G protein βγ subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25 ^Mm

Abstract

Ras-GRF1 has been implicated as a Ras-specific guanine nucleotide exchange factor (GEF), which mediates calcium- and muscarinic receptor-triggered signals in the brain. Although a Dbl homology domain known as a motif conserved among GEFs that target Rho family GTP-binding proteins exists in Ras-GRF1, GEF activity toward Rho family proteins has not been observed. Here we show that Ras-GRF1 exhibits Rac1-specific GEF activity when recovered from cells overexpressing G protein beta gamma subunits (Gbeta gamma). Substitution of conserved amino acids within the Dbl homology domain abolished this activity. Activation of the Rac pathway in the cell was further evidenced by synergistic activation of the stress kinase JNK1 by Ras-GRF1 and Gbeta gamma, which is sensitive to inhibitory action of dominant-negative Rac1(17N). In addition, association of Ras-GRF1 with Rac1(17N) was demonstrated by coimmunoprecipitation. Evidence for the involvement of tyrosine kinase(s) in Gbeta gamma-mediated induction of Rac1-specific GEF activity was provided by the use of specific inhibitors. These results suggest a role of Ras-GRF1 for regulating Rac-dependent as well as Ras-dependent signaling pathways, particularly in the brain functions.