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14-3-3 Proteins in Cell Regulation Affinity purification of diverse plant and human 14-3-3-binding partners F. C. Milne, G. Moorhead, M. Pozuelo Rubio, B. Wong, A. Kulma, J. E. Harthill, D. Villadsen, V. Cotelle and C. MacKintosh' MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD I 5EH, Scotland, U.K. Abstract the discovery that 14-3-3s inhibit phosphorylated nitrate reductase [4,5]. This mechanism is triggered by a nutrient-sensing pathway that functions to inhibit nitrate assimilation in the dark, or whenever photosynthesis is blocked. There are at least two physiological reasons to co-ordinate nitrate reductase activity with photosynthesis. First, rates of nitrate assimilation must not exceed the supplies of reductant or carbohydrate that are required to combine with the reduced nitrogen to form amino acids. Both the reductant and carbohydrate are produced by photosynthesis. Secondly, the product of nitrate reductase, nitrite, is very toxic. Photosynthesis is required to produce the reductant to convert nitrite into ammonia, which means that there is a danger of nitrite poisoning in the dark. Poisoning is prevented, however, by rapid phosphorylation of nitrate reductase and its inhibition by 14-3-3 proteins. In these ways, 14-3-3s can be considered to minimize the major costs and potential hazards associated with nitrate assimilation [6,7]. More recently, further plant proteins that bind to 14-3-3s in a phosphorylation-dependent manner have been identified, and it is striking that many of these targets are involved in primary sugar, amino acid and energy metabolism, including glutamine synthetases, an isoform of trehalose-phosphate synthase, sucrose-phosphate synthase and A T P synthase /3 [8-111. These findings indicate that 14-3-3s are likely to play key roles in determining the balance of carbon and nitrogen assimilation, storage and distribution in higher plants. T h e biochemical studies needed to identify the relevant regulatory phosphorylation and 14-3-3-binding sites on these proteins are underway, preliminary to investigating the global physiological functions of 14-3-3s in plant metabolism. Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants. Our results also suggest that the current perception that 14-3-3s bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen because of the intensity of research on mammalian signalling and for technical reasons. 14-3-3s in regulation of light-coupled metabolism in plants 14-3-3s are a highly conserved family of proteins that generally bind to phosphopeptide motifs in diverse target proteins, and play central regulatory roles in all eukaryotic cells. Recently, 14-3-3 proteins have emerged as components of the signalling mechanisms that regulate light-coupled metabolic fluxes in leaves. Green leaves have the remarkable ability to use A T P and reductants generated in the light to fix CO, into sugars, and assimilate nitrate and carbohydrate into amino acids. These lightcoupled biosynthetic processes are highly responsive to the environment and physiological status of the plant [1,2]. Detailed measurements of enzyme activities and metabolites have revealed complicated and shifting regulatory relationships among the biosynthetic pathways. Subsets of metabolites and enzymes activities display distinctive diurnal variations, whose exact patterns are determined by signals from phosphorylated sugars, p H sensors, malate, nitrate and hormones [3]. T h e first identified role for 14-3-3s in the regulation of light-coupled metabolism came with 14-3-3 affinity chromatography of plant proteins One procedure that proved very useful for identifying novel plant 14-3-3-binding proteins is 14-3-3 affinity chromatography, followed by matrix-assisted laser-desorption ionization-time- Key words 14-3-3 proteins, nitrate reductase, vesicle trafficking 'To whom correspondence should be addressed (e-mail c rnackintosh(u1dundee ac uk) 379 0 2002 Biochemical Society Biochemical Society Transactions (2002) Volume 30, part 4 differences in the types of 14-3-3-binding partners found in the Plant and Animal Kingdoms seemed odd, given the extremely high conservation of 14-3-3 sequences and the common mechanism of interaction of 14-3-3s with phosphopeptide motifs in target proteins. Are 14-3-3s really specialized in signalling in mammalian cells, or have perceptions been biased by the intensity of research on mammalian signalling and for technical reasons? One potential bias is that there are only a few reports of 14-3-3 proteins being used as bait in yeast two-hybrid screens [15], although 14-3-3s are often picked up with other proteins as bait (e.g. [16-201 and many others). This may be because 14-3-3 proteins exist as dimers and binding of 14-3-3 bait to endogenous or recombinant 14-3-3s can occur to the exclusion of other interactions [21-231. Deleting the dimerization site in the 14-3-3 bait may be useful. Certainly, truncated monomeric forms of 14-3-3s can bind Raf-1 [24], although results from yeast two-hybrid screens with monomeric 14-3-3s as baits have not been reported widely. Interaction cloning from bacterial expression libraries, which of-flight (MALDI-TOF) tryptic mass fingerprinting of purified proteins (Figure 1) [8]. Generally, protein-protein chromatography is not widely used because non-specific binding is a common problem. An important step in the 14-3-3 affinity chromatography procedure, though, is the specific elution of bound proteins by competition with a synthetic phosphopeptide carrying the canonical 14-3-3-binding motif, ARAApSAR (where pS is phosphoserine). In this way, the pool of purified proteins is highly enriched in proteins that bind directly to the phosphopeptide-binding site on 14-3-3s. 14-3-3 affinity purification of human proteins of signalling, metabolism, vesicle trafficking and chromatin function One reason that we were intrigued to find 14-3-3s as regulators of enzymes in primary plant metabolism is that the majority of the known 14-3-3binding proteins in mammalian cells are enzymes of oncogenic signalling pathways and cell-cycle regulators (reviewed in [12-141). T h e apparent Figure I 14-3-3 affinity chromatography Cell and tissue extracts are prepared in buffers containing protein phosphatase inhibitorst o maintain the phosphorylation and 14-3-3-binding capability of target proteins. The proportion of any particular target protein that binds t o the column depends on the proportion that is phosphorylated under the conditions of cell extraction, and competition for the target protein between endogenous 14-3-3 proteins in the extract and immobilized 14-3-3 proteins on the column. After extensive washing in salt and with control peptides, binding proteins are eluted from the column with a synthetic 14-3-3-binding phosphopeptide. The elution pool contains proteins that bind directly and indirectly to 14-3-3s.Those proteins that bind directly to 14-3-3s can be identified by binding to 14-3-3s in overlay assays. - Plant/hurnan cell extract 14-3-3 column ‘=I C A -R-A- A- pS- A-P- A Washes NaCl 0.5M Control peptides Analys is SOS-PAGE 14-3-3 overlays Binding +/- dephosphorylation Mass spec fingerprinting + Western blotting 0 2002 Biochemical Society 3 80 14-3-3 Proteins in Cell Regulation 5 is another common method for identifying protein-protein interactions, is not suitable if 14-3-3binding proteins have to be phosphorylated. Therefore, to determine whether or not 14-3-3s have related functions in both plants and mammals, we applied 14-3-3 affinity chromatography to purify phosphorylated proteins from human HeLa cells and bovine brain. Of course, it may be that this method has its own bias, in that it is easier to identify those purified proteins that are more abundant. Nevertheless, we have recently purified many mammalian proteins by this method. As the purified proteins are being identified and characterized, it seems striking that all the proteins purified to date fall into four categories : signalling components, enzymes of primary metabolism, proteins of vesicle and protein trafficking, and proteins that regulate chromatin function. Interestingly, the 14-3-3-affinity purified proteins do include mammalian counterparts of known plant 14-3-3-binding proteins, such as the mitochondria1 A T P synthase p subunit (B. Wong, M. Pozuelo Rubio and C. MacKintosh, unpublished work). In addition to the evolutionary aspects, these findings have other implications. For example, from the phenotypes of Dvosophila and yeast mutants, 14-3-3s have been implicated in neuronal differentiation, learning and memory [25,26], regeneration of damaged tissue [27] and vesicle trafficking [28,29]. T h e rationale for these phenotypes has often been discussed in terms of 14-3-3 interactions with signalling pathways. Our findings suggest that 14-3-3s regulate not only upstream signalling events but participate more directly in cellular events such as vesicle trafficking and metabolism. 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 References I 2 3 4 26 Bryce, J. H. and Hill, S. A. ( I 999) in Plant Biochemistry and Molecular Biology (Lea, P. J. and Leegood, R. C., eds), pp. 1-28, John Wiley and Sons, Chichester Lea, P. J. ( I 999) in Plant Biochemistry and Molecular Biology (Lea, P. J, and Leegood, R. C., eds), pp. 163- 192, John Wiley and Sons, Chichester Stitt. M., Muller, C., Matt, P., Gibon, Y., Carillo, P., Morcuende, R., Scheible, W.-R. and Krapp, A. (2002) J. EXP. Bot. 53, 959-970 Bachmann. M., Huber, J. L., Liao, P. C., Gage, D. A. and Huber, S. C. ( I 996) FEBS Lett, 387, 127- I3 I 27 28 29 Moorhead. G., Douglas, P., Momce, N.,Scarabel, M., Aitken, A. and MacKintosh, C. ( I 996) Cum Biol. 6, 1104-1 I13 MacKintosh, C. and Meek, S. E. (200 I) Cell. Mol. Life Sci. 58,205-2 I 4 Kaiser, W. M. and Huber, S. C. (2001) J. Exp. Bot. 52, I98 I -I 989 Moorhead, G.. Douglas, P.. Cotelle, V., Harthill, J., Momce, N., Meek, S., Deiting, U., Stitt, M., Scarabel, M., Aitken. A. and MacKintosh, C. (1999) Plant J. 18. 1-1 2 Toroser, D., Athwal, G. S. and Huber, S. C. ( I 998) FEBS Lett. 435, I 10- I I4 Finnernan. J. and Schjoemng,J. K. (2000) Plant J.24, 171-181 Bunney, T. D.. van Walraven, H. S. and de Boer, A. H. (200 I ) Proc. Natl. Acad. Sci. U.S.A. 98, 42494254 van Hemert, M. J., Steensma, H. Y. and van Heusden, G. P. (2001) Bioessays 23,936-946 Muslin, A. J. and Xing, H. (2000) Cell. Signal. 12, 703-709 Roberts, M. R. (2000) Cum. Opin. Plant Biol. 3, 400405 Yan, J., Wang, J. and Zhang. H. (2002) Plant J. 29, 193-202 Li. S., Janosch, P., Tanji, M., Rosenfeld, G. C.. Wayrnire, J. C., Mischak H., Kolch, W. and Sedivy, J. M. ( I 995) EMBO J. 14, 685-696 Furlanetto, R. W., Dey, B. R., Lopaczynski, W. and Nissley, S. P. ( I 997) Biochem. J. 327,765-77 I Domer, C., Ullrich, A., Haring, H. U. and Lammers, R. ( I 999) J. Biol. Chem. 274, 33654-33660 Pierrat, B., Bo, M., Hinz, W., Simonen, M., Erdmann, D., Chiesi, M. and Heim, J. (2000) Eur. J. Biochem. 267, 2680-2687 Jeanclos, E. M., Lin, L., Treuil, M. W.. Rao, J, DeCoster, M. A. and Anand, R. (2001) J. Biol. Chem. 276, 28281-28290 Xiao, 6.. Srnerdon, S. 1.. Jones, D. H., Dodson, G. G., Soneji. Y., Altken, A. and Garnblin, S. J. ( I 995) Nature (London) 376, 188- I9 I Liu. D., Bienkowska. J., Petosa, C., Collier, R. J., Fu. H. and Liddington, R. ( I 995) Nature (London) 376, I9 I - I94 Wu, K.. Lu, G., Sehnke, P. and Fed. R. J. (1997) Arch. Biochern. Biophys. 339, 2-8 Lu, Z. J., Zhang. X. F., Rapp. U. and Avruch. J. ( I 995) J. Biol. Chem. 270,2368 1-23687 Philip, N.,Acevedo, S. F. and Skoulakis. E. M. (2001) J. Neurosci. 2 I,84 17-8425 Skoulakis, E. M. and Davis, R L. ( I 998) Mol. Neurobiol. 16, 269-284 Namikawa, K., Su, Q., Kiryu-Seo, S. and Kiyama, H. ( I 998) Brain Res. Mol. Brain Res. 55, 3 15-320 Gelperin, D., Weigle, J., Nelson, K., Roseboom, P., Irie, K., Matsumoto, K. and Lemmon, S. ( I 995) Proc. Natl. Acad. Sci. U.S.A. 92, I 1539- I I543 Roth, D., Birkenfeld, J. and Betz, H. (1999) FEBS Lett. 460, 41 1 4 1 6 Rect2ived I0 April 2002 38 I 0 2002 Biochemical Society