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14-3-3 Proteins Associate with Cdc25 Phosphatases DOUGLAS S

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14-3-3 Proteins Associate with Cdc25 Phosphatases DOUGLAS S Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7892-7896, August 1995 Cell BioIlogy 14-3-3 proteins associate with cdc25 phosphatases DOUGLAS S. CONKLIN, KONSTANTIN GALAKTIONOV, AND DAVID BEACH* Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 Communicated by Marc W Kirschner, Harvard Medical School, Boston, MA, May 19, 1995 ABSTRACT The cdc25 phosphatases play key roles in cell onset of mitosis by cdc2-cyclin B phosphorylation (27). Since cycle progression by activating cyclin-dependent kinases. Two cdc25C phosphatase activity stimulates cdc2 kinase activity, members of the 14-3-3 protein family have been isolated in a phosphorylation by cdc2-cyclin B may result in the formation yeast two-hybrid screen designed to identify proteins that of an auto-amplification loop with increased cdc25C activity interact with the human cdc25A and cdc25B phosphatases. further activating cdc2 kinase activity. cdc25A, which plays a Genes encoding the human homolog ofthe 14-3-3e protein and crucial role early in the cell cycle (28), has been shown to be the previously described 14-3-3f8 protein have been isolated in phosphorylated by cdk2-cyclin E at the G1/S transition (29). this screening. 14-3-3 proteins constitute a family of well- Recently, the Raf-1 protooncogene kinase has been found to conserved eukaryotic proteins that were originally isolated in form a tight complex with cdc25 phosphatases both in somatic mammalian brain preparations and that possess diverse cells and in Xenopus oocytes (30). Raf-1-dependent kinase biochemical activities related to signal transduction. We activity phosphorylates and stimulates cdc25A activity. This present evidence that indicates that cdc25 and 14-3-3 proteins constitutes a major link between mitogenic signaling and cell physically interact both in vitro and in vivo. 14-3-3 protein does cycle control. not, however, affect the phosphatase activity of cdc25A. Raf-1, To identify other potential regulators of cdc25, a yeast which is known to bind 14-3-3 proteins, has recently been two-hybrid protein interaction screening was undertaken to shown to associate with cdc25A and to stimulate its phos- identify proteins that interact with the human cdc25 phos- phatase activity. 14-3-3 protein, however, has no effect on the phatases. Two interacting clones were found to encode 14-3-3 cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate isoforms.t These proteins are members of a family of proteins the association ofcdc25 with Raf-1 in vivo, participating in the that are known to associate with components of several signal linkage between mitogenic signaling and the cell cycle ma- transduction pathways, including the Raf-1 kinase cascade. chinery. This interaction is likely to play some role in linking mitogenic signaling to the regulation of cdc25 activity. In eukaryotic cells, the cyclin-dependent kinases (cdks) regu- late cellular functions critical to passage through successive phases of the cell cycle (1). The activity of these kinases is MATERIALS AND METHODS regulated both by association with ancillary proteins and by Two-Hybrid Screens. Saccharomyces cerevisiae strains HF7C phosphorylation. Foremost among cdk-associated proteins are (31) and L40 (32) were used as hosts for two-hybrid vectors. the cyclins, which function as positive regulatory subunits for HeLa cell two-hybrid cDNA libraries were gifts of G. Hannon specific members of the cdk family. Several recently described (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). cdk inhibitors, p15 (2), p16 (3), p21 (4-6), and p27 (7, 8), also cdc25A and cdc25B were fused to the sequence encoding the physically interact with cdks. The expression of these proteins GAL4 DNA-binding domain of pGBT9. Yeast cells were increases in response to cellular damage or extracellular simultaneously transformed with GAL4 DNA-binding domain growth inhibitory signals, resulting in the inhibition of kinase (GAL4BD) fusion plasmids and GAL4 transcriptional activa- activity. tion domain (GAL4AD) fusion plasmids. Leu+ Trp+ transfor- cdk activity is also regulated by phosphorylation. Phosphor- mants, which contain both GAL4BD and GAL4AD fusion ylation of a threonine residue within the catalytic domain of plasmids, were streaked on plates with or without histidine to cdks by the cdk-activating kinase (CAK) is required for kinase determine whether the encoded proteins interacted. Yeast activity (9). Inhibitory phosphorylation of threonine and ty- cells were grown in YPD (2% glucose/2% peptone/1% yeast rosine residues in the ATP-binding site of most cdks inhibits extract) or synthetic minimal media (2% glucose/0.67% yeast kinase activity (10). These inhibitory sites are conserved nitrogen base, with appropriate auxotrophic supplements) by between fission yeast and several metazoan cdks and are found using standard techniques. Plasmids carrying sequences en- near the N termini, usually at residues Thr-14 and Tyr-15 (11). coding Ras and Raf-1 fusions (33) were gifts of L. VanAelst The phosphorylation state of Thr-14 and Tyr-15 residues of (Cold Spring Harbor Laboratory). fission yeast cdc2 is a major determinant of the onset of mitosis Binding Experiments. A fusion of maltose-binding protein and is controlled by the opposing actions of the weel/mikl (MBP) and 14-3-313 for expression in bacteria was constructed kinases and the cdc25 phosphatase (12-15). weel kinases are by removing the open reading frame from pGBT9-14-3-3,B by now known from many species (16-18) and have been shown using PCR with primers to flanking DNA. The resulting to phosphorylate Tyr-15 (12, 13). cdc25 phosphatases, al- fragment was inserted into the EcoRI and Pst I sites of though weakly related to the main family of protein phos- pBluescript KS (-) (Stratagene), producing plasmid p14-3- phatases, have been shown to specifically dephosphorylate 3KS-. In vitro translation and DNA sequence analysis con- Thr-14 and Tyr-15 (15, 19-23). Yeasts have a single cdc25 gene firmed that the wild-type 14-3-31 open reading frame had been (16, 24), whereas human cells possess three related cdc25 subcloned without the introduction of errors. The MBP-14- genes: CDC25A, -B, and -C (25, 26). The activity of the cdc25 phosphatases is also regulated by 3-3,B fusion was constructed by inserting the entire 14-3-313 phosphorylation. In human cells, cdc25C is activated at the Abbreviations: cdk, cyclin-dependent kinase; MBP, maltose-binding protein; GST, glutathione S-transferase. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U20972). Downloaded by guest on September 26, 2021 7892 Cell Biology: Conklin et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7893 open reading frame into pMAL-c2 (New England Biolabs). Ag/ml, chymostatin at 1 Ag/ml, 1 mM benzamidine, and 0.5 Construction of the glutathione S-transferase (GST)-cdc25A mM phenylmethanesulfonyl fluoride). A soluble extract was fusion has been described (25). Each protein was purified on prepared by repeated passage of cells through a 26 gauge either glutathione-Sepharose or amylose-Sepharose columns needle followed by the removal of cellular debris by centrifu- by using standard procedures. Purified proteins were incu- gation. Hexahistidine-tagged human 14-3-313 was prepared bated at approximate final concentrations of 500 nM in 50 mM from insect cells infected with a baculovirus expressing the Tris-HCl, pH 7.5/150 mM NaCl/0.5% Nonidet P-40 for 30 min entire 14-3-313 protein fused in frame to the six-histidine tag on ice. Complexes were subsequently precipitated by the sequence of pAcSGHisNT-A (PharMingen). addition of 50 Al of a 1:1 slurry of glutathione-Sepharose and the above buffer. Beads were washed four times with the above buffer, heated to 95°C in sample buffer (55), and subjected to RESULTS SDS/PAGE and immunoblotting. The immunoblot was Isolation of cdc25A-Binding Proteins. A two-hybrid screen- probed with anti-MBP-antibody (New England Biolabs). ing was undertaken to identify proteins that physically interact Staphylococcal protein A coupled with horseradish peroxidase with the human cdc25 phosphatases. Human cdc25A and HRP (Amersham) and the ECL reagent (Amersham) were cdc25B genes, fused in frame to the GAL4 DNA-binding used to detect bound antibody in all immunoblots. domain vector pGBT9, were each cotransformed into yeast Immunoprecipitation and Antibody Production. HeLa cells, with a HeLa cell-derived cDNA library contained in the GAL4 grown in suspension to a density of 2 x 106 cells per ml, were transcriptional activation domain vector, pGADGH. Thirty- pelleted by centrifugation at 1000 x g for 10 min, washed three four clones interacted with the cdc25 constructs sufficiently to times with phosphate-buffered saline, and lysed in a hypotonic allow growth of the transformed host on media lacking histi- lysis buffer (10 mM Tris HCl, pH 7.5/5 mM MgCl2/25 mM dine and to express detectable levels of 13-galactosidase activ- NaF/1 mM EGTA/1 mM dithiothreitol/1 mM sodium or- ity. thovanadate containing aprotinin at 10 jig/ml, leupeptin at 10 Among these positives were two members of the 14-3-3 ,mg/ml, pepstatin at 0.5 ,ug/ml, chymostatin at 1 ,ug/ml, 1 mM family of proteins. One of 10 interaction-positive clones found benzamidine, and 0.5 mM phenylmethanesulfonyl fluoride). with cdc25A encoded the previously identified (34) 14-3-313 Soluble proteins from 1.5 x 109 cells were subjected to isoform (Fig. 1). Eight of 24 interaction-positive clones found immunoprecipitation with S jig of affinity-purified anti-14- with cdc25B encoded the human homolog of the mouse 3-33 antibody, anti-cdc25A antibody, or preimmune rabbit 14-3-3E isoform (35).
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