STUDIES OF PERINUCLEAR AND NUCLEAR TRANSLOCATION OF THE RAF-1 PROTEININ RODENT FIBROBLASTS

Raf-1, A-Raf and B-Raf comprise a small family of highly conserved ser ine/threonine protein kinases, whose activities play a fundamental rol e in the control of proliferation and differentiation. The best studie d family member, Raf-1, is expressed ubiquitously and constitutively, and its activity is regulated by post-translational mechanisms. Raf-1 can be activated by many signals that include growth factors, tumor pr omoters, inflammatory cytokines, calcium mobilization, DNA damaging ag ents, and oxygen radicals. Ras-mediated translocation of Raf-1 to the plasma membrane is a crucial step in its activation process, and is th ought to facilitate phosphorylation by membrane-bound kinases. Raf-1 h as also been reported to undergo intracellular redistribution followin g its activation: to the perinuclear space in murine NIH3T3 cells and rat hepatic Ito cells, and into the nucleus in gerbil hippocampal pyra midal cells and human MO7 leukemia cells. In contrast to the transloca tion to the plasma membrane, the perinuclear and/or nuclear translocat ion of Raf-1 has not been investigated in detail. In this paper, we re port an examination of the subcellular localization of endogenous Raf- 1 in a fibroblastic cell line (Raf-1) commonly used in transformation assays. Using the methods of cellular fractionation as well as in situ immunofluorescence, we show that no detectable movement of Raf-1 to t he perinuclear or nuclear space can be observed. Tethering of activate d Raf to the plasma membrane does not interfere with its transforming activity. (C) 1998 Elsevier Science B.V.