Braf mutations are associated with high levels of phosphorylated RKIP in melanoma cell lines: Potential prognostic significance

BRAF proto-oncogene is a serine-threonine protein kinase that functions as an immediate downstream effector of RAS. Activated BRAF mutations are found at high frequency in melanoma, where they occur at a frequency of 50-70%. The mutation increases protein kinase activity, resulting in constitutive BRAF/MEK/ERK signaling. Raf kinase inhibitor protein (RKIP) is a inhibitor of the MAP kinase cascade because it dissipates the Raf-1/MEK interaction, thereby preventing activation of MEK by Raf-1 and downstream signal transduction. RKIP expression is decreased in melanoma and absent in melanoma metastases. We hypothesized that BRAF mutations, through their activation of PKCs, will result in the phosphorylation and inactivation of RKIP and potentiation of ERK 1/2 activation, metastasis, and drug resistance. We have examined a large panel of melanoma cell lines with no mutations and with BRAF mutations using a tissue microarray technology. The expression of both RKIP and phospho (p) RKIP was examined and protein expression was performed by IHC and analyzed. Our results revealed that the majority (75%) of RKIP expression was in its phosphorylated form in cell lines with BRAF mutations. We suggest that in melanoma cell lines with BRAF mutations, GRK2 is inactivated through its association with pRKIP and, thus, maintaining the activating signals mediated by GPCRs.