Abstract Establishing the mechanism by which S100B affects ERK and its downstream signaling provided insight into how S100B affects the progression of malignant melanoma and could aid in developing new pharmacological drugs. S100B is a 21.5 kDa symmetric homodimer that is highly conserved and expressed in a number of tissues and cell lines, including melanocytes. Generally, low levels of S100B have trophic effects, while higher levels can have dire consequences, as is the case in human malignant melanoma. S100B is an effective and widely used prognostic marker for malignant melanoma, with its increased level in serum being predictive of disease stage, increased recurrence, and low overall survival. More recently, S100B has been investigated as a potential contributor to cancer progression, which may be related to how it impacts cell signaling, including the MAPK pathway (BRAF-MEK-ERK). To further evaluate its significance, S100B knock-down clones were created from the WM115 melanoma cell line, and a positive correlation was found between S100B expression and cell viability, as measured by MTT assays. It was also discovered that cells with suppressed S100B expression showed significantly lower levels of ERK phosphorylation. Likewise, over-expression of S100B in the human melanoma cell line, 501-MEL, showed the reciprocal effect, with the introduction of high levels of S100B leading to increased cell viability and ERK phosphorylation. However, the phosphorylation status of ERK does not translate to all of its downstream targets. For example, increased RSK phosphorylation was observed in the S100B knock-down clones, and correspondingly, RSK phosphorylation was decreased with over-expression of S100B. Additionally, over-expression of a mutant S100B construct (E31A + E72A) that was incapable of binding calcium yielded neither effect, indicating that the effect of S100B on RSK phosphorylation was calcium-dependent. To determine if S100B interacted directly with RSK, pull-down assays were performed next. Consistent with the calcium-mutant data, RSK was detected in S100B pull-downs in the presence of calcium, but not in the presence of the calcium chelator EDTA. Changes in RSK localization was also observed, where RSK was enriched in the nucleus of WM115 cells when S100B was knocked down and diffuse in control cells. Together these data are consistent with a mechanism in which elevated S100B binds directly to RSK, preventing its phosphorylation by ERK and its subsequent translocation to the nucleus. Thus, the calcium-binding protein S100B affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously decreasing phosphorylated RSK. Together, these two effects of S100B on MAPK signaling could impact cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2164. doi:1538-7445.AM2012-2164