Cell BioIlogy 14-3-3 proteins associate with cdc 25 phosphatases

Content Provider	Semantic Scholar
Author	Conklin, Douglas S. Galaktionov, Konstantin I. Beach, David S.
Copyright Year	2005
Abstract	The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog ofthe 14-3-3e protein and the previously described 14-3-3f8 protein have been isolated in this screening. 14-3-3 proteins constitute a family of wellconserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery. In eukaryotic cells, the cyclin-dependent kinases (cdks) regulate cellular functions critical to passage through successive phases of the cell cycle (1). The activity of these kinases is regulated both by association with ancillary proteins and by phosphorylation. Foremost among cdk-associated proteins are the cyclins, which function as positive regulatory subunits for specific members of the cdk family. Several recently described cdk inhibitors, p15 (2), p16 (3), p21 (4-6), and p27 (7, 8), also physically interact with cdks. The expression of these proteins increases in response to cellular damage or extracellular growth inhibitory signals, resulting in the inhibition of kinase activity. cdk activity is also regulated by phosphorylation. Phosphorylation of a threonine residue within the catalytic domain of cdks by the cdk-activating kinase (CAK) is required for kinase activity (9). Inhibitory phosphorylation of threonine and tyrosine residues in the ATP-binding site of most cdks inhibits kinase activity (10). These inhibitory sites are conserved between fission yeast and several metazoan cdks and are found near the N termini, usually at residues Thr-14 and Tyr-15 (11). The phosphorylation state of Thr-14 and Tyr-15 residues of fission yeast cdc2 is a major determinant of the onset of mitosis and is controlled by the opposing actions of the weel/mikl kinases and the cdc25 phosphatase (12-15). weel kinases are now known from many species (16-18) and have been shown to phosphorylate Tyr-15 (12, 13). cdc25 phosphatases, although weakly related to the main family of protein phosphatases, have been shown to specifically dephosphorylate Thr-14 and Tyr-15 (15, 19-23). Yeasts have a single cdc25 gene (16, 24), whereas human cells possess three related cdc25 genes: CDC25A, -B, and -C (25, 26). The activity of the cdc25 phosphatases is also regulated by phosphorylation. In human cells, cdc25C is activated at the onset of mitosis by cdc2-cyclin B phosphorylation (27). Since cdc25C phosphatase activity stimulates cdc2 kinase activity, phosphorylation by cdc2-cyclin B may result in the formation of an auto-amplification loop with increased cdc25C activity further activating cdc2 kinase activity. cdc25A, which plays a crucial role early in the cell cycle (28), has been shown to be phosphorylated by cdk2-cyclin E at the G1/S transition (29). Recently, the Raf-1 protooncogene kinase has been found to form a tight complex with cdc25 phosphatases both in somatic cells and in Xenopus oocytes (30). Raf-1-dependent kinase activity phosphorylates and stimulates cdc25A activity. This constitutes a major link between mitogenic signaling and cell cycle control. To identify other potential regulators of cdc25, a yeast two-hybrid protein interaction screening was undertaken to identify proteins that interact with the human cdc25 phosphatases. Two interacting clones were found to encode 14-3-3 isoforms.t These proteins are members of a family of proteins that are known to associate with components of several signal transduction pathways, including the Raf-1 kinase cascade. This interaction is likely to play some role in linking mitogenic signaling to the regulation of cdc25 activity. MATERIALS AND METHODS Two-Hybrid Screens. Saccharomyces cerevisiae strains HF7C (31) and L40 (32) were used as hosts for two-hybrid vectors. HeLa cell two-hybrid cDNA libraries were gifts of G. Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). cdc25A and cdc25B were fused to the sequence encoding the GAL4 DNA-binding domain of pGBT9. Yeast cells were simultaneously transformed with GAL4 DNA-binding domain (GAL4BD) fusion plasmids and GAL4 transcriptional activation domain (GAL4AD) fusion plasmids. Leu+ Trp+ transformants, which contain both GAL4BD and GAL4AD fusion plasmids, were streaked on plates with or without histidine to determine whether the encoded proteins interacted. Yeast cells were grown in YPD (2% glucose/2% peptone/1% yeast extract) or synthetic minimal media (2% glucose/0.67% yeast nitrogen base, with appropriate auxotrophic supplements) by using standard techniques. Plasmids carrying sequences encoding Ras and Raf-1 fusions (33) were gifts of L. VanAelst (Cold Spring Harbor Laboratory). Binding Experiments. A fusion of maltose-binding protein (MBP) and 14-3-313 for expression in bacteria was constructed by removing the open reading frame from pGBT9-14-3-3,B by using PCR with primers to flanking DNA. The resulting fragment was inserted into the EcoRI and Pst I sites of pBluescript KS (-) (Stratagene), producing plasmid p14-33KS-. In vitro translation and DNA sequence analysis confirmed that the wild-type 14-3-31 open reading frame had been subcloned without the introduction of errors. The MBP-143-3,B fusion was constructed by inserting the entire 14-3-313 Abbreviations: cdk, cyclin-dependent kinase; MBP, maltose-binding protein; GST, glutathione S-transferase. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. U20972). 7892 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 92 (1995) 7893 open reading frame into pMAL-c2 (New England Biolabs). Construction of the glutathione S-transferase (GST)-cdc25A fusion has been described (25). Each protein was purified on either glutathione-Sepharose or amylose-Sepharose columns by using standard procedures. Purified proteins were incubated at approximate final concentrations of 500 nM in 50mM Tris-HCl, pH 7.5/150mM NaCl/0.5% Nonidet P-40 for 30 min on ice. Complexes were subsequently precipitated by the 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 SDS/PAGE and immunoblotting. The immunoblot was probed with anti-MBP-antibody (New England Biolabs). Staphylococcal proteinA coupled with horseradish peroxidase HRP (Amersham) and the ECL reagent (Amersham) were used to detect bound antibody in all immunoblots. Immunoprecipitation and Antibody Production. HeLa cells, grown in suspension to a density of 2 x 106 cells per ml, were pelleted by centrifugation at 1000 x g for 10 min, washed three times with phosphate-buffered saline, and lysed in a hypotonic lysis buffer (10 mM Tris HCl, pH 7.5/5 mM MgCl2/25 mM NaF/1 mM EGTA/1 mM dithiothreitol/1 mM sodium orthovanadate containing aprotinin at 10 jig/ml, leupeptin at 10 ,mg/ml, pepstatin at 0.5 ,ug/ml, chymostatin at 1 ,ug/ml, 1 mM benzamidine, and 0.5 mM phenylmethanesulfonyl fluoride). Soluble proteins from 1.5 x 109 cells were subjected to immunoprecipitation with S jig of affinity-purified anti-143-33 antibody, anti-cdc25A antibody, or preimmune rabbit IgG. Immunoprecipitations were carried out essentially as previously described (3). Antibody against human 14-3-3,3 was raised against purified MBP-14-3-313 fusion protein in rabbits and affinity purified on a column containing Sepharose 4B-coupled, hexahistidinetagged human 14-3-3,3 (see below). Antibodies against Raf-1 (C20) were purchased from Santa Cruz Biotechnology. Antibodies against cdc25A have been described (30). Samples for detection of Raf-1 and cdc25A were analyzed on 7.5% polyacrylamide gels. Samples for detection of 14-3-3 were analyzed on 12.5% gels. Baculovirus Infections. A baculovirus which directs the expression of the entire 14-3-313 protein was constructed with the vector pLV1393 and the Baculo-gold kit (PharMingen). This was used to infect Spodoptera frugiperda (Sf9) cells grown in monolayer at a multiplicity of infection of 5, either alone or in combination with viruses directing the expression of cdc25A and Raf-1. Labeling with [35S]methionine indicated that approximately equal amounts of each protein were produced in both doubly and triply infected cells. Insect cells were lysed at 60-72 h after infection in an NP40 lysis buffer (50 mM Tris-HCl, pH 7.5/150 mM NaCl/0.5% Nonidet P-40/1 mM dithiothreitol containing 1 mM sodium orthovanadate, aprotinin at 10 jig/ml, leupeptin at 10 ,ug/ml, pepstatin at 0.5
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