Activation of c‐Raf‐1 by Ras and Src through different mechanisms: activation in vivo and in vitro

The c‐Raf‐1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G‐protein‐linked receptors. c‐Raf‐1 binds to the proto‐oncogene Ras in a GTP‐dependent manner, but the exact mechanism of activation of c‐Raf‐1 by Ras is still unclear. We have established a system to study the activation of c‐Raf‐1 in vitro. This involves mixing membranes from cells expressing oncogenic H–RasG12V, with cytosol from cells expressing epitope‐tagged full‐length wild‐type c‐Raf‐1. This results in a fraction of the c‐Raf‐1 binding to the membranes and a concomitant 10‐ to 20‐fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K‐Ras.GTP, but not non‐farnesylated K–Ras.GTP or farnesylated K‐Ras.GDP, was able to activate c‐Raf‐1 to the same degree as intact H–RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation‐permissive conditions, activation of c‐Raf‐1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c‐Raf‐1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c‐Raf‐1, and in vitro, activation of c–Raf–1 by Src requires the presence of ATP. Therefore we propose that activation of c‐Raf‐1 by Ras or by Src occurs through different mechanisms.

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