Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1.
Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.
Kiyono
M
M
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Kato
J
J
Kataoka
T
T
Kaziro
Y
Y
Satoh
T
T
D002460:Cell Line; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000378:metabolism*
D020663:ras Guanine Nucleotide Exchange Factors; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human; D020764:cdc42 GTP-Binding Protein