raf-1 retains sufficient activity to phosphorylate MEK under some conditions (such as those typically employed in direct kinase assays). Our findings that B-Rafcat S728A retained catalytic activity when measured in a direct kinase assay in vitro support this interpretation. By contrast, when 14-3-3depleted Raf-1 activity is assayed in a coupled reaction, 14-3-3-depleted Raf-MEK complexes may fail to engage MAP kinase in a productive configuration, or the MEK-MAP kinase complex may not be engaged by the 14-3-3-depleted Raf-1. Because there is growing evidence from studies in both yeast and mammalian systems that elements of the MAP kinase signaling module are associated with "scaffolding" proteins (51), the data may be reconciled by proposing a scaffolding role for 14-3-3 in coupling Raf to MEK and MAP kinase, which is only apparent in vivo and under coupled in vitro assay conditions. No consensus 14-3-3 interaction motifs are present within MEK, MAP kinase, or the MP-1 protein sequences, and no interaction of 14-3-3 with MEK has been observed with yeast two-hybrid analyses (32), suggesting that any scaffolding effect would have to be mediated through nonconsensus 14-3-3 interaction motifs (52,53) or mediated indirectly through an additional protein.
Our observation that inhibition of 14-3-3 binding correlates with loss of B-Raf biological activity was made using the isolated catalytic domain of B-Raf, and it was possible that the loss of activity would not be relevant within the context of the full-length B-Raf protein. However, we also observed that expression of the S728A mutation in the full-length B-Raf protein attenuated NGF-stimulated PC12 cell differentiation (Fig. 7). Similarly, in the Xenopus oocyte system, we observed that expression of B-Raf S728A protein blocked progesterone-induced maturation. Moreover, a biochemical analysis of progesterone-treated oocytes revealed that the B-Raf S728A protein appeared to exert an inhibitory influence by blocking the activation of endogenous MEK and MAP kinase (Fig. 5). The B-Raf S728A protein may thus be acting in a dominant inhibitory manner in oocytes to titrate out positive activators of the endogenous MEK and MAP kinase enzymes in a manner similar to the effect of a dominant-negative Raf-1 protein (34).
It has been suggested that by virtue of their ability to dimerize, the 14-3-3 proteins may contribute to Raf-1 catalytic activity by forming an intramolecular bridge between the Raf-1 N-terminal regulatory domain and C-terminal catalytic domain. Addition of recombinant 14-3-3 to Raf-1 that had been stripped of 14-3-3 was able to reconstitute Raf activity only if 14-3-3 could dimerize (50). These findings were interpreted as a requirement for 14-3-3 dimers to interact with both the Nterminal and C-terminal Raf-1 domains to maintain the activated Raf-1 in an "open" conformation (50). Our results suggest an alternative role for 14-3-3 in the regulation of B-Raf catalytic activity. Rather than mediating an intramolecular bridge between the N-and C-terminal domains, our data would suggest that dimeric 14-3-3 molecules are necessary to couple B-Raf to downstream effector complexes.