A glutamic acid residue at position 31 of Ras protein is essential to the signal transduction for neurite outgrowth of PC12 cells and the stimulation of GTPase activity by GAPRas.
The roles of residues at positions 23-31 adjacent to the 'effector region' and residues at positions 61-65 in a phosphoryl binding loop of the human c-Ha-ras protein were studied by changing each residue of the normal (Gly-12 type) and oncogenic (Val-12 type) Ras proteins to the corresponding residue of the K-rev-1 protein. Firstly, the signal-transducing activities of the mutant Ras proteins of Val-12 type were examined by analysis of their ability to induce neurite outgrowth of phaeochromocytoma (PC12) cells upon expression of the mutant ras gene. Thus, replacement of Glu-31 by Lys was found to impair the signal-transducing activity of the oncogenic Ras protein. Furthermore, it was shown that expression of the Gly-12----Val/Glu-31----Lys mutant Ras protein in PC12 cells suppresses neurite outgrowth induced either by microinjection of the oncogenic Ras protein or by addition of nerve growth factor to the medium. As for the Glu-31----Lys mutant Ras protein (Gly-12 type), the GTPase activity in the presence of GTPase-activating protein for Ras (GAPRas) is much lower than that of the normal Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal Ras protein. Therefore, Gly-31 is one of the determinants for the signal transduction and the correct interaction with GAPRas. On the other hand, the GTPase activity of the Gln-61----Thr mutant Ras protein (Gly-12 type) is negligibly low both in the absence and in the presence of GAPRas.
Shirouzu
M
M
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Fujita-Yoshigaki
J
J
Ito
Y
Y
Koide
H
H
Nishimura
S
S
Yokoyama
S
S
D000595:Amino Acid Sequence; D000818:Animals; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry; D020690:GTPase-Activating Proteins; D005971:Glutamates / Q000737:chemistry; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016716:PC12 Cells; D011506:Proteins / Q000737:chemistry; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000502:physiology; D051381:Rats; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D020668:rap GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D005971:Glutamates; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins; D008239:Lysine