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American Society of Plant Biologists
Transcription Factors Dial 14-3-3 for Nuclear Shuttleneckardt{at}aspb.org
14-3-3 proteins are a highly conserved family of phosphoserine binding proteins found in all eukaryotes. The name "14-3-3" comes from the specific ion exchange chromatography elution profiles and starch gel electrophoresis migration patterns of a group of proteins originally isolated from mammalian brain tissue. 14-3-3 proteins play key roles in many diverse physiological processes that involve regulation by phosphorylation. A number of enzymes involved in fundamental processes of plant physiology are regulated in part via interactions with 14-3-3s, including nitrate reductase (NR), sucrose phosphate synthase, starch synthase, glutamate synthase, ATP synthase, and ascorbate peroxidase (Finnie et al., 1999
14-3-3 target binding proteins often are key members of signal transduction cascades or complexes, and they have been found to interact with transcription factors and/or other proteins that affect transcription. For example, in mammals and yeast, interaction with 14-3-3 proteins appears to be critical for the function of Raf-1 kinase, which activates a mitogen-activated protein kinase cascade that mediates the transcription of genes involved in mitogenesis and cell differentiation (Aitken, 1996  ), and of histone deacetylases that interact with transcription factors to repress gene expression (Grozinger and Schreiber, 2000).
14-3-3 proteins have been found to bind specifically with TATA box binding proteins from plants as well as other eukaryotes and to activate GAL4-dependent
Crystal structure analysis and binding studies have shown that 14-3-3s are sequence-specific phosphoserine binding proteins that function as homodimers or heterodimers (Xiao et al., 1995 ; Muslin et al., 1996). The most highly conserved regions of the polypeptides form a large negatively charged channel at the dimerization interface, which provides the binding site for target phosphoproteins. 14-3-3s are not involved in simple phosphorylation/dephosphorylation reactions of target proteins; rather, they appear to interact with their phosphoserine targets in a way that alters the stability, activity, and/or localization of the target protein within the cell.
Binding of 14-3-3 proteins sometimes has been shown to influence the proteolysis or stability of the target protein. For example, binding of 14-3-3 proteins does not appear to activate Raf-1 directly; rather, it may act to stabilize an activatable conformation of Raf-1 (Morrison, 1994
Interactions with 14-3-3 proteins also have been found to alter the intracellular localization of certain target proteins. May and Soll (2000)
In this issue of The Plant Cell, Igarashi et al. (pages 2483–2497) show that 14-3-3 proteins regulate the intracellular localization of REPRESSION OF SHOOT GROWTH (RSG), a transcription factor from tobacco that controls the expression of a gibberellin biosynthesis enzyme. RSG is expressed ubiquitously in plant organs, suggesting that some form of post-transcriptional or post-translational modification controls its activity (Fukazawa et al., 2000 ). A mutant version of RSG that had lost the ability to bind 14-3-3s, as a result of a single amino acid change of serine residue 114 to alanine (RSG-S114A), was localized exclusively in the nucleus (Figure 1)
and exhibited higher transcriptional activation capability than wild-type RSG in transient expression assays in tobacco protoplasts. Similar results were observed with wild-type or mutant RSG-S114A constructs fused with green fluorescent protein (GFP) and expressed in transgenic tobacco plants.
Igarashi et al. further found that wild-type RSG is not localized statically in the cytosol but is capable of shuttling in and out of the nucleus. This was shown in experiments using leptomycin B, an inhibitor of nuclear export that acts by inhibiting Crm1/Exportin1, a receptor that mediates the nuclear export of proteins carrying a nuclear export sequence. Treatment of leaf epidermal cells with leptomycin B caused rapid relocalization of RSG from the cytosol to the nucleus, suggesting that RSG normally continually shuttles between the nucleus and the cytoplasm. The results suggest that 14-3-3 proteins are involved in the regulation of RSG transport into and/or out of the nucleus and that this regulation may play a key role in controlling the activity of RSG as a transcriptional activator and regulator of gibberellic acid (GA) biosynthesis (Figure 2) .
This model leads to the hypothesis that inducers of GA biosynthesis should have a direct or indirect effect on the phosphorylation status and 14-3-3 binding capacity of RSG. Thus, the investigation of 14-3-3 binding properties might reveal more details of the regulation of GA levels in plants.
Interestingly, a comparable mode of action was found for the interaction of 14-3-3 proteins with the Cdc25 protein in Xenopus cells. The dual specificity phosphatase Cdc25 dephosphorylates specific sites on Cdc2, which forms part of the maturation-promoting factor complex responsible for triggering the entry into mitosis. Binding of 14-3-3 proteins to Cdc25 suppresses its ability to induce entry into mitosis. Kumagai and Dunphy (1999)
The results of Igarashi et al. and of Kumagai and Dunphy (1999)
The interaction of 14-3-3 proteins with plant NR is well characterized and provides a good example of what may be a general characteristic of 14-3-3 interactions: a primary role in the tight control of complex regulatory pathways. Plant NR is subject to rapid changes in activity in response to a wide variety of environmental factors that involve Ca2+, phosphorylation, interactions with 14-3-3 proteins, regulation of proteolysis, and coordination with photosynthetic rate (reviewed by MacKintosh and Meek, 2001 ). NR cycles between inactive and active forms, and inactivation occurs via phosphorylation by a specific NR kinase. However, phosphorylation alone does not alter NR activity; rather, it creates a binding site for 14-3-3 proteins, and it is 14-3-3 binding to NR that inhibits activity. Coordination of NR activity with photosynthesis is regulated in part via the interaction with 14-3-3 proteins; extracellular sugars influence the phosphorylation status and the binding of 14-3-3 proteins to NR, and 14-3-3 binding also may influence the proteolysis of NR. The complex and extremely rapid regulation of NR activity is believed to minimize the major costs and possible hazards associated with the synthesis of amino acids from nitrogenous minerals and sugars. The conversion of nitrate to amino acids requires the reducing power of a considerable amount of NADH and NADPH. Furthermore, nitrite generated in the cytosol via NR activity is highly toxic. Photosynthesis normally produces enough reductant to eliminate nitrite, but nitrite poisoning might pose a threat to the cell if photosynthesis were inhibited. 14-3-3 interactions often form part of such tightly regulated processes.
14-3-3 proteins usually are members of reasonably large gene families. For example, there are 15 family members in the Arabidopsis genome, and at least 12 of these are expressed (Rosenquist et al., 2001
Despite the high degree of conservation of the phosphoserine site and evidence that 14-3-3 proteins function as dimers, there is some evidence that not all 14-3-3 proteins require phosphoserine binding or dimerization. Pan et al. (1999) As with the regulation of NR activity, hormonal regulation of biochemical pathways usually is found to be highly complex and tightly regulated and it may involve rapid shifts in enzyme activities and/or regulation of gene expression. 14-3-3 protein interactions appear to be a universal fine-tuner of biochemical regulation, perhaps offering a highly flexible and somewhat variable mode of action that can be applied to a variety of metabolic processes that require the tight control of numerous inputs and outputs.
Aitken, A. (1996). 14-3-3 and its possible role in co-ordinating multiple signalling pathways. Trends Cell Biol. 6, 341–347.[CrossRef][Web of Science][Medline] Cotelle, V., Meek, S.E., Provan, F., Milne, F.C., Morrice, N., and MacKintosh, C. (2000). 14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells. EMBO J. 19, 2869–2876.[CrossRef][Web of Science][Medline]
DeLille, J.M., Sehnke, P.C., and Ferl, R.J. (2001). The Arabidopsis 14-3-3 family of signaling regulators. Plant Physiol. 126, 35–38. Finnie, C., Borch, J., and Collinge, D.B. (1999). 14-3-3 proteins: Eukaryotic regulatory proteins with many functions. Plant Mol. Biol. 40, 545–554.[CrossRef][Web of Science][Medline]
Freed, E., Symons, M., Macdonald, S.G., McCormick, F., and Ruggieri, R. (1994). Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. Science 265, 1713–1716.
Fukazawa, J., Sakai, T., Ishida, S., Yamaguchi, I., Kamiya, Y., and Takahashi, Y. (2000). REPRESSION OF SHOOT GROWTH, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 12, 901–915.
Grozinger, C.M., and Schreiber, S.L. (2000). Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization. Proc. Natl. Acad. Sci. USA 97, 7835–7840.
Igarashi, D., Ishida, S., Fukazawa, J., and Takahashi, Y. (2001). 14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG. Plant Cell 13, 2483–2497.
Kumagai, A., and Dunphy, W.G. (1999). Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev. 13, 1067–1072.
Liao, J., and Omary, M.B. (1996). 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility factor. J. Cell Biol. 133, 345–357. MacKintosh, C., and Meek, S.E.M. (2001). Regulation of plant NR activity by reversible phosphorylation, 14-3-3 proteins and proteolysis. Cell. Mol. Life Sci. 58, 205–214.[CrossRef][Web of Science][Medline] Malerba, M., and Bianchetti, R. (2001). 14-3-3 protein-activated and autoinhibited forms of plasma membrane H+-ATPase. Biochem. Biophys. Res. Commun. 286, 984–990.[Medline]
May, T., and Soll, J. (2000). 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants. Plant Cell 12, 53–63.
Morrison, D. (1994). 14-3-3: Modulators of signaling proteins? Science 266, 56–57. Morsomme, P., and Boutry, M. (2000). The plant plasma membrane H+-ATPase: Structure, function and regulation. Biochim. Biophys. Acta 1465, 1–16.[Medline] Muslin, A.J., Tanner, J.W., Allen, P.M., and Shaw, A.S. (1996). Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897.[CrossRef][Web of Science][Medline]
Pan, S., Sehnke, P.C., Ferl, R.J., and Gurley, W.B. (1999). Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex. Plant Cell 11, 1591–1602. Rosenquist, M., Sehnke, P., Ferl, R.J., Sommarin, M., and Larsson, C. (2000). Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity? J. Mol. Evol. 51, 446–458.[Web of Science][Medline]
Rosenquist, M., Alsterfjord, M., Larsson, C., and Sommarin, M. (2001). Data mining the Arabidopsis genome reveals fifteen 14-3-3 genes: Expression is demonstrated for two out of five novel genes. Plant Physiol. 127, 142–149.
Schultz, T.F., Medina, J., Hill, A., and Quatrano, R.S. (1998). 14-3-3 proteins are part of an abscisic acid–VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1. Plant Cell 10, 837–847. Xiao, B., Smerdon, S.J., Jones, D.H., Dodson, G.G., Soneji, Y., Aitken, A., and Gamblin, S.J. (1995). Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways. Nature 376, 188–194.[CrossRef][Medline]
Zhao, X., Ito, A., Kane, C.D., Liao, T.S., Bolger, T.A., Lemrow, S.M., Means, A.R., and Yao, T.P. (2001). The modular nature of histone deacetylase HDAC4 confers phosphorylation-dependent intracellular trafficking. J. Biol. Chem. 276, 35042–35048. This article has been cited by other articles:
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14-3-3 PROTEINS AND THE...
14-3-3 MODE OF ACTION
-glucuronidase reporter gene expression when translationally fused with the GAL4 DNA binding domain in a plant transient expression system (Pan et al., 1999
, whereas nuclear export involves the activity of the receptor Crm1/Exportin1, which recognizes one or more nuclear export sequences. Cdc25 has several putative nuclear export sequences, and mutational studies showed that some of these sequences might play a role in localization mediated by 14-3-3s. The authors next asked whether the binding of Importin-
