Studies of perinuclear and nuclear translocation of the Raf-1 protein in rodent fibroblasts.
Raf-1, A-Raf and B-Raf comprise a small family of highly conserved serine/threonine protein kinases, whose activities play a fundamental role in the control of proliferation and differentiation. The best studied 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 promoters, inflammatory cytokines, calcium mobilization, DNA damaging agents, and oxygen radicals. Ras-mediated translocation of Raf-1 to the plasma membrane is a crucial step in its activation process, and is thought to facilitate phosphorylation by membrane-bound kinases. Raf-1 has also been reported to undergo intracellular redistribution following its activation: to the perinuclear space in murine NIH3T3 cells and rat hepatic Ito cells, and into the nucleus in gerbil hippocampal pyramidal cells and human MO7 leukemia cells. In contrast to the translocation to the plasma membrane, the perinuclear and/or nuclear translocation of Raf-1 has not been investigated in detail. In this paper, we report an examination of the subcellular localization of endogenous Raf-1 in a fibroblastic cell line (Rat-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 the perinuclear or nuclear space can be observed. Tethering of activated Raf to the plasma membrane does not interfere with its transforming activity.
Prouty
S M
SM
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA.
Maroo
A
A
Maucher
C
C
Mischak
H
H
Kolch
W
W
Sedivy
J M
JM
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005849:Gerbillinae; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007938:Leukemia; D051379:Mice; D019908:Proto-Oncogene Proteins c-raf / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017966:Pyramidal Cells / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D019908:Proto-Oncogene Proteins c-raf