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-ras 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 posttranslational processing of ras p21 is important for the MAP kinase activation.