Analysis of guanine nucleotide bound to ras protein in PC12 cells

Abstract

The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21 (Val-12) but also proto-oncogenic p21(Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTPγS [(1987) Mol. Cell. Biol. 7, 4553–4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd²⁺, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val-12) was mostly present in the GTP-bound form, whereas the endogenous p21(Gly-12) was in the GDP-bound form. These results indicate again that p21·GTP induces the morphological differentiation of PC12 cells.