Oncogene (2005) 24, 3535–3540
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SHORT REPORT
RKIP downregulates B-Raf kinase activity in melanoma cancer cells Sungdae Park1, Miranda L Yeung1, Sandy Beach1, Janiel M Shields2 and Kam C Yeung*,1 1
Medical College of Ohio, Department of Biochemistry & Cancer Biology, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2
The Raf-MEK-ERK protein kinase cascade is a highly conserved signaling pathway that is pivotal in relaying environmental cues from the cell surface to the nucleus. Three Raf isoforms, which share great sequence and structure similarities, have been identified in mammalian cells. We have previously identified Raf kinase inhibitor protein (RKIP) as a negative regulator of the Raf-MEKERK signaling pathway by specifically binding to the Raf-1 isoform. We show here that RKIP also antagonizes kinase activity of the B-Raf isoform. Yeast two-hybrid and coimmunoprecipitation experiments indicated that RKIP specifically interacted with B-Raf. Ectopic expression of RKIP antagonized the kinase activity of B-Raf. We showed that the effects of RKIP on B-Raf functions were independent of its known inhibitory action on Raf-1. The expression levels of RKIP in melanoma cancer cell lines are low relative to primary melanocytes. Forced expression of RKIP partially reverted the oncogenic B-Raf kinase-transformed melanoma cancer cell line SK-Mel-28. The low expression of RKIP and its antagonistic action on B-Raf suggests that RKIP may play an important role in melanoma turmorgenesis. Oncogene (2005) 24, 3535–3540. doi:10.1038/sj.onc.1208435 Published online 14 March 2005 Keywords: B-Raf; melanomas; RKIP
RKIP (Raf kinase inhibitor protein) is a member of an evolutionarily conserved group of proteins called PEBP (phosphatidylethanolamine-binding protein). In our previous studies we identified RKIP as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase (MAPK) cascade initiated by Raf-1 (Yeung et al., 1999). Recently, we showed that the expression levels of RKIP were downregulated in prostate and breast carcinoma cell lines as well as in primary and metastasized prostate tumors (Chatterjee et al., 2004). To examine whether the downregulation of RKIP is a general phenomenon that can be applied to other types of cancer, we evaluated the RKIP protein expression levels in nine different
*Correspondence: KC Yeung, Department of Biochemistry & Cancer Biology, Medical College of Ohio, BHSB Rm 469, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA; E-mail:
[email protected] Received 23 July 2004; revised 3 November 2004; accepted 3 December 2004; published online 14 March 2005
melanoma-derived cancer cell lines by Western blot analysis using an anti-RKIP antibody. All melanoma cell lines, except for the PMWK cells, were derived from vertical growth phase tumors. The PMWK cells were derived from an early stage radial growth phase tumor. The SK-Mel-28 cells were from a primary tumor and the SK-Mel-24 cells were from a lymph node metastasis. Regardless of their origins, we observed a consistent reduction in RKIP protein expression levels in all melanoma cell lines as compared to primary melanocytes (Figure 1a). To verify the Ras/B-Raf mutational status of the cell lines used in out study, we sequenced the PCR amplified genomic DNA for missense mutations. We found that the reduction of RKIP expression occurred independently of the mutational status of N-Ras and B-Raf (Figure 1a). We next investigated at which level the expression of RKIP was regulated. Northern blot analysis demonstrated a significant decrease of RKIP mRNA in all melanoma cell lines studied when compared to the primary melanocytes (Figure 1b). Our results therefore suggest that the expression of RKIP is regulated at the level of RNA. To investigate the biological consequences of RKIP downregulation in melanoma, we restored the expression levels of RKIP by retroviral transduction in the melanoma cell line SK-Mel-28. One of the salient features of cancer cell lines like SK-Mel-28 is their ability to form colonies in soft agar. We transduced SK-Mel-28 cells with either RKIP or an empty vector control and measured the anchorageindependent growth of the cells in soft agar. We observed a more than 66% decrease in the number of colonies formed by cells transduced with RKIP as compared with an empty vector control (Figure 1c). We also observed a drastic reduction in the phosphorylation of MEK, a direct downstream substrate of the Raf family of protein kinases, in RKIP-transduced cells (Figure 1c, inset). It has been demonstrated that Ras and its directly targeted downstream Raf signaling pathway play an important role in human melanoma. Recently, activating mutations have been identified in the coding region of the BRAF genetic locus (Davies et al., 2002). SK-Mel-28 cells were derived from a primary melanoma and have been shown to harbor a gain-of-function mutation (V599E) in the BRAF allele (Davies et al., 2002). We have previously shown that knockdown of BRAFV599E expression downregulated the Raf-MEK-ERK signaling and completely reverted the transformed phenotype of SK-Mel-28 melanoma
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(manuscript submitted). B-Raf is a member of the Raf protein kinase family including A-Raf and Raf-1, which are structurally and functionally very similar (Mercer and Pritchard, 2003). Thus, since SK-Mel-28 cells are dependent on the continued expression of oncogenic B-Raf V599E and forced overexpression of RKIP significantly downregulated the Raf-MEK-ERK signaling and reduced the ability of these cells to grow in soft agar, we reasoned that RKIP, which is known to inhibit Raf-1 activity, may be inhibiting the kinase activity of B-Raf. To test this possibility, we examined whether RKIP interacts with B-Raf. We expressed both B-Raf and RKIP in Saccharomyces cerevisiae and studied their interaction using of the yeast two-hybrid system. B-Raf
was expressed as a fusion protein with the DNA-binding domain of Gal4. This domain enables B-Raf to enter the nucleus and bind to DNA containing Gal4-binding sites. RKIP was expressed as a fusion protein with the acidic activation domain (AAD) of VP16. The interaction between the two fusion proteins brings the AAD of VP16 into the proximity of the promoter and activates transcription of two Gal4-binding sites bearing His3 and b-gal reporter genes. Yeast expressing both Gal4-B-Raf and RKIP-AAD grew on a His dropout plate supplemented with 10 mM 3-aminotriazole (3-AT) and was b-gal positive. The interaction was specific, as expression of either fusion with NC2 or empty vector controls did not confer Histidine prototrophy or b-gal expression on the yeast (Figure 2a and data not shown). NC2 is a transcription factor composed of tightly associated a and b subunits (Mermelstein et al., 1996). In support of our yeast 2-hybrid data, the interaction of B-Raf with RKIP was also observed in mammalian cells by coimmunoprecipitation. RKIP and myc-tagged B-Raf were transiently coexpressed in COS-1 cells. Cell lysates were immunoprecipitated with a myc tag-specific antibody, and coimmunoprecipitation of RKIP was visualized by immunoblotting with an RKIP-specific antibody. Clearly detectable amounts of RKIP were coimmunoprecipitated with B-Raf (Figure 2b). The coimmunoprecipitation of RKIP with B-Raf is specific because RKIP was not found in the anti-myc immunoprecipitates when myc-Rab24 was used in the transfection (Figure 2b, right panel). Approximately equal amounts of myc-tagged proteins were immunoprecipitated from the transfected cells lysates (Figure 2b, upper panel). The interaction between B-Raf and RKIP was also observed between endogenous proteins. An B-Raf antiserum coimmunoprecipitated RKIP from PC12 cells lysate. The coimmunoprecipitation was specific as a negligible amount of RKIP was detected when antiNC2b antiserum was used (Figure 2c).
Figure 1 Expression of RKIP in melanoma cancer cells. (a) Comparison of RKIP expression in melanoma cancer cell lines and primary melanocytes by Western blot analysis. NP-40 extracted cell lysates (30 mg) were immunoblotted using polyclonal anti-RKIP antibodies. The same membrane was reblotted with an anti-actin serum (Sigma; A5444) as a loading control. The mutational status of B-Raf and Ras are indicated by ‘ þ ’ (constitutively active mutant) or ‘’ (wild type). (b) Comparison of RKIP expression in melanoma cancer cell lines and primary melanocytes by Northern blot analysis. Total RNA (20 mg) was resolved over a 1.3% formaldehyge–agarose gel, transferred to Hybond-N (Amersham) and hybridized to 32P-labeled cDNA of RKIP. The same membrane was re-hybridized to 32P-labeled cDNA of GAPDH as a loading control. (c) Ectopic expression of RKIP inhibits B-Raf-mediated anchorage-independent growth of SK-Mel-28 cells in soft agar. SK-Mel-28 cells stably overexpressing ectopic RKIP were assayed for their ability to proliferate in growth medium containing 0.3% agar and the formation of multicellular colonies photographed at 40 after staining with MTT and quantitated after 21 days from plates in triplicate (lower panel). Data shown are representative of three independent experiments and are expressed as mean colony number per plate 7s.d. Photomicrographs on the right of (c) are of the colonies shown at 400 magnification. Insets in (c, lower panel) Immunoblot analysis to examine the expression of HAtagged RKIP and MEK proteins Oncogene
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Figure 2 RKIP interacts with B-Raf. (a) RKIP interacts with B-Raf but not with control bait in the yeast two-hyrid system. The yeast two-hybrid analysis was performed as previously described (Yeung et al., 1999). (b) Coimmunoprecipitation of B-Raf with RKIP. CMV expression vector with cloned Rat RKIP cDNA was transfected into COS-1 cells with the myc-tagged B-Raf or myctagged Rab24 expression plasmids. Cells were lysed with TBST (20 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100). Whole-cell extracts were immunoprecipitated (IP) with anti-myc monoclonal antibody (9E10). Immunoprecipitates were washed two times with the lysis buffer and separated by SDS– PAGE, transferred to the membrane, and the expression levels of myc-tagged B-Raf or RKIP were monitored by immunoblotting (IB) with the indicated antibodies. Asterisks marked the proteins that crossreacted with the anti-RKIP Ab. (c) Coimmunoprecipitation of endogenous RKIP and B-Raf. Total cell lysate was prepared from cycling PC12 cells by sonication in PBS. In all, 100 mg of the cell lysate was immunoprecipitated with 4 mg of polyclonal antibodies against either B-Raf or NC2a proteins at 41C for 1 h. Immunoprecipitates were washed twice with TBST and the bound proteins were analyzed with the indicated Abs by IB
It has been demonstrated that MEK is the direct downstream substrate of all three Raf isoforms and RKIP is the physiological inhibitor of the Raf-1 isoform (Kolch, 2000). To test whether RKIP also interfered with the kinase activity of B-Raf, we examined the effect of RKIP expression on the activation phosphorylation of MEK. Previous studies have demonstrated that in mouse embryonic fibroblast (MEF) cells B-Raf is the major MEK kinase, while A-Raf shows low MEK activity (Huser et al., 2001; Mikula et al., 2001; Mercer et al., 2002). To rule out the possibility that the observed effect from RKIP on MEK1 phosphorylation was due to its reported inhibitory action on Raf-1 kinase, our studies were performed in Raf-1 knockout (KO) MEF cells (Mikula et al., 2001). As shown in Figure 3a, the MEF þ / þ parental cells expressed detectable Raf-1 expression, while the / cells do not. Both cell types expressed comparable levels of B-Raf. It has been reported that deletion of both alleles of the Raf-1 gene
has no effect on MEK1 kinase activity in MEF cells (Huser et al., 2001; Mikula et al., 2001). We ectopically expressed RKIP in Raf-1/ MEF cells by retroviral transduction. To measure MEK activation, a pool of viral-infected MEF cells were made quiescent by serum starvation and then stimulated by serum. We monitored the extent of MEK activation phosphorylation utilizing phospho-MEK-specific antibodies. Consistent with previously published reports, the activation of MEK in the KO cells was normal and comparable to that of the wild-type cells (data not shown). Infection of KO cells with an empty vector control did not affect the activation of MEK following serum stimulation. However, we observed a substantial reduction in MEK phosphorylation in cells infected with retrovirus expressing RKIP (Figure 3b). The effects of RKIP on MEK phosphorylation were specific as we did not observe any alteration in the levels of JNK phosphorylation after the RKIP-infected Raf-1/ MEF cells were treated with a known activator (sorbital) of the JNK signaling pathway (Figure 3c). Our results suggest that RKIP inhibits the kinase activity of B-Raf in MEF cells. To corroborate studies of overexpressing RKIP, we designed several siRNAs targeting the coding region of RKIP to downregulate the expression of endogenous RKIP RNA in Raf-1 KO cells. The designed siRNAs are highly specific, as they have no effect on the expression of other genes including the highly related RKIP paralogue, mPEBP2 (Figure 3d, and data not shown). Consistent with overexpression studies, downregulation of endogenous RKIP with siRNAs increased MEK phosphorylation in Raf-1 KO MEF cells. Importantly, the increase in MEK phosphorylation directly correlated with the effectiveness of the suppression (Figure 3d and e). To examine directly the effect of RKIP on B-Raf kinase activities, we transiently expressed myc-tagged BRaf in COS-1 cells in different combinations with Ras and RKIP as shown in Figure 3f. The myc-tagged B-Raf was subsequently purified by immunoprecipitation and its kinase activities were examined by in vitro kinase assay bacterial recombinant kinase inactive MEK as a substrate. As expected, cotransfection of Ras robustly activated using the kinase activities of B-Raf (Figure 3g). In line with studies with Raf-1/ MEF cells, the activation of B-Raf by Ras was diminished when cells were cotransfected with RKIP (Figure 3g, compare lanes 3 and 5). In addition to the inhibition of Rasinduced activation, RKIP also inhibited the basal activities of B-Raf (Figure 3g, compare lanes 2 and 4). To determine whether B-Raf kinase is a direct target of RKIP, we examined the effect of bacterial recombinant RKIP on the kinase activity of B-Raf kinase in an in vitro linked kinase assay reconstituted with immunoaffinity-purified B-Raf, and bacterial-purified recombinant ERK, MEK, and myelin basic protein (MBP) as substrates. The bacterial-purified MEK and ERK had very little kinase activities toward MBP in the absence of active B-Raf (Figure 3h, compare lanes 1 and 2). Upon the addition of active B-Raf, we observed a marked increase in MBP phosphorylation resulting from the Oncogene
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sequential activation of MEK and ERK (Figure 3h, compare lanes 1 and 2). In agreement with results of the transient transfection experiments, we observed reproducible inhibitory effects of recombinant RKIP on B-Raf activity (Figure 3h). Next, we examined the effect of RKIP on neurite outgrowth in PC12 cells. B-Raf is highly expressed in PC12 cells and activation of B-Raf kinase activities induces neurite outgrowth in PC12 cells (Cowley et al., 1994; Hagemann and Rapp, 1999). Unlike transient activation assays with Ras, the induction of neurite outgrowth accommodates the complexity of a cellular response induced by substantial activation of the MEKERK signaling pathway mediated by B-Raf. PC12 cells were transfected with kinase constitutively active B-Raf (B-RafED) in the presence or absence of an RKIP expression vector and neurite outgrowth was determined by microscopy. Productively transfected cells were visualized by coexpression of EGFP using fluorescence microscopy. Consistent with previously published reports, about 17% of the B-RafED-transfected PC12 possessed outgrowth of neurites that were at least two cell bodies or greater in length (Zhang and Guan, 2000).
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On the contrary, we observed a 71% reduction in the number of PC12 cells with neurites when RKIP was coexpressed (Figure 3f). Recently, we showed that the expression levels of RKIP were low in prostate and breast cancer cell lines (Chatterjee et al., 2004). In our present study, we show that the expression levels of RKIP are also low in nine different melanoma cell lines derived from both primary and metastasized tumors. The mechanism responsible for the downregulation of RKIP expression is not yet known. We showed by Northern blot analysis that the steady-state RKIP mRNA was decreased in melanoma. At present we do not know whether this decrease is a result of differences in message stability or transcription initiation. Activating mutations in B-Raf are found in a majority of human malignant melanomas (Davies et al., 2002). Importantly, it has been demonstrated that dysregulated activities of B-Raf are important for the maintenance of the transformed phenotype (Hingorani et al., 2003; Sumimoto et al., 2004; Wellbrock et al., 2004). In this study, we showed that restoration of RKIP expression levels in SK-Mel-28 cells harboring the B-Raf V599E-activating mutation yielded a phenotype
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that was reminiscent of knocking down the activated BRaf allele with specific siRNA. Our results therefore suggest that RKIP and B-Raf may act on the same pathway leading to cellular transformation. Alternatively, RKIP may act antagonistically by inhibiting the kinase activities of B-Raf. Several lines of evidence presented in this communication suggest that B-Raf is a direct target of RKIP. First, we showed that B-Raf interacted with RKIP. Second, expression of RKIP in COS-1 cells inhibited both the basal as well as activated B-Raf kinase activities. Third, we showed that the bacterial recombinant RKIP specifically inhibited the signaling cascade initiated by B-Raf in vitro. Finally, we showed that RKIP inhibited the B-Raf-mediated differentiation of PC12 cells. RKIP was initially identified as a physiologically negative regulator of Raf-1 kinase. Recently, it was demonstrated that oncogenic B-Raf mutants were capable of hetero-oligomerizing and transactivating Raf-1 kinase (Weber et al., 2001; Wan et al., 2004). It
is therefore possible that the observed B-Raf inhibition resulted from its known effects on Raf-1. Several observations suggest that this is a very unlikely event. According to their in vitro kinase activities, the more than 30 known tumor-associated B-Raf mutants can be grouped into three groups: high, intermediate, and low (Wan et al., 2004). Although most B-Raf mutants can activate Raf-1 kinase, only the low activity mutants require Raf-1 for B-Raf-mediated MEK activation phosphorylation. Both the V599E and the B-RafED are highly active and do not require Raf-1 for their activities (Wan et al., 2004). Furthermore, our results with the Raf-1 knockout MEF unequivocally demonstrated that RKIP was capable of inhibiting MEK kinase activities in a Raf-1 null genetic background. It has recently been shown that mutations in the RasRaf signaling pathway play a pivotal role in the progression of malignant melanoma. While approximately 9% of melanomas have activating N-RAS mutations, more than 60% of melanomas contain
Figure 3 RKIP interferes with the kinase activities of B-Raf. (a) Western analysis of B-Raf, Raf-1, and RKIP expression in Raf-1 þ / þ and / MEF. Whole-cell lysates (20 mg) were prepared as described previously (Yeung et al., 2000) and immunoblotted using polyconal anti-B-Raf, Raf-1, RKIP, and tubulin antibodies. (b) RKIP inhibits the serum-activated phosphorylation of MEK in Raf-1/ MEF. RKIP was ectopically expressed in Raf-1/ MEF cells by retrovirus infection as previously described (Chatterjee et al., 2004). RKIP or empty vector control-transduced Raf-1/ MEF were made quiescent by culturing overnight in 0.5% FBS. Quiescent cells were subsequently stimulated with 20% FBS for 15 min before harvesting. Whole-cell lysates were prepared as described above and immunoblotted using phosho-specific MEK polyclonal antibodies (Cell signaling). The same membrane was stripped and reblotted with an anti-MEK monoclonal antibody (Santa Cruz Biotech). (c) RKIP has no detectable effect on sorbitol-induced activation phosphorylation of JNK in Raf-1/ MEF. Asynchronous cycling Raf-1/ MEF cells were treated with Sorbital (0.5 M) for 30 min. Whole-cell lysates were prepared and immunoblotted using phospho-specific JNK polyclonal antibodies (Cell signaling). The same membrane was stripped and reblotted with an anti-JNK polyclonal antibody (Cell signaling) to visualize the total amount of JNK protein. (d) Specific downregulation of RKIP expression by RKIP siRNA expression vectors. siRNA expression vectors pLLsi186, pLLsi279, and pLLsi369, which targeted three different regions of the mouse RKIP-coding sequence, were transfected into Raf-1/ MEF together with either FLAG-tagged mouse RKIP or FLAG-tagged mouse RKIP paralogue, mpebp2 expression vectors. After 3 days, whole-cell lysates were prepared and immunoblotted using anti-FLAG and tubulin antibodies. The sense oligonucleotides encoding the mouse siRNA are: TAC TCT ACA CCC TGG TCC TCT TCA AGA GAG AGG ACC AGG GTG TAG AGT TTT TTT C (si186), TGG GTA ATG ACA TTA GCA GTT TCA AGA GAA CTG CTA ATG TCA TTA CCC TTT TTT C (si279), and TGG TGT ACG AGC AGG AAC AGT TCA AGA GAC TGT TCC TGC TCG TAC ACC TTT TTT C (si279). The sense and antisense oligonucleotides were annealed and cloned into the siRNA expression vector pLL3.7 (Rubinson et al., 2003). (e) Downregulation of RKIP expression in Raf-1/ MEF cells stimulates MEK phosphorylation. Raf-1/ MEF cells were cotransfected with the indicated siRNA and the HA-tagged MEK expression vectors. After 2 days, whole-cell lysates were prepared and immunoblotted using phosho-specific MEK polyclonal antibodies (Cell signaling) and anti-RKIP antibody. The same membrane was stripped and reblotted with an anti-MEK monoclonal antibody (Santa Cruz Biotech). Owing to the tag, the transfected version of the MEK can be identified from the endogenous MEK due to a difference in size. (f) Western blot analysis of transfected COS-1 cells. COS-1 cells were transfected in 6 cm plates with 2.0 mg DNA per well using DEAE-Dextran-chloroquine. At 24 h after transfection, cells were collected and lysed in TBST supplemented with protease and phosphatase inhibitors. Whole-cell lysates (20 mg) were immunoblotted with antibodies as indicated. (g) RKIP suppresses basal and activated B-Raf kinase activities. Cell lysates (30–50 mg) from COS-1 transfected with the indicated plasmid DNA were incubated with 2.5 mg 9E10 antibody at 41C for 1 h. Then, 20 ml protein G was added, and incubated for an additional 1 h at 41C. Immunoprecipitates were washed with TBST buffer twice, and with kinase buffer (20 mM Tris (pH 7.4), 20 mM KCl, 10 mM MgCl2) twice. For in vitro kinase assays, immunoprecipitates were incubated with recombinant kinase negative MEK-1 and 20 mM ATP in 30 ml kinase buffer at room temperature for 30 min. Samples were boiled for 5 min in SDS-sample loading buffer, resolved by 12% SDS–PAGE, and then transferred to PVDF membrane for Western blot analysis. The activity of myc-tagged B-Raf was measured by phosphorylation of kinase negative MEK. (h) Recombinant RKIP protein inhibits B-Raf kinase in vitro. COS-1 cells were transfected in 6 cm plates with 1.0 mg DNA using Fugene (Roche). At 24 h after transfection, cells were collected and lysed as described above. Cleared cell lysates were incubated with 5 mg 9E10 (for myc-B-Raf) or 12CA5 (for kinase negative Ha-B-Raf) at 41C for 1 h. Then, protein G beads were added, and incubated for another hour. Immunoprecipitates were washed with RIPA buffer twice, and with kinase buffer twice. The protein G beads bound B-Raf immunoprecipitates from one 6 cm transfected cells were aliquoted and were used for 10 kinase reactions. For in vitro linked kinase assays, immunoprecipitates were preincubated with 0.25 mM His-MEK-1, 0.5 mM His-ERK-2, 5 mM dephosphorylated MBP (Upstate), and increasing amounts of purified RKIP at room temperature for 10 min. Then, 2 mM ATP and 5 mCi [g32P]ATP were added, and incubated at room temperature for 30 min. Reactions were terminated by boiling in SDS-loading buffer, separated by 12% SDS– PAGE, blotted on PVDF membrane, and subsequently subjected to autoradiography. Phosphorylation was quantitated using a PhosphoImager. (i) RKIP inhibits B-Raf-mediated PC12 differentiation. PC12 cells were cotransfected with pcDNA-HA-B-RafED and the pCMV-GFP reporter construct using lipofectamine 2000 (Invitrogen). The indicated percentages represent the ratios between green fluorescent protein (GFP)-positive cells undergoing neurite outgrowth and the total number of GFP-positive transfected cells. The experiments were repeated in triplicate Oncogene
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oncogenic mutations in the B-Raf allele (Carr and Mackie, 1994; Davies et al., 2002). The occurrence of activated Ras and strongly activating mutations in B-Raf alleles in the same tumor are generally mutually exclusive, suggesting that B-Raf can be a main effector in oncogenesis. Indeed, knockdown experiments with B-Raf allele-specific siRNA demonstrated that mutated B-Raf was required for the later stage of melanoma progression (Hingorani et al., 2003; Wellbrock et al., 2004). Paradoxically, it has been found that B-Raf mutations usually occur early and that about 80% of premalignant nevi lesions contained V599E mutations without elevated levels of MEK-ERK activities (Mercer and Pritchard, 2003). These observations suggest that the mechanism by which MEK/ERK remains inactive in nevi in spite of the presence of oncogenic B-Raf may be that Raf inhibitors, such as RKIP, are not lost until a
later stage melanoma oncogenesis. In this communication, we showed that the expression levels of RKIP were low in melanoma cell lines and that restoration of RKIP expression reverted the oncogenic B-Raf kinase-transformed phenotype of the SK-Mel-28 cells. The low expression of RKIP and its antagonistic action on B-Raf suggests that RKIP may play an important role in determining the outcome of oncogenic B-Raf mutations in melanoma oncogenesis. Acknowledgements We thank K-L Guan and William Maltese for plasmid constructs, M Baccarini for the Raf-1/ MEF cells. We also thank John Sedivy for his continued support and Walter Kolch for sharing results on RKIP expression in melanoma before publication. This work was supported by NIH grants to KCY (R01 GM64767).
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