Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo

14-3-3 proteins bind their targets through a specific serine/threonine-phos phorylated motif present on the target protein. This binding is a crucial s tep in the phosphorylation-dependent regulation of various key proteins inv olved in signal transduction and cell cycle control, We report that treatme nt of COS-7 cells with the phosphatase inhibitor calyculin A induces associ ation of 14-3-3 with a 55-kDa protein, identified as the intermediate filam ent protein vimentin, Association of vimentin with 14-3-3 depends on viment in 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 am ino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do n ot bind vimentin in vivo or in vitro, indicating that a stable complex requ ires the binding of a 14-3-3 dimer to two sites on a single vimentin polype ptide. 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 pr otooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vit ro, 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 p rocesses that require 14-3-3.