CONSTITUTIVE EFFECTOR REGION ON THE C-TERMINAL SIDE OF SWITCH-I OF THE RAS PROTEIN

The ''switch I'' region (Asp(30)-Asp(38)) of the Ras protein takes remarkably different conformations between the GDP- and GTP-bound forms and coincides with the so-called ''effector region.'' As for a region on the C-terminal side of switch I, the V45E and G48C mutants of Pas failed to promote neurite outgrowth of PC12 cells (Fujita-Yoshigaki, J., Shirouzu, Pd, Koide, H., Nishimura, S., and Yokoyama, S. (1991) FEBS Lett 294, 187-190). In the present study, we performed alanine-scanning mutagenesis within the region Lys(42)-Ile(55) Of Pas and found that the K42A, I46A, G48A, E49A, and L53A mutations significantly reduced the neurite-inducing activity. This is an effector region by definition, but its conformation is known to be unaffected by GDP --> GTP exchange. So, this region is referred to as a ''constitutive'' effector (E(c)) region, distinguished from switch I, a ''switch'' effector (E(s)) region. The E(c) region mutants exhibiting no neurite-inducing activity were found to be correlatably unable to activate mitogen-activated protein (MAP) kinase in PC12 cells. Therefore, the E(c) region is essential for the MAP kinase activation in PC12 cells, whereas mutations in this region only negligibly affect the binding of Ras to Raf-1 (Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, Il., Toyama, Y., Yamasaki, H., Fuhrman, S. A., Villafranca, E., Kaziro, Y., and Yokoyama, S. (1994) Oncogene 9, 2153-2157).