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Abstract
Ras-GRF1/CDC25(Mm) has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca(2+) influx and G protein betagamma subunit (Gbetagamma) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gbetagamma-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gbetagamma. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1 in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gbetagamma to regulate Ras-GRF1.
Highlights
The catalytic domain conserved among guanine nucleotide exchange factor (GEF) that target Ras is designated the CDC25 homology domain, which was originally identified as a region in the Saccharomyces cerevisiae Cdc25 protein essential for the function as an upstream regulator of Ras [1]
We describe that Src elicits tyrosine phosphorylation of Ras-GRF1 in living cells and in a cell-free system, allowing Ras-GRF1 to act as a GEF for Rac
Tyrosine Phosphorylation of Ras-GRF1 in v-Src-co-transfected and platelet-derived growth factor (PDGF)-stimulated Cells—As a first step to manifest a possible involvement of Src in the regulation of Ras-GRF1, Src-dependent tyrosine phosphorylation of Ras-GRF1 was examined by the use of a transient expression system in 293 cells
Summary
(Received for publication, October 6, 1999, and in revised form, December 4, 1999). Mari Kiyono, Yoshito Kaziro, and Takaya Satoh‡ From the Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan. Tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of G␥ to regulate Ras-GRF1. Ras-GRF1 functions as a Rac-GEF in response to signals mediated by G␥ [22], while Ras-GRF2 shows constitutive and Ca2ϩ-stimulated Rac-GEF activity [23]. We describe that Src elicits tyrosine phosphorylation of Ras-GRF1 in living cells and in a cell-free system, allowing Ras-GRF1 to act as a GEF for Rac. GEF activity toward other Rho family members, Rho and Cdc, remained undetectable upon Src-dependent tyrosine phosphorylation. Ras-GEF activity was unaffected when phosphorylated by Src. Considering the observation that G␥ induces Rac-specific GEF activity of Ras-GRF1 in a tyrosine phosphorylation-dependent manner, Src may be involved in G protein-coupled receptor stimulation of Ras-GRF1
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