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Vol. 19, Issue 7, 2907-2915, July 2008
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*Molecular Oncology Program, H. Lee Moffitt Cancer and Research Institute, Tampa, FL 33612; and Johnnie B. Byrd, Sr. Alzheimer's Center and Research Institute, Tampa, FL 33613
Submitted July 18, 2007;
Revised March 10, 2008;
Accepted April 16, 2008
Monitoring Editor: Yixian Zheng
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Morrison and Cutler, 1997). All three Raf proteins are detected in most cell types at various expression levels (Storm et al., 1990; Luckett et al., 2000; Wojnowski et al., 2000). Cell culture and gene knockout studies in mice suggest that the three Raf proteins have overlapping and unique regulatory functions (Hagemann and Rapp, 1999). Targeted disruption of B-Raf or C-Raf genes in mice is embryonic lethal, whereas A-Raf–/– mice die shortly after birth (Pritchard et al., 1996; Wojnowski et al., 1997, 1998, 2000). Because all Raf proteins are expressed in most embryonic and adult tissues, differences in their subcellular localization and protein interactions may account for their unique functions.
Of all three Rafs, B-Raf is the major activator of MEK (MAPK/ERK kinase; Catling et al., 1994; Jaiswal et al., 1994; Huser et al., 2001), even in cells where its expression is low (Reuter et al., 1995). This is consistent with B-Raf having a higher affinity than the other Rafs, for MEKs 1 and 2 (Papin et al., 1998) and being more efficient at phosphorylating MEKs under in vitro conditions (Marais et al., 1997; Papin et al., 1998). B-Raf contains structural features that may contribute to its being a potent MEK activator. In contrast to A- and C-Rafs, B-Raf possesses a "phosphomimetic" aspartate (D449) at the position equivalent to tyrosine-341 in C-Raf and is constitutively phosphorylated at residue S446, which is equivalent to S338 of C-Raf (Wellbrock et al., 2004). These differences contribute to B-Raf's high basal kinase activity and allow it to be "primed" for activation. In addition, B-Raf contains a long extended N-terminal region not present in the other Rafs, which may facilitate protein interactions with downstream components of the MAPK cascade.
The details of B-Raf activation have been best studied in the context of mitogen stimulation involving Ras interaction, membrane translocation, protein interaction, and site-specific phosphorylations (Mercer and Pritchard, 2003). Phosphorylation is a critical step for B-Raf activation. Ras-mediated activation of B-Raf requires the phosphorylation of conserved residues Thr599 and Ser602 within the activation segment of the kinase domain (Zhang and Guan, 2000). However, B-Raf can be activated independent of Ras mechanisms. For instance, in certain cell types, cyclic AMP and the small GTPase Rap1 have been shown to activate B-Raf (Ohtsuka et al., 1996; Vossler et al., 1997; Garcia et al., 2001). Recently, we demonstrated that B-Raf is activated at mitosis in Xenopus cycling egg extracts, which is essential for mitotic activation of the MAPK pathway (Borysov et al., 2006). In turn, mitotic roles for MAPK have been demonstrated for spindle formation, normal mitotic progression, and activation of the spindle assembly checkpoint (Minshull et al., 1994; Takenaka et al., 1997; Guadagno and Ferrell, 1998; Horne and Guadagno, 2003; Zhao and Chen, 2006). Thus, understanding how B-Raf activation is regulated at mitosis is integral to understanding its roles at mitosis.
In this study, we investigate the mechanism through which B-Raf activation is regulated at mitosis. Using the Xenopus egg extract system to recapitulate mitotic activation of the MAPK cascade, we provide evidence that supports a key role for Cdk1/cyclin B in regulating the activation of B-Raf at mitosis. We propose that Cdk1 targets a conserved N-terminal activating site in B-Raf, which is essential for mediating activation of the B-Raf/MEK/MAPK cascade during mitosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To cycle S-phase–arrested egg extracts into a stable M-phase state, glutathione-S-transferase (GST) nondegradable sea urchin 
90 cyclin B1 chimeric protein was added to a final concentration of 75–100 nM. Cytostatic factor (CSF)-arrested Xenopus egg extracts were prepared as described (Murray, 1991). The MEK specific inhibitor U0126 was used at a final concentration of 50 µM in Xenopus egg extracts as previously described (Horne and Guadagno, 2003). Kapil Bhalla generously provided Flavopiridol that was used in Xenopus egg extracts at 10 µM to inhibit mitotic Cdk1.
Immunoblot Analysis
Primary antibodies used for Western blots include: mouse monoclonal anti-phospho ERK (extracellular signal–regulated kinase; T202/Y204; Cell Signaling, Beverly, MA), mouse monoclonal myc-tag (Cell Signaling); rabbit polyclonal anti-B-Raf (Santa Cruz Biotechnology, Santa Cruz, CA; sc9002), anti-ERK2 and anti-phospho B-Raf (T599/S602; Santa Cruz); and a mouse monoclonal anti-Cdk1 (Calbiochem, La Jolla, CA). Rabbit anti-Xenopus MEK was prepared by Zymed Laboratories (South San Francisco, CA) against an N-terminal 16-amino acid sequence of Xenopus MEK1. Sheep anti-Xenopus cyclin B1 antibodies were a generous gift from James Maller (University of Colorado). Secondary antibodies to species-specific alkaline phosphatase–conjugated IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and anti-rabbit IgG (Sigma, St. Louis, MO) were detected with the CDP-Star chemiluminescence substrate (Roche Diagnostics, Alameda, CA).
B-Raf and Cdk1 Activity Assays
MEK kinase activity of B-Raf immunoprecipitates was measured in an in vitro–linked kinase assay as described (Guan et al., 2000). Briefly, purified B-Raf immunocomplexes were incubated with 1.0 µg of recombinant unactive GST-MEK1 (Upstate Biotechnology, Lake Placid, NY) in 25 µl of reaction buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM DTT, 5 mM NaF, 1 mM Na3VO4, and 0.1 mM ATP) for 20 min at room temperature. The reaction mix was briefly centrifugated to pellet immunocomplexes. Next, 20 µl of the supernatant was mixed with 10 µl of the reaction buffer containing 9.0 µg of recombinant unactive GST-ERK, and the reaction was continued for another 15 min. Finally, 3-µl aliquot of the reaction was mixed with 30 µl of reaction buffer containing 50 µg of myelin basic protein (MBP) and 5 µCi of [
-32P]ATP and incubated for another 10 min at room temperature. The kinase reaction was stopped with SDS sample buffer, separated on 15% SDS-PAGE, and transferred to a PVDF membrane. The levels of MBP phosphorylation was visualized by autoradiography and quantified by using ImageQuant software. Cdk1/cyclin B activity was measured in an in vitro histone H1 kinase assay as described (Borysov et al., 2006).
Immunodepletion and Immunoprecipitation
B-Raf immunodepletion and immunoprecipitation procedures were performed as described (Borysov et al., 2006). For coimmunoprecipitation studies, 20 µl of Xenopus egg extracts were mixed with 4 µg of anti-Cdk1 (Calbiochem) or 6 µg of anti-cyclin B1 antibodies. Extracts were diluted 1:1 in EB buffer (80 mM β-glycerol phosphate, pH 7.3, 15 mM MgCl2, 20 mM EGTA, 25 mM NaF, 1 mM Na3VO4) supplemented with 0.1% Triton X-100 and 10 µg/ml each pepstatin, leupeptin, and chymostatin. The immune complexes were recovered on protein A agarose beads (Sigma) and washed three times with EB buffer containing 0.1% Triton X-100.
Generation and Precipitation of Wild-Type and Mutant Xenopus B-Raf Proteins
Myc-tagged Xenopus B-Raf phosphorylation mutants were generated by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Site-directed mutations were confirmed by DNA sequencing of both strands performed by the Molecular Biology Core Facility at Moffitt Cancer Center. Wild-type (WT) and (S144A, S144D, S144E, S329A) mutant myc-B-Raf proteins were translated from in vitro–transcribed mRNAs in B-Raf–depleted CSF-arrested Xenopus egg extracts supplemented with rabbit reticulocyte lysate using protocols as previously described (Borysov et al., 2006). Levels of recombinant myc-B-Raf proteins, relative to B-Raf protein found in 1 µl of egg extract, were assessed by Western blot analysis. Generally, the recombinant myc-B-Raf protein mixtures were diluted 1:10 or 1:15 into S- or M-phase egg extracts. To isolate myc-tagged B-Raf proteins from egg extracts, 10–20-µl aliquots of egg extracts containing myc-B-Raf protein were diluted 1:20 in buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 25 mM β-glycerophosphate, 10 mM MgCl2, 10% glycerol, 5 mM EGTA, 1 mM DTT, 1 mM Na2VO4, 5 mM NaF, 0.1% Triton X-100, and 10 µg/ml each pepstatin, leupeptin, and chymostatin) and incubated on ice with myc-tag monoclonal antibodies (Cell Signaling) for 2 h. Protein A Sepharose beads were added, and incubation was continued for another 12–16 h with gentle inversion at 4°C. Immunocomplexes were washed twice with the same buffer and twice with the reaction buffer (25 mM HEPES, pH 7.5, 25 mM β-glycerophosphate, 10 mM MgCl2, 5 mM EGTA, 1 mM DTT, 1 mM Na2VO4, and 5 mM NaF) before accessing by Western analysis or applying to in vitro kinase assays (see below).
In Vitro B-Raf Phosphorylation Assays
Purified human (His)6-B-Raf (Upstate Biotechnology, Lake Placid, NY) was incubated in the absence or presence of 100 U of active recombinant Cdk1/cyclin B (New England Biolabs, Beverly, MA) in 30 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.1 mM ATP, and 0.01% Brij 35) with radioactive [-32P]ATP (5 µCi/reaction) for 30 min at 30°C. Reactions were stopped by the addition of SDS sample buffer and heating at 95°C for 5 min and separated by SDS-PAGE, and B-Raf phosphorylation was visualized by autoradiography. For experiments in Figure 6B, recombinant Xenopus kinase-dead myc-tagged B-Raf proteins were immunoprecipitated (IPed) from S-phase extracts and subjected to in vitro kinase reactions with 100 U of active Cdk1/cyclin B complexes as described above.
Phosphatase Treatment of B-Raf Immunoprecipitates
To dephosphorylated B-Raf IPed complexes, 50 U of recombinant lambda protein phosphatase (Upstate Biotechnology) was added to a reaction mixture containing 50 µl of phosphatase buffer as previously described (Borysov et al., 2006). B-Raf immunoprecipitates (±phosphatase treatment) were washed three times with 40 volumes of buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2) containing phosphatase inhibitors (25 mM NaF and 10 mM Na3VO4). B-Raf immunoprecipitates were analyzed either by Western blots or an in vitro–linked kinase assay to measure B-Raf–associated kinase activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Part of this phosphorylation stems from a negative feedback loop involving ERK to regulate the inactivation of B-Raf. However, the mechanism responsible for mediating the activation of B-Raf during mitosis was not addressed. On the basis of the importance of phosphorylation in regulating the activity of Raf kinases (Chong et al., 2003), we postulated that it might also play a role in regulating mitotic activation of B-Raf. Therefore, we isolated active B-Raf complexes from Xenopus M-phase egg extracts and incubated them with lambda protein phosphatase before measuring B-Raf activity by an in vitro–linked kinase assay. The results show that dephosphorylation of mitotic B-Raf abolished its kinase activity (Figure 1). In addition, the basal activity of B-Raf isolated from S-phase extracts was also sensitive to phosphatase treatment. This may reflect the dependence of B-Raf basal activity on a constitutive phosphorylation within the N-region (Marais et al., 1997; Tran et al., 2005). Thus, our data indicate that phosphorylation is critical for regulating M-phase–specific activation of B-Raf.
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). Both of these residues are conserved in Xenopus B-Raf (Thr-633 and Ser-636, respectively) and therefore we asked whether they played a role during mitotic activation of B-Raf. Briefly, recombinant WT or (T633A/S636A) mutant myc epitope–tagged B-Raf proteins were added to a B-Raf–depleted S-phase Xenopus egg extract that was then cycled into a stable mitotic state with nondegradable cyclin B. The myc-tagged B-Raf WT or (T633A/S636A) mutant proteins were isolated from S- and M-phase extracts to assess for B-Raf kinase activity by an in vitro–linked kinase assay. From this analysis it appears that the absence of both phosphorylation sites had little effect on mitotic activation of B-Raf compared with WT myc-B-Raf (Figure 2A). Next, we tested whether the nonphosphorylatable myc-B-Raf mutant could rescue MAPK activation at mitosis in B-Raf–depleted Xenopus egg extracts. The B-Raf–depleted extracts were supplemented with buffer, WT, or nonphosphorylatable (T633A/S636A) B-Raf and cycled into mitosis with nondegradable cyclin B. The depletion of B-Raf blocked mitotic activation of MAPK, which was rescued by the addition of WT B-Raf protein as shown previously (Borysov et al., 2006). Similar to WT, the nonphosphorylatable B-Raf (T633A/S636A) mutant was able to restored MAPK activation in M-phase Xenopus egg extracts (Figure 2B). Taken together, our data indicate that the conserved phosphorylation sites Thr-633 and Ser-636 are not required for mitotic activation of the B-Raf/MEK/MAPK cascade.
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) or partial activation. In contrast, B-Raf activity was markedly increased at M-phase by 70–80 min after the addition of nondegradable cyclin B (Figure 3A). Therefore, we conclude that Cdk1 activation (40–50 min) precedes the mitotic activation of B-Raf (70 min). Consistent with this, previous results in Xenopus cycling extracts show sequential activation of Cdk1, B-Raf, and MAPK during mitosis (Borysov et al., 2006). Is Cdk1/cyclin B activity necessary for maintenance of B-Raf activity during mitosis? To address this question, M-phase–arrested extracts were treated with Flavopiridol, a Cdk-specific inhibitor (Shapiro, 2004). Abolishing Cdk1 activity in M-phase–arrested extracts led to a substantial reduction of B-Raf activity (Figure 3B), suggesting that the enzymatic activity of Cdk1/cyclin B is required to regulate B-Raf activity during mitosis. Together, these results indicate that Cdk1/cyclin B triggers (directly or indirectly) mitotic activation of B-Raf.
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-32P]ATP. Reaction products were separated by SDS-PAGE, electrotransferred onto a PDVF membrane, and detected by autoradiography. A modest amount of B-Raf 32P-labeling was detected without the addition of Cdk1, which is probably due to autophosphorylation (Figure 5A). Importantly, 32P-labeling of B-Raf substantially increased in the presence of active Cdk1, suggesting that Cdk1 can directly mediate phosphorylation of B-Raf under in vitro conditions.
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Cdk1 Directly Phosphorylates Xenopus B-Raf In Vitro at a Conserved Serine-144 Residue
Cdk1 is a serine/threonine proline-directed protein kinase that preferentially phosphorylates substrates containing the consensus motif S/T-P-X-K/L (Nigg, 1991). The N-terminal region of Xenopus B-Raf contains two preferential Cdk1 consensus SPXK sites that are perfectly conserved among vertebrates (Figure 6A). Further comparison among other Raf family members shows that the two Cdk1 consensus sites are specific for B-Raf because they are not found in either Raf-1 (C-Raf) or A-Raf. Site-directed mutagenesis and in vitro Cdk1 kinase reactions were performed to test directly whether either of the serine residues (Ser-144 and Ser-328) within the two Cdk1 consensus sites are possible targets for Cdk1/cyclin B phosphorylation. Kinase-dead (KD) versions of WT and nonphosphorylatable B-Raf mutants (S144A, S328A, and S144A/S328A) were used for in vitro Cdk1 kinase reactions. The presence of active Cdk1 led to robust labeling of B-Raf KD over background levels (minus Cdk1) as shown in Figure 6B. Phosphorylation of B-Raf mutant S144A, but not S328A, was abolished in the presence of Cdk1. Thus, our data demonstrate that under in vitro conditions Ser-144 of Xenopus B-Raf is the primary target of Cdk1 phosphorylation.
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), we asked whether the B-Raf S144A mutant was capable of rescuing this effect when added back to B-Raf–depleted extracts. For this experiment, recombinant WT or S144A mutant B-Raf proteins were introduced into B-Raf–depleted extracts (Figure 7B) that were then driven into M-phase by the addition of recombinant nondegradable GST-cyclin B. Aliquots of extracts were collected every 20 min for analysis of MAPK activation by immunoblotting with phospho-MAPK antibodies. As expected, depletion of endogenous B-Raf abrogated activation of MAPK in M-phase–arrested extracts, which could be rescued by adding back recombinant WT myc-B-Raf (Figure 7C). In contrast, the addition of an equivalent amount of nonphosphorylatable S144A myc-B-Raf mutant failed to rescue MAPK activation at mitosis in B-Raf–depleted extracts. Taken together, our data suggest that phosphorylation of Ser-144 is critical for mitotic activation of B-Raf and enables B-Raf to activate the MAPK cascade in Xenopus M-phase egg extracts. Finally, we asked whether phosphorylation of the Ser-144 residue was sufficient for B-Raf activation. To test this, phospho-mimetic mutants (S144D and S144E) were generated by site-directed mutagenesis and assessed for kinase activity in Xenopus egg extracts. If phosphorylation of Ser-144 is sufficient, then the phospho-mimetic B-Raf mutant should be constitutively active in both S-phase and mitotic egg extracts. Our results showed, instead, that the kinase activities of the recombinant myc-tagged B-Raf S144D or S144E mutants were regulated at M-phase similarly to the WT myc-B-Raf (Figure 8A). Consistent with this, add-back of the B-Raf S144D mutant to Xenopus egg extracts depleted of endogenous B-Raf led to MAPK activation during mitosis but not S-phase (Figure 8B). Taken together, we conclude that phosphorylation of B-Raf at Ser-144 residue is required but not sufficient for mitotic activation of B-Raf.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, the mitotic mechanism for the activation of B-Raf was investigated in Xenopus egg extracts. We show here that a conserved Cdk1 consensus site (S144PQK) in Xenopus B-Raf is required for mediating its activation during mitosis. Consistent with this, Cdk1/cyclin B was found to associate with B-Raf in Xenopus cycling egg extracts during entry into mitosis. Furthermore, Cdk1 directly phosphorylated B-Raf under in vitro conditions, and mutation of Ser-144 to an alanine residue completely abolished the phosphorylation of B-Raf by Cdk1. Together, we propose that Cdk1 participates in the mitotic activation of the B-Raf/MEK/MAPK pathway through its direct phosphorylation of a novel activating site.
Conserved Thr-599 and Ser-602 Sites Are Dispensable for Mitotic B-Raf Activation
Phosphorylation plays an important role in regulating the activity and function of Raf kinases. B-Raf sites regulated by phosphorylation have been well characterized during mitogen stimulation of the Ras-Raf-MEK-ERK pathway in mammalian tissue culture cells. In this context, Ras-dependent phosphorylation of conserved residues Thr-599 and Ser-602 in human B-Raf is essential for its activation (Zhang and Guan, 2000, 2001). The results from our studies suggest that these two phosphorylation sites are dispensable for mitotic activation of B-Raf in Xenopus egg extracts (Figure 2A). In fact, the recombinant myc-B-Raf (T633A/S636A) mutant protein worked as well as the WT version of B-Raf in restoring mitotic activation of the MAPK cascade in Xenopus egg extracts depleted of endogenous B-Raf protein (Figure 2B). Thus, Thr-633 and Ser-636 residues of Xenopus B-Raf are dispensable for mitotic activation of the B-Raf/MEK/MAPK pathway, indicating that a Ras-independent signaling mechanism controls mitotic activation of B-Raf.
Biochemical Link between Cdk1/cyclin B and B-Raf
The results presented in this study indicate a biochemical link between Cdk1 and B-Raf activation during mitosis. First, we showed that addition of recombinant cyclin B to Xenopus S-phase egg extracts triggered sequential activation of Cdk1 and B-Raf (Figure 3A). This is consistent with previous studies that showed sequential activation of Cdk1, B-Raf, and p42 MAPK in Xenopus cycling egg extracts (Guadagno and Ferrell, 1998; Borysov et al., 2006). Likewise, blocking Cdk1 activity in M-phase Xenopus egg extracts resulted in a marked reduction of B-Raf activity (Figure 3B), further supporting that Cdk1 acts upstream of B-Raf. The association B-Raf and Cdk1/cyclin B complexes during M-phase in Xenopus egg extracts (Figure 4) indicates a direct regulatory link between the two protein kinases. Indeed, we showed that Cdk1 can mediate phosphorylation of B-Raf, both in M-phase Xenopus egg extracts (Figure 5B) and under in vitro conditions (Figure 5A). Furthermore, mutagenesis analysis demonstrated that Cdk1 phosphorylates B-Raf at a conserved Cdk1 preferential site (S144PQK; Figures 6). Therefore, B-Raf is a direct target of Cdk1/cyclin B. Conversely, cyclin B is phosphorylated at its cytoplasmic retention sequence by MAPK to allow for nuclear entry of Cdk1/cyclin B complexes at the onset of mitosis (Walsh et al., 2003), indicating that both signaling pathways can regulate each other during the cell cycle.
During meiosis of frog oocyte development, Cdk1 phosphorylates the germ cell–specific MEK kinase, c-Mos, at Ser-3 residue, and this was shown to be critical for the stabilization and activation of c-Mos (Castro et al., 2001; Yue and Ferrell, 2006). Subsequent to fertilization, c-Mos is targeted for degradation and remains essentially undetectable throughout the mitotic cell cycles (Watanabe et al., 1989, 1991; Borysov et al., 2006). We propose that during the mitotic cell cycle Cdk1 then plays a role to regulate B-Raf for transient activation of the MAPK cascade during mitosis. Whether B-Raf is regulated by Cdk1 at mitosis in somatic cells remains to be tested, but immunofluorescence studies in tissue culture cells indicate that Cdk1/cyclin B1 (Riabowol et al., 1989; Rattner et al., 1990; Jackman et al., 2003) and B-Raf (Borysova and Guadagno, unpublished results) localize to similar spindle structures during mitosis including the centrosomes and kinetochores. Importantly, physiological functions for B-Raf in regulating spindle formation and the spindle checkpoint arrest have been revealed in recent studies that target its expression by siRNA (Borysova and Guadagno, unpublished data) or ectopically express the constitutively active B-RafV600E mutant (Cui and Guadagno, 2007). In agreement with these findings, similar mitotic functions have been attributed to MAPK (Takenaka et al., 1997; Wang et al., 1997; Horne and Guadagno, 2003). Thus, we propose that mitotic regulation of B-Raf has important implications for coordinating mitotic events through the MAPK pathway.
B-Raf Activation at Mitosis Requires Phosphorylation at Ser-144
How does Cdk1 regulate B-Raf? On the basis of our data, we propose that Cdk1 directly phosphorylates a novel activating site on B-Raf. Two potential Cdk1 consensus phosphorylation sites (S144PQK and S328PSK) reside in B-Raf, but not Raf-1 or A-Raf (see Figure 6A). In this study, we show Cdk1 phosphorylates B-Raf at Ser-144, but not Ser-328, under in vitro conditions (Figure 6B). Mutation of Ser-144 demonstrates that it positively regulates mitotic activation of B-Raf (Figure 7A) and subsequent activation of the MAPK cascade (Figure 7C) in Xenopus egg extracts. In contrast, the Ser-328 residue does not appear to be required for B-Raf activation during mitosis (see Supplementary Figure S1). The conservation of the S144PQK motif in B-Raf, but not other Raf members, among vertebrates may help explain why B-Raf is required for controlling mitotic activation of the MAPK pathway in Xenopus egg extracts. Indeed, Raf-1, which lacks this Cdk1 preferential consensus site, is not essential for mitotic activation of MAPK cascade in Xenopus egg extracts (Yue and Ferrell, 2004; Borysov et al., 2006). Hence, our studies reveal a novel regulatory site in the N-terminal region of B-Raf that is essential for promoting its activation in the context of mitosis.
The molecular basis for how phosphorylation at this Cdk1 phosphorylation site contributes to mitotic activation of B-Raf remains to be addressed in future studies. Current models of Raf activation suggest that regulatory phosphorylations promote conformational changes that open the C-terminal kinase domain from the inhibitory N-terminal regulatory domain (Kolch, 2000). Therefore, we speculate that the presence of a negative charge at the Serine-144 residue may promote, at least in part, the aforementioned intramolecular changes necessary for B-Raf activation. Alternatively, phosphorylation at Ser-144 creates a binding site for a B-Raf-interacting protein that, in turn, promotes mitotic activation of B-Raf. In addition, the analysis of the phospho-mimetic B-Raf mutants (S144E or S144D) described in this study (Figure 8) indicates that phosphorylation of Ser-144 alone may not be sufficient to mediate B-Raf activation. Therefore, it is possible that other regulatory step(s) are required for mitotic activation of B-Raf. This would be consistent with our observations showing a delay between Cdk1 and B-Raf activation (Figure 2A).
In summary, we identify a new B-Raf regulatory site that facilitates mitotic activation of the B-Raf/MEK/MAPK pathway. A role for Cdk1/cyclin B as a mitotic activator of B-Raf opens a new perspective for understanding how B-Raf signaling is regulated during mitosis, which is quite distinct from its activation during cell cycle entry via Ras (Roovers and Assoian, 2000). The ability of B-Raf to respond to mitotic signals from Cdk1/cyclin B would allow for temporal and spatial regulation of MAPK signaling at spindle structures during M-phase progression.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Thomas Guadagno (Thomas.Guadagno{at}moffit.org)
Abbreviations used: CR, conserved regions; CSF, cytostatic factor; GST, glutathione S-transferase; IP, immunoprecipitation; IPed, immunoprecipitated; WT, wild type.
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