Induction of Rac-Guanine Nucleotide Exchange Activity of Ras-GRF1/CDC25Mm following Phosphorylation by the Nonreceptor Tyrosine Kinase Src

Abstract

Ras-GRF1/CDC25Mm 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 ␤␥ subunit (G␤␥) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by G␤␥-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 vSrc was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by G␤␥. 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 G␤␥ to regulate Ras-GRF1.

Epidermal growth factor differentially augments Gi‐mediated stimulation of c‐Jun N‐terminal kinase activity

“…Members of the guanine nucleotide exchange factors (GEFs) family are capable of regulating the small GTPase activities, which in turn activate the downstream JNK pathway (Fan et al, 1998). GEF proteins such as Ras-GRF1, Ras-GRF2 and Vav isoforms can be stimulated through multiple mechanisms, including phosphorylation by Src family tyrosine kinases (Kiyono et al, 2000), binding of Ca 2 þ to their CaM-like regions (Fan et al, 1998) and conformational changes induced by interaction with PI3K-phosphorylated lipids (Das et al, 2000). Hence, members of the GEF family may serve as modulators to integrate various inputs (such as Gbg/Src family tyrosine kinases, PI3K and Ca 2 þ /CaM) from G i and EGF signaling, and transmit stimulatory signals directly to small GTPases-mediated JNK pathway (Figure 7).…”

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

“…The p21 GTPases themselves are regulated by guanine nucleotide exchange factors (GEFs) that turn them on, GTPase activating proteins (GAPs) that turn them off, and guanine nucleotide dissociation inhibitors that can function as sequestering agents (Suetsugu and Takenawa, 2003;Burridge and Wennerberg, 2004;Raftopoulou and Hall, 2004). The activities of many of these proteins have been found to be regulated by FAK and Src-family kinases (Hildebrand et al, 1996;Han et al, 1997;Kiyono et al, 2000;Haskell et al, 2001;Tu et al, 2003;Zhai et al, 2003;Stacey et al, 2004).…”

“…The p21 GTPases themselves are regulated by guanine nucleotide exchange factors (GEFs) that turn them on, GTPase activating proteins (GAPs) that turn them off, and guanine nucleotide dissociation inhibitors that can function as sequestering agents (Suetsugu and Takenawa, 2003;Burridge and Wennerberg, 2004;Raftopoulou and Hall, 2004). The activities of many of these proteins have been found to be regulated by FAK and Src-family kinases (Hildebrand et al, 1996;Han et al, 1997;Kiyono et al, 2000;Haskell et al, 2001;Tu et al, 2003;Zhai et al, 2003;Stacey et al, 2004).The pathways of activation and signaling from the p21-activated protein kinase (PAK) typify the sophisticated mechanisms developed by cells to respond to extracellular cues (Bokoch, 2003). In response to cell attachment and/or growth factor stimulation, PAK is activated and signals to Galisteo et al, 1996;Lu et al, 1997;Sells et al, 1997;Kiosses et al, 2002), the Cdc42/Rac GEF PIX (Bagrodia et al, 1998;Zhao et al, 2000a), the paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT) family of ArfGAPs Premont et al, 2000;Brown et al, 2002;Manabe Ri et al, 2002), and the molecular scaffold protein paxillin Zhao et al, 2000b;Hashimoto et al, 2001;West et al, 2001;Brown et al, 2002).…”

Sites of Phosphorylation by Protein Kinase A in CDC25Mm/GRF1, a Guanine Nucleotide Exchange Factor for Ras

“…The latter modification was found to concern serine/threonine (6,16) and very recently also tyrosine residues (17). The nonreceptor tyrosine kinase Src can induce on CDC25Mm a Rac1-GEF activity (18), showing that this protein is an important element for cross-regulation of Ras and Rac-dependent pathways (19), as has been reported for SOS (20) and GRF2 (21). Taken together, these results illus-trate the complexity of the upstream activation of CDC25Mm, as well as the many aspects that remain to be clarified.…”