RKIP downregulates B-Raf kinase activity in melanoma cancer cells

Abstract

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-MEK-ERK 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. 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 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...

Snail is a repressor of RKIP transcription in metastatic prostate cancer cells

“…The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. (Chatterjee et al, 2004;Park et al, 2005). Consistent with its demonstrated inhibitory effect on Raf and NF-kB signaling, we and others have shown that the expression levels of RKIP are downregulated in a number of tumors, including highly metastatic prostate, breast and colon cancer, hepatocellular carcinoma, melanomas and insulinomas (Fu et al, 2003;Chatterjee et al, 2004;Schuierer et al, 2004Schuierer et al, , 2006Zhang et al, 2004;Hegan et al, 2005;Al-Mulla et al, 2006;Lee et al, 2006).…”

Human melanoma cells expressing V600E B-RAF are susceptible to IGF1R targeting by small interfering RNAs

“…There was no evidence of similar responses in V600E transfectants, providing no clear basis for the observed survival inhibition. Similarly there was no detectable change in the phosphorylation status of p38 MAPK (not shown), and no change in levels of RAF kinase inhibitor protein (RKIP; Figure 4), a recently reported negative regulator of RAF-1 and B-RAF kinases (Park et al, 2005). However, six days after induction of IGF1R gene silencing there was clear suppression of Akt phosphorylation in all three transfected CHL-1 cell populations (Figure 4b).…”

Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells

“…Whether spindle checkpoint functions of RKIP are exerted through Raf-1 or B-Raf signaling remains uncertain since RKIP can inhibit either Raf-1, 42,43 or B-Raf signaling. 44 Depletion of Raf-1 was shown to rescue the RKIP depletion phenotype whereas Raf-1 overexpression mimicked the RKIP depletion phenotype, 13 suggesting that RKIP might exert its effects through Raf-1. In contrast, overexpression of a constitutively active B-Raf mutant (B-Raf V600E ) in melanoma cells delays mitotic progression by hyper-activation of the mitotic checkpoint.…”