The ERK pathway is a major determinant in the control of diverse cellular processes, such as proliferation, survival, and motility. This pathway is often upregulated in human cancers. The exact role of each ERK molecule in these biological and pathological processes is not fully determined. An efficient strategy for revealing these roles is to activate each ERK individually, without activation of the upstream Ras-Raf-MEK cascade. This could be achieved via expression of intrinsically active ERKs. Such intrinsically active variants were generated in our lab for the yeast orthlog of ERK, Mpk1. The mutants were identified via an unbiased genetic screen that isolated Mpk1 molecules that function biologically in the absence of their upstream activators. Equivalent mutations were inserted to the human ERKs and only one mutation (R65S in ERK2; R84S in ERK1) rendered ERKs intrinsically active in vitro. The mechanism of action of the other biologically active variants remains obscure. Here we further elucidate the biochemical mechanisms and the biological effects of these variants, as well as of other known mutants. Two "catalytic" mutants were studied, ERK2R65S, and a mutant carrying the "Gatekeeper"-related mutation, ERK2I84A, identified by Natalie Ahn's group. Mutations located at the two conserved docking domains were also examined: a mutation at the CD domain (the Sevenmaker mutation, D319N) and mutations at the DEF pocket, that exhibit opposite biological effects; ERK2Y261A, shown as a biological loss of function mutation and ERK2Y261C, found in our screen as a biological gain of function mutation. ERK and Mpk1 molecules carrying those mutations and combination of the different mutations were assayed in yeast, in mammalian cells and in vitro as recombinant proteins. Our preliminary results revealed surprising insights on the involvement of the CD and DEF domains in catalysis and autophosphorylation and also legitimate the mutants for studying the biology of ERKs.