title: Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo.
abstract: 14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.
authors.affiliation: Diabetes Unit, the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
authors.forename: G
authors.initials: G
authors.lastname: Tzivion
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Luo
authors.forename: J
authors.initials: J
authors.lastname: Avruch
mesh_terms: D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D008387:Marine Toxins; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000378:metabolism / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D014746:Vimentin; C059041:calyculin A; D014446:Tyrosine 3-Monooxygenase; D010749:Phosphoprotein Phosphatases