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Calyculin A-induced Vimentin Phosphorylation Sequesters 14-3-3 and Displaces Other 14-3-3 Partners in Vivo

2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês

10.1074/jbc.m001207200

ISSN

1083-351X

Autores

Guri Tzivion, Zhijun Luo, Joseph Avruch,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

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. 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. intermediate filament polyacrylamide gel electrophoresis polyvinylidene difluoride high pressure liquid chromatography epidermal growth factor N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine extracellular signal-regulated kinase The 14-3-3 protein family consists of seven highly conserved proteins of 28–31 kDa that naturally assemble as homodimers or heterodimers and bind to a wide array of intracellular proteins (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (350) Google Scholar). Among the proteins known to associate with 14-3-3 in vivoare proteins that function in cell cycle control (e.g.Cdc25 (2Conklin D.S. Galaktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (246) Google Scholar) and Wee1 (3Honda R. Ohba Y. Yasuda H. Biochem. Biophys. Res. Commun. 1997; 230: 262-265Crossref PubMed Scopus (45) Google Scholar)), signal transduction (e.g.Raf (4Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (358) Google Scholar), phosphatidylinositol 3-kinase (5Bonnefoy-Berard N. Liu Y. Willebrand M.V. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (134) Google Scholar), and BAD (6Zha J. Harda H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar)), transcriptional regulation (e.g. FKHRL1 (7Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5512) Google Scholar)), and cell structure (e.g. keratins (8Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (180) Google Scholar)). Genetic alterations in 14-3-3 proteins have been shown to impair a variety of biologic functions, such as cell cycle timing (9Zeng Y. Forbes K.C. Wu Z. Moreno S. Piwnica-Worms H. Enoch T. Nature. 1998; 395: 507-510Crossref PubMed Scopus (308) Google Scholar) and cell cycle arrest in response to DNA damage in yeast (10Ford J.C. al-Khodairy F. Fotou E. Sheldrick K.S. Griffiths D.J. Carr A.M. Science. 1994; 265: 533-535Crossref PubMed Scopus (306) Google Scholar), photoreceptor development in theDrosophila eye (11Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (125) Google Scholar), and the Ras/Raf signaling pathway in various organisms (4Freed E. Symons M. Macdonald S.G. McCormick F. Ruggieri R. Science. 1994; 265: 1713-1716Crossref PubMed Scopus (358) Google Scholar, 11Chang H.C. Rubin G.M. Genes Dev. 1997; 11: 1132-1139Crossref PubMed Scopus (125) Google Scholar). The ability of 14-3-3 to bind target proteins has been shown in many instances to depend on the phosphorylation of specific sites on the target protein (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (350) Google Scholar), and studies with synthetic phosphopeptides have defined two preferred 14-3-3 binding motifs as RSXpSXP or RXXXpSXP (where pS stands for phosphoserine) (12Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-898Abstract Full Text Full Text PDF PubMed Scopus (1205) Google Scholar, 13Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1372) Google Scholar). The existence of additional variations in the sequence of 14-3-3 binding sites is probable inasmuch as 14-3-3 partners such as phosphatidylinositol 3-kinase (5Bonnefoy-Berard N. Liu Y. Willebrand M.V. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (134) Google Scholar), tyrosine hydroxylase (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (350) Google Scholar), and keratins (14Ku N.O. Liao J. Omary M.B. EMBO J. 1998; 17: 1892-1906Crossref PubMed Scopus (196) Google Scholar) do not contain either of these motifs. Association with 14-3-3 may positively or negatively regulate the function of the partner; thus, the binding of 14-3-3 results in activation of tyrosine hydroxylase (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (350) Google Scholar) but inhibition of phosphatidylinositol 3-kinase (5Bonnefoy-Berard N. Liu Y. Willebrand M.V. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (134) Google Scholar) and Cdc25 (15Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1198) Google Scholar); a continuous association with 14-3-3 is necessary for physiologic regulation of Raf kinase activity (12Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-898Abstract Full Text Full Text PDF PubMed Scopus (1205) Google Scholar, 16Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (186) Google Scholar, 17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar). The biochemical mechanisms by which 14-3-3 proteins modify the function of the target protein may vary for each partner; for example, in the case of yeast Cdc25 (18Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (509) Google Scholar) and FKHRL1 (7Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5512) Google Scholar) the association with 14-3-3 (Rad24 in yeast) results in their exclusion from the nucleus. The ability of the proapoptotic protein BAD to bind and sequester the antiapoptotic factors BCL2 and BCLxL is inhibited by the phosphorylation of BAD and subsequent binding of 14-3-3 (6Zha J. Harda H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar). The 14-3-3 proteins have multiple functions in the regulation of c-Raf-1; the binding of 14-3-3 to Raf stabilizes the inactive state in unstimulated cells but is also required to maintain the active conformation of Raf generated after growth factor stimulation (17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar). The intermediate filament (IF)1 network provides a rigid framework that supports cell architecture during interphase (19Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1310) Google Scholar,20Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (848) Google Scholar). IF proteins undergo disassembly and major reorganization during cell cycle progression (21Klymkowsky M.W. Curr. Opin. Cell Biol. 1995; 7: 46-54Crossref PubMed Scopus (102) Google Scholar). Inasmuch as IF proteins constitute at least 1% of total cell protein, IF disassembly creates a large pool of soluble IF proteins. The assembly/disassembly of IFs is regulated by dephosphorylation/phosphorylation of the filaments on their exposed N- and/or C-terminal domains (22Inagaki M. Matsuoka Y. Tsujimura K. Ando S. Tokui T. Takahashi T. Inagaki N. Bioessays. 1996; 18: 481-487Crossref Scopus (161) Google Scholar). A variety of protein (Ser/Thr) kinases likely to phosphorylate IFs in vivo have been proposed, including protein kinase Cs, protein kinase A, CaMKII, and Cdc2 kinase. In BHK-21 cells, the IF proteins vimentin and desmin undergo a major increase in phosphorylation (to a stoichiometry of approximately 2 mol of phosphate/mol of protein) concomitant with filament disassembly that occurs as the cells move from G2 to M (23Chou Y.-H. Bischoff J.R. Beach D. Goldman R.D. Cell. 1990; 62: 1063-1071Abstract Full Text PDF PubMed Scopus (297) Google Scholar, 24Chou Y.-H. Ngai K.-L. Goldman R.D. J. Biol. Chem. 1991; 266: 7325-7328Abstract Full Text PDF PubMed Google Scholar). Incubation of these cells with the protein phosphatase inhibitor calyculin A results in a rapid disassembly of IFs, in parallel with their hyperphosphorylation, both at sites already partially phosphorylated in interphase and at mitosis-specific sites (25Eriksson J.E. Brautigan D.L. Vallee R. Olmsted J. Fujiki H. Goldman R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11093-11097Crossref PubMed Scopus (165) Google Scholar). The epithelial IF protein keratin K18 associates with 14-3-3 proteins in a cell cycle- and phosphorylation-dependent manner, reaching a peak during late S-G2/M phase (8Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (180) Google Scholar, 14Ku N.O. Liao J. Omary M.B. EMBO J. 1998; 17: 1892-1906Crossref PubMed Scopus (196) Google Scholar). We now report that calyculin A treatment of COS cells induces the binding of vimentin to 14-3-3-, accompanied by the displacement of virtually all other 14-3-3 partners, including Raf-1. 14-3-3-Raf complex disassembly results in a complete inhibition of Raf kinase activity, which is, however, completely overcome by 14-3-3 overexpression in vivo or by the addition of recombinant 14-3-3 to the cell extract in vitro. These results suggest that IF phosphorylation may serve to regulate 14-3-3 availability to key proteins involved in intracellular signaling and consequently regulate their function. GST-wt-14-3-3ζ-(1–245), GST-nt-14-3-3-(1–140), GST-ct-14-3-3-(139–245), and GST-dm-14-3-3 (E5K, L12Q/A13Q/E14R, Y82Q, K85N, E87Q) for expression in mammalian cells were in the pEBG vector. Myc-wt-14-3-3-(1–245) and Myc-c-Raf-1 were in the pMT2 vector. GST, GST-wt-14-3-3, and GST-dm-14-3-3 were expressed in Escherichia coliusing pGEX-KG vector. The construction of these vectors is described elsewhere (17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar, 26Luo Z. Zhang X. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29Luo Z.-J. Tzivion G. Belshow P.J. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (209) Google Scholar). COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For transient expression of proteins, cells were transfected either with the DEAE-dextran method using 5–10 μg of DNA/10-cm dish or using LipofectAMINE (Life Technologies, Inc.) with the indicated amounts of DNA, following the manufacturer's instructions. For cell stimulation, 24–48 h after transfection cells were serum-deprived for 24 h prior to the addition of the stimulator. Serum-deprived cells were washed once with medium lacking methionine/cysteine for [35S]Met labeling or lacking phosphate for 32P labeling and incubated for 30 min in the same media to deplete intracellular sources. Cells were labeled by incubation in the presence of 0.5 mCi/ml35S or 32P for 4 or 2 h, respectively. Cells were lysed in ice-cold extraction buffer containing 50 mm Tris-CL, pH 7.5, 100 mm NaCl, 1% Triton X-100, 1 mmdithiothreitol, 1 mm EDTA, 1 mm EGTA, 2 mm Na3VO4, 50 mmβ-glycerophosphate, and a mixture of proteinase inhibitors (Amersham Pharmacia Biotech). Cell lysates were incubated at 4 °C for 90 min with the appropriate antibody precoupled to protein A/G-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for immunoprecipitation or with GSH-Sepharose beads (Amersham Pharmacia Biotech) for GSH affinity purification. The beads were washed twice with the extraction buffer, twice with extraction buffer containing 0.5m LiCl, and twice with buffer A (40 mm Tris-Cl, pH 7.5, 0.1 mm EDTA, 5 mm MgCl2, and 2 mm dithiothreitol). The proteins were either eluted directly in SDS-sample buffer or served for additional experiments. Cell extracts were incubated with 300 μm synthetic phosphopeptide corresponding to c-Raf-1 amino acids 613–627 containing a canonical 14-3-3 binding motif, LPKINRSApSEPSLHR, or with a 300 μm concentration of the same peptide in its unphosphorylated form for 30 min at 4 °C prior to protein purification. Prokaryotic recombinant GST or GST-14-3-3 fusion variants precoupled to GSH-Sepharose beads were incubated with the various cell extracts or with the lysyl C digest of32P-labeled vimentin at 4 °C for 30–60 min in the presence or absence of phosphopeptide as indicated. The beads were washed twice with extraction buffer, twice with extraction buffer containing 0.5 m LiCl, and twice with buffer A. The associated proteins were eluted either with GSH or with SDS-sample buffer as indicated. For the phosphatase/kinase-treated vimentin binding assays, GST-14-3-3 fusions were labeled using the biotinylation kit from Amersham Pharmacia Biotech. Following anti-Myc immunoprecipitation, the Myc-Raf-containing beads were incubated in buffer A (100 μl final volume) supplemented with 100 μmATP, 10 μCi of [γ-32P]ATP, and 0.3 μg of prokaryotic recombinant GST-MEK-1 for 20 min at 30 °C and for an additional 30 min after the addition of 2 μg of prokaryotic recombinant kinase-inactive ERK-2 (K52R). Samples were separated on SDS-PAGE and transferred to PVDF membranes. The 32P content in ERK-2 was quantified by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA) (data are presented as PhosphorImager units × 103). To survey the array of cellular polypeptides that interact with 14-3-3ζ in vivo, GST-14-3-3 associated proteins, extracted from [35S]Met-labeled COS cells, were resolved on SDS-PAGE (Fig. 1 A). A large number of cellular polypeptides copurified with full-length GST-14-3-3 but not with control GSH beads (Fig. 1 A, comparelanes 1 and 2) or with GST-nt-14-3-3-(1–140), which lacks the carboxyl-terminal target protein-binding domain (26Luo Z. Zhang X. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) (Fig. 1 A, compare lanes 2 and 4). The amino-terminally truncated GST-ct-14-3-3-(139–245), which lacks the 14-3-3 dimerization domain (26Luo Z. Zhang X. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), did not associate with endogenous 14-3-3 (Fig. 1 A, lane 3) but maintained the ability to bind several other endogenous polypeptides and to bind cotransfected Myc-Raf (Fig. 1 A,lanes 5 and 6). The addition of the phosphatase inhibitor calyculin A to COS cells resulted in an upshift of several of the 14-3-3-associated proteins (Fig. 1 A, compare lane 2 with lane 8) and induced the association of a 55-kDa polypeptide with full-length 14-3-3 but not with either of the 14-3-3 fragments (Fig. 1 A, compare lane 8 with lanes 9and 10). Since following calyculin A treatment the 55-kDa polypeptide became the most abundant [35S]methionine-labeled 14-3-3 partner, it was purified (Fig. 1 B) and subjected to in-gel digestion with lysyl C. Two prominent peptides isolated by reverse phase HPLC were analyzed by automated Edman degradation, each yielding amino acid sequence identical to human vimentin (Fig. 1 C). Confirming this identification, anti-vimentin immunoblot detected a time-dependent, calyculin A-induced association of vimentin with recombinant GST-14-3-3, in vivo (Fig. 1 D). Reciprocally, calyculin A induced the appearance of both GST-14-3-3 and endogenous 14-3-3 in anti-vimentin immunoprecipitates (Fig.1 E). Immobilized prokaryotic recombinant GST 14-3-3 was able to bind in vitro vimentin from extracts of calyculin A-treated cells but not from extracts of control or EGF- or 12-O-tetradecanoylphorbol-13-acetate-treated cells (Fig.1 F). Exposure of COS cells to heat shock, okadaic acid, sodium vanadate, and extracellular hypertonicity also failed to induce vimentin/14-3-3 association (Fig.2 C and data not shown), pointing to a specific effect of calyculin A, probably trough inhibition of protein phosphatase-1. Calyculin A, in contrast to okadaic acid, is able to enter cells rapidly and inhibit both protein phosphatase-1 and protein phosphatase 2A while okadaic acid inhibits preferentially protein phosphatase 2A (27Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar).Figure 2Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires 14-3-3 dimerization and its phosphopeptide-binding domain. A, vimentin immunoprecipitated from COS cells treated for 45 min with carrier (lanes 9 and 10) or calyculin A (150 nm; lanes 1–6) was incubated for 30 min at 30 °C with buffer alone (lanes 1, 2,6, and 9) or with buffer containing calf intestinal alkaline phosphatase (Amersham Pharmacia Biotech) (ALK; 20 units, lanes 3 and4), protein phosphatase 2A (PP2; 200 milliunits,lane 5), or a protein kinase A/AKT combination (20 units and 2 μg, respectively; lane 10) andp-nitrophenylphosphate (PNPP; 50 mm,lane 4). After washes, the immunoprecipitates were incubated for 30 min at 30 °C with vehicle (lane 1), biotinylated recombinant GST-14-3-3 (10 μg; lanes 2–5), or biotinylated recombinant GST-ct-14-3-3 (10 μg; lane 6), washed, and separated on SDS-PAGE. The bound biotinylated 14-3-3 proteins were detected by blotting with avidin-horseRadish peroxidase and ECL (upper panel). The lower panel shows equal recovery of vimentin. Lanes 7 and 8 contain 5% input of biotinylated GST-14-3-3 and GST-ct-14-3-3, respectively. B, COS cells transfected with empty vector or with GST-14-3-3 were treated with calyculin A (100 nm, 30 min) or vehicle as indicated. Cell extracts were incubated in the absence or presence of 300 μm synthetic phosphopeptide corresponding to the 14-3-3 binding sequence of Raf (PP) or in the presence of a 300 μm concentration of the unphosphorylated peptide (P) at 4 °C for 1 h followed by anti-vimentin immunoprecipitation. Shown are anti-14-3-3 immunoblot (upper panel) and a Coomassie Blue stain (lower panel). C, COS cells transiently expressing wild type GST-14-3-3 (wt, lanes 1–4), dimerization-deficient 14-3-3 mutant GST-dm-14-3-3 (dm,lanes 5–8), GST-ct-14-3-3 (ct,lanes 9 and 10), or GST-nt-14-3-3 (nt, lanes 11 and 12) were treated with calyculin A (150 nm, 30 min), okadaic acid (3 μm, 30 min), or heat shock (42 °C, 45 min) or left untreated (−). The GST fusions were recovered on GSH-Sepharose beads and assayed for coassociated vimentin by an anti-vimentin immunoblot (upper panel). The recovery of GST fusions was determined by anti-GST (Upstate Biotechnology, Inc., Lake Placid, NY) immunoblot of the stripped membrane (lower panel). D, vimentin immunoprecipitated from32P-labeled COS cells treated for 60 min with calyculin A (0.3 μm) was resolved on SDS-PAGE and transferred to PVDF membrane. The 32P-labeled vimentin band was excised and subjected to endoproteinase Lys-C cleavage. The digest was absorbed on immobilized wild type prokaryotic recombinant GST-14-3-3 (wt, lanes 1 and 3) or on the dimerization-deficient 14-3-3 mutant GST-dm-14-3-3 (dm,lanes 2 and 4) in the absence (lanes 1 and 2) or presence of 300 μm phosphopeptide (lanes 3 and4). Bound polypeptides were separated on a 15% SDS-PAGE using the Tris Tricine buffer system, stained with Coomassie Blue (left half), and subjected to autoradiography (right half).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ability of the phosphatase inhibitor calyculin A to induce the association of vimentin with 14-3-3 suggested that vimentin phosphorylation is involved; however, since vimentin does not contain a "perfect" 14-3-3 binding motif (19Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1310) Google Scholar), we tested the role of vimentin phosphorylation in 14-3-3 binding. Incubation of vimentin extracted from calyculin A-treated cells with alkaline phosphatase or protein phosphatase 2A completely prevents vimentin binding to 14-3-3 in vitro (Fig. 2 A, compare lane 2 with lanes 3and 5). The specificity of the inhibition was demonstrated by the ability of the alkaline phosphatase inhibitorp-nitrophenylphosphate (PNPP) to prevent the inhibition (Fig. 2 A, lane 4). In support of this finding, phosphorylation of vimentin extracted from control, untreated cells with protein kinase A/AKT combination (two kinases previously shown to phosphorylate several proteins on 14-3-3 recognition sites (1Aitken A. Trends Cell Biol. 1996; 6: 341-347Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 6Zha J. Harda H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar, 7Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5512) Google Scholar)) induced 14-3-3 binding to vimentin (Fig.2 A, compare lane 9 withlane 10). These results demonstrate that vimentin phosphorylation mediates and is required for 14-3-3 binding. To further support our findings and to demonstrate that 14-3-3 binding to vimentin is mediated by the 14-3-3 phosphopeptide-binding region, we used a synthetic phosphopeptide corresponding to c-Raf-1 amino acids 613–627, a canonical 14-3-3 binding motif previously shown to dissociate Raf 14-3-3 complexes (12Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-898Abstract Full Text Full Text PDF PubMed Scopus (1205) Google Scholar, 17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar), to dissociate 14-3-3/vimentin complexesin vitro (Fig. 2 B). It is notable that only the phosphopeptide is able to dissociate 14-3-3-vimentin complexes, whereas the same peptide in the unphosphorylated state has no effect (Fig.2 B). Yaffe et al. (13Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1372) Google Scholar) demonstrated that the presence of two or more phosphoserine-containing 14-3-3 binding motifs on a single synthetic phosphopeptide increases the apparent affinity for 14-3-3 by 30-fold over the same peptide containing a single phosphoserine. In view of the absence of "optimal" 14-3-3 binding motifs on vimentin and the inability of the amino-terminally truncated, dimerization-deficient GST-ct-14-3-3-(139–245) polypeptide to bind vimentin (Fig. 1 A), we hypothesized that the coligation of two phosphoserines on a single vimentin polypeptide by a 14-3-3 dimer might be necessary to confer sufficient avidity for a stable association. Consistent with this idea, several forms of dimerization-deficient 14-3-3, although able to bind c-Raf-1 efficiently in vivo (Fig. 1 A and Refs. 17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar and 26Luo Z. Zhang X. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) were unable to bind vimentin in vivo (Fig. 2 C) orin vitro (Fig. 2, A and D). The requirement for a 14-3-3 dimer for vimentin binding is thus consistent with the view that phosphovimentin in calyculin A-treated cells contains two or more low affinity 14-3-3 binding sites, neither of which is sufficient by itself to bind 14-3-3, whereas their coligation by a 14-3-3 dimer enables stable association. To identify regions in vimentin required for 14-3-3 binding, a lysyl C digest of32P-vimentin, isolated from calyculin A-treated COS cells, was adsorbed to immobilized prokaryotic recombinant GST-14-3-3 or dimerization-deficient (dm) 14-3-3 mutant (Fig.2 D). A single 32P-labeled peptide fragment corresponding to approximately 25% of the applied 32P was retained by the wild type 14-3-3, whereas no 32P-peptide was retained by the monomeric 14-3-3 (Fig. 2 D, comparelanes 1 and 2); retention of the vimentin 32P-peptide was inhibited by the synthetic Raf phosphopeptide (Fig. 2 D, lane 3). The lysyl C32P-peptide that binds to 14-3-3 exhibits an apparent molecular mass of 14 kDa, consistent only with one predicted lysyl C fragment, corresponding to the vimentin amino-terminal region (amino acids 1–96). These results suggest that the vimentin phosphorylation sites that mediate binding to 14-3-3 are located in this amino-terminal segment. We noticed that the progressive increase in GST-14-3-3/vimentin association in vivo that occurs with prolonged exposure of cells to calyculin A (300 nm, 60 min) is accompanied by the displacement of most other 14-3-3 partners (Fig. 3,A and B), when low levels of GST-14-3-3 are expressed, but not with high level GST-14-3-3 expression (Fig.3 A). Inasmuch as the identity of specific endogenous 14-3-3-associated polypeptide bands is not known, Myc-Raf was coexpressed with GST-14-3-3 to serve as a reporter (Fig.3 B). Calyculin A pretreatment abrogates completely the association of Myc-Raf with 14-3-3 (Fig. 3 B, comparelane 1 with lane 4 andlane 2 with lane 5); a similar inhibition is observed for the association of endogenous c-Raf-1 with GST 14-3-3 in calyculin A-treated cells (Fig.3 C, compare lane 2 withlane 4). The loss of the 14-3-3/Raf association and 14-3-3 association with other targets caused by calyculin Ain vivo is not due to a modification of c-Raf-1 or the other target proteins that impair their ability to bind 14-3-3, since Raf and the other targets from extracts of untreated or calyculin A-treated cells bind equally well to prokaryotic recombinant GST 14-3-3 in vitro (Fig. 3, D and E). Thus, a more feasible explanation for the calyculin A-induced inhibition of 14-3-3 association with Raf and other target proteins in vivo is that the phosphovimentin that accumulates in the calyculin A-treated cells simply outcompetes and displaces Raf and the other 14-3-3 partners. It has previously been shown that 14-3-3 serves as an indispensable cofactor for Raf kinase activity (16Thorson J.A., Yu, L.W. Hsu A.L. Shih N.Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell. Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (186) Google Scholar, 17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar). This, together with the ability of calyculin A treatment to disrupt 14-3-3/Raf complexes in vivo, implied that the regulation of Raf kinase activity in calyculin A-treated cells would be impaired. Thus, we decided to use Raf kinase activity as a functional read-out assay for 14-3-3 availability in the cell. Incubation of serum-deprived COS cells with calyculin A caused a slight increase in Raf kinase activity but inhibited by 95% the EGF-induced activation of Raf (Fig. 4 A). To confirm that this low activity was due to 14-3-3 limitation in vivo, we examined the ability of purified prokaryotic recombinant 14-3-3 to restore the activity of Raf extracted from calyculin A-treated cells (Fig. 4 B). Consistent with earlier findings, the addition of recombinant 14-3-3 to Raf from unstimulated cells (Fig.4 B, compare lane 2 withlane 8) or EGF-treated cells (Fig. 4 B, compare lanes 3 and 4 withlane 9) had no effect on Raf kinase activity. In contrast, the addition of 14-3-3 to Raf extracted from calyculin A-treated cells greatly increased Raf kinase activity (Fig.4 B, no EGF, compare lane 5 withlane 10; with EGF, compare lane 6 with lanes 11 and 12). The ability of 14-3-3 to restore Raf activity in extracts of calyculin A-treated cells was completely blocked by the Raf synthetic phosphopeptide (Fig. 4 B, lane 13), demonstrating that 14-3-3 ability to reactivate Raf requires the 14-3-3 phosphopeptide binding domain. Moreover, a dimerization-deficient 14-3-3 mutant, which is unable to reactivate Raf deactivated in vitro after phosphopeptide-induced displacement of native 14-3-3 (17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar), is also unable to activate Raf extracted from calyculin A-treated cells (Fig. 4 B, lane 14). To further ascertain whether the inability of EGF to activate Raf in calyculin A-treated cells is due to a limitation in the availability of endogenous 14-3-3, increasing amounts of GST-14-3-3 were cotransfected together with Myc-Raf, and the effect of calyculin A pretreatment on EGF-stimulated Raf activation was assayed (Fig. 4 C). Transfection of as little as 10 ng/plate of pEBG-GST-14-3-3 DNA was able to substantially overcome the calyculin A-induced inhibition of Raf activation, and at 160 ng/plate, transfection resulted in complete restoration of Raf activity to levels seen with EGF stimulation (Fig.4 C). The requirement for wild type 14-3-3 is shown by the inability of the pEBG vector (i.e. GST alone; Fig.4 C, lanes 2–4) as well as the pEBG-encoded dimerization-deficient mutant 14-3-3 (Fig. 4 C,lanes 17–19), each added at 640 ng/plate, to increase Raf activity in calyculin A-treated cells. It is notable that the expression of recombinant 14-3-3 does not alter Raf activity in cells not exposed to calyculin A, whether unstimulated (Fig.4 C, compare lane 2 withlanes 5, 8, 11, and14) or treated with EGF (Fig. 4 C, comparelane 3 with lanes 6,9, 12, and 13), confirming that 14-3-3 overexpression does not activate Raf per se. Rather, 14-3-3 overexpression specifically overcomes the inhibition caused by calyculin A pretreatment. These results demonstrate that the calyculin A-induced vimentin binding to 14-3-3 results in limitation in 14-3-3 availability to Raf and in altered regulation of its kinase activity. 14-3-3 proteins have been found to specifically associate with and participate in the regulation of many proteins including several key proteins involved in cellular signaling and cell cycle control. The present work identifies vimentin as an additional, regulated, 14-3-3 interactor. Vimentin undergoes extensive phosphorylation during cell cycle progression, and the inhibition of protein phosphatase 1 and 2A by calyculin A causes a rapid increase in vimentin phosphorylation comparable in magnitude with that occurring during mitosis, although with a somewhat different site specificity (25Eriksson J.E. Brautigan D.L. Vallee R. Olmsted J. Fujiki H. Goldman R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11093-11097Crossref PubMed Scopus (165) Google Scholar). The ability of calyculin A treatment to promote sufficient phosphorylation of endogenous vimentin to enable it to bind a very large fraction of cellular 14-3-3 raises two intriguing possibilities. First, it suggests that 14-3-3 may contribute to the regulation of vimentin phosphorylation/dephosphorylation and/or to the ability of vimentin to participate in filament formation. Second, the high stoichiometry of cellular vimentin phosphorylation achieved during specific phases of the cell cycle may bind sufficient cellular 14-3-3 to limit its availability, thereby modulating the function of other 14-3-3-dependent proteins. Previous work has shown that the binding of 14-3-3 to phosphoproteins, such as Raf, can diminish their susceptibility to protein (Ser/Thr) phosphatase action (28Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (175) Google Scholar). Thus, the association of 14-3-3 with phosphovimentin may alter vimentin dephosphorylation. As regards the impact of 14-3-3 on intermediate filament assembly, Liao et al. (8Liao J. Omary M.B. J. Cell Biol. 1996; 133: 345-357Crossref PubMed Scopus (180) Google Scholar) demonstrated that binding of 14-3-3 to keratin K18 could interfere with its assembly into filaments in vitro. 14-3-3 binding to K18 depends on keratin Ser33phosphorylation; however, this phosphorylation is not sufficient by itself for conferring association (14Ku N.O. Liao J. Omary M.B. EMBO J. 1998; 17: 1892-1906Crossref PubMed Scopus (196) Google Scholar). Since the sequence around keratin Ser33 does not represent a strong 14-3-3 motif, it seems that another 14-3-3 binding site(s) on K18 may collaborate with the Ser33 site to support 14-3-3 binding. This possibility is strengthened by our finding that vimentin, which, like K18, does not contain an optimal high affinity 14-3-3 biding site, can form a stable association only with dimeric 14-3-3 and not with various 14-3-3 monomers. The importance of 14-3-3 dimerization in 14-3-3 function has not gained much attention; the functional role usually proposed for the 14-3-3 dimer is to allow the bridging of two different proteins (29Luo Z.-J. Tzivion G. Belshow P.J. Marshall M. Avruch J. Nature. 1996; 383: 181-185Crossref PubMed Scopus (209) Google Scholar, 30Marshal C.J. Nature. 1996; 383: 127-128Crossref PubMed Scopus (100) Google Scholar, 31Braselmann S. McCormick F. EMBO J. 1995; 14: 4839-4848Crossref PubMed Scopus (177) Google Scholar). For proteins that lack high affinity 14-3-3 binding sites, such as vimentin, keratin K18, and Wee1 (3Honda R. Ohba Y. Yasuda H. Biochem. Biophys. Res. Commun. 1997; 230: 262-265Crossref PubMed Scopus (45) Google Scholar), the coligation of two lower affinity sites by the 14-3-3 dimer may be necessary to enable stable target protein recognition and binding. The role of 14-3-3 dimerization extends, however, beyond the ability to bridge two different proteins and enable binding to single proteins with only low affinity sites. We recently showed that a 14-3-3 dimer is necessary to support Raf activity (17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar) despite the fact that Raf contains two high affinity binding sites for 14-3-3, each of which is independently sufficient to stably bind 14-3-3 (17Tzivion G. Luo Z.-J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (394) Google Scholar, 32Rommel C. Radziwill G. Lovric J. Noeldeke J. Heinicke T. Jones D. Aitken A. Moelling K. Oncogene. 1996; 12: 609-619PubMed Google Scholar). The present report extends those findings by demonstrating that not only is the dimerization-deficient 14-3-3 unable to support the reactivation of Raf inactivated by 14-3-3 displacement in vitro, it is also unable to support an active configuration of Raf achieved in vivo in calyculin A-treated cells. Along similar lines, we recently observed that the ability of 14-3-3 to modulate the DNA binding of the Caenorhabditis elegans transcription factor DAF-16 requires a dimeric 14-3-3 and two independent 14-3-3 binding sites on the DAF-16 polypeptide. 2C. M. Cahill, G. Tzivion, N. Nasrin, S. Ogg, J. Dore, G. Ruvkun, and M. Alexander-Bridges, submitted for publication. The finding that vimentin phosphorylation and its concomitant binding to 14-3-3 proteins results in disassembly of Raf-14-3-3 complexes and other 14-3-3-protein complexes suggests that the association of phosphovimentin with 14-3-3 may have significance in the regulation of 14-3-3 availability besides a role in vimentin dephosphorylation/disaggregation. This intriguing possibility implies that phosphorylation of vimentin and perhaps other IF polypeptides (e.g. keratin K18) may affect various intracellular signaling and cell cycle control pathways by modifying 14-3-3 availability, as observed with Raf in this paper. Further evaluation of the cellular distribution of the different 14-3-3 isoforms under varying physiologic conditions and during different stages of the cell cycle and the effect of IF phosphorylation on this distribution is needed to ascertain whether variation in the availability of 14-3-3 by IFs serves as a regulatory factor in cellular signaling and cell cycle control. We thank J. Prendable for preparation of the manuscript and W. L. Lane and the Harvard Microchemistry facility for microsequence analysis. PP2 was a kind gift from D. L. Brautigan.

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