The MAPK kinase MEK1 is a central component of the Extracellular signal-Regulated Kinase (ERK) cascade. Its proper function is required for the regulation of a variety of cellular processes. Recently, activating mutations of MEK1 were found in different cancers and shown to be the driver of cellular transformation in these cases. These mutations included mainly the substitutions of Lys57 to Asn (K57N) and Asp67 to Asn (D67N). In order to understand the mechanism of transformation by MEK1 mutants, we undertook to study their signaling properties. Transient overexpression of both mutants resulted in increased proliferation of cells. Western blot analysis revealed that K57N and to some extent D67N mutants had increased phosphorelation of both their activatory Ser (218 and 222) and also Ser298. Interestingly, the overexpressed mutants only marginally affected the activities of the downstream targets, indicating a possible ERK1/2-independent mechanism of transformation. Indeed, the D67N mutant exhibited different subcellular localization, and was much more abundant in the nucleus, regardless of stimulation. In addition, both mutants resulted in increased phosphorylation of Elongation Factor 2 (EF2). The aberrant cross-talk with other signaling pathways, the increased phosphorylations and the nuclear localization, are likely to contribute to carcinogenesis by these mutants.