The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase

The point-mutated active form of ras p21 is known to activate mitogen-activated protein (MAP) kinase/ extracellular signal-regulated kinase (ERK) in intact mammalian cells and Xenopus oocytes, although the direct target molecule of ras p21 remains to be identified. To elucidate the role of the post-translational processing of ras p21 for the MAP kinase activation, we established the cell-free system in which ras p21 activated MAP kinase. The guanosine 5′-(3-O-thio)triphosphate (GTPγS) bound form of post-translationally processed Ki-ras 4B p21 activated MAP kinase in the cytosol fraction of Xenopus oocytes, but the GTPγS bound form of post-translationally unprocessed Ki-ras 4B p21 or the GDP bound form of processed or unprocessed Ki-ras 4B p21 was far less effective. The GTPγS bound form of processed Ki-ros 4B p21 activated recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes, but the unprocessed protein was far less effective. These results provide a complete biochemical assay for ras p21 to activate MAP kinase in a cell-free system and indicate that all the elements downstream of ras p21 necessary for the MAP kinase activation are cytosolic and that the post-translational processing of ras p21 is important for the MAP kinase activation.