Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras.
Ras is a small GTPase that cycles between an inactive GDP-bound and an active GTP-bound form. A large variety of ligands that stimulate cell surface receptors induce the activation of Ras. Thus far, this activation could only be measured by the increase of GTP bound to Ras, which was precipitated from radio-labelled cell extract. We have used the minimal Ras-binding domain (RBD) of Raf1 (aa 51-131) to identify in vivo activated Ras. This novel method is based on the observation that RBD binds RasGTP in vitro with a Kd of 20 nM whereas the affinity between RBD and RasGDP is three orders of magnitude lower. Here we show that the Gst-RBD fusion protein precipitates transfected RasL61 (RasGTP) but not RasN17 (RasGDP) from cell lysates. In addition, we demonstrate for two different cell lines that the increase in RasGTP is reflected by an increase in Ras bound to Gst-RBD. From these results we conclude that the minimal Ras-binding domain of Raf1 is an excellent activation specific-probe for Ras.
de Rooij
J
J
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Bos
J L
JL
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D009414:Nerve Growth Factors / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D009457:Neuroglia; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D007328:Insulin; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020558:GTP Phosphohydrolases; D018631:ras Proteins