Glucocorticoid receptor interaction with 14-3-3 and Raf-1, a proposed mechanism for cross-talk of two signal transduction pathways.
The glucocorticoid receptor (GR) functions as a ligand-dependent transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. We have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-1, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 m salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.
Widén
C
C
Department of Medical Nutrition, Karolinska Institutet, NOVUM, S-141 86 Huddinge, Sweden.
Zilliacus
J
J
Gustafsson
J A
JA
Wikström
A C
AC
D048948:14-3-3 Proteins; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008024:Ligands; D008099:Liver / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D012965:Sodium Chloride / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*
D048948:14-3-3 Proteins; D008024:Ligands; D011965:Receptors, Glucocorticoid; D012965:Sodium Chloride; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf