SHORTREPORT RKIPdownregulatesB-Rafkinaseactivityinmelanomacancercells Sungdae Park 1 , Miranda L Yeung 1 , Sandy Beach 1 , Janiel M Shields 2 and Kam C Yeung* ,1 1 Medical College of Ohio, Department of Biochemistry & Cancer Biology, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA; 2 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 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)asanegativeregulatoroftheRaf-MEK- ERK signaling pathway by specifically binding to the Raf-1isoform.WeshowherethatRKIPalsoantagonizes kinase activity of the B-Raf isoform. Yeast two-hybrid and coimmunoprecipitation experiments indicated that RKIPspecificallyinteractedwithB-Raf.Ectopicexpres- sion of RKIP antagonized the kinase activity of B-Raf. WeshowedthattheeffectsofRKIPonB-Raffunctions wereindependentofitsknowninhibitoryactiononRaf-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 playanimportantroleinmelanomaturmorgenesis. 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 ( phosphatidyl ethanolamine- 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 down- regulated 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 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 melano- cytes (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 muta- tions. 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 demon- strated 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 conse- quences 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 anchorage- independent 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 Received 23 July 2004; revised 3 November 2004; accepted 3 December 2004; published online 14 March 2005 *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: kyeung@mco.edu Oncogene (2005) 24, 3535–3540 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc