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 (GTP gamma S) bound form of post-translationally processed Ki-ras 4B p21 activated MAP kinase in the cytosol fraction of Xenopus oocytes, but the GTP gamma S 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 GTP gamma S 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 post-translational processing of ras p21 is important for the MAP kinase activation.
Department of Biochemistry, Kobe University School of Medicine, Japan.
T
T
Itoh
K
K
Kaibuchi
T
T
Masuda
T
T
Yamamoto
Y
Y
Matsuura
A
A
Maeda
K
K
Shimizu
Y
Y
Takai
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014981:Xenopus
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D016283:Proto-Oncogene Proteins p21(ras)