Plant 14-3-3s: Omnipotent Metabolic Phosphopartners?

Paul C. Sehnke , and Robert J. Ferl

The authors are at the Program in Molecular and Cellular Biology, Department of Horticultural Sciences, University of Florida, Fifield Hall, Gainesville, FL 32611, USA. E-mail: robferl{at}ufl.edu

The regulation of metabolic processes in eukaryotic systems is quite simply a matter of life and death. These dynamics take on even further relevance in plants because the sessile nature of most plants precludes movement to more favorable environments as a remedy for local distresses or nutrient imbalances. Therefore, the process by which signal perception leads to adaptation must be expedient to meet diverse demands that range from metabolite assimilation (such as nitrogen uptake) to product storage in sink tissues (such as starch accumulation) to maintenance of turgor pressure (such as that involved in stomatal regulation). The underlying theme for these processes is the response to environmental stimuli: light, water content, and nutrients, often on a diurnal schedule. The main goal of these processes is the efficient adaptation of metabolism within the parameters dictated by the environment. In plants, several key metabolic enzymes are regulated by kinase-specific phosphorylation as an endpoint of signal transduction. It now appears, however, that several of these major metabolic pathways use two-step regulation: protein phosphorylation, followed by physical partnering with proteins that bind to specific phosphoserine-phosphothreonine motifs to regulate accurate enzymatic activity. Surprisingly, these proteins are members of the 14-3-3 family of proteins, first identified in animal brain tissue and now considered to be rather ubiquitous partners in signal transduction cascades (1).

Many metabolic enzymes are phosphorylated as they become activated or inactivated to complete the transduction of a signal to an action. The classic example in plants is that of nitrate reductase (NR) (2). Light and nutrient availability regulate NR in order to make efficient use of the nitrogen present at any given time. Biochemically, NR activity is regulated via the two-step process: (i) specific serine phosphorylation, and (ii) binding of an inhibitory protein to this regulatory phosphoserine moiety of NR (Fig. 1). Thus, phosphorylated NR is active; however, the binding of the NR inhibitory protein causes a conformational change that renders the enzyme inactive. NR becomes reactivated upon dephosphorylation by a microcystin-sensitive phosphatase and release of the inhibitory protein. This inhibitor protein was revealed to be a member of the 14-3-3 family, and is closely related to the 14-3-3 proteins that bind phosphoserine residues of proteins in several animal kinase activation cascades (3, 4).  

View larger version (31K): [in this window] [in a new window]   Fig. 1. 14-3-3 involvement in the regulation of metabolic enzymes as well as in the regulation of signal transduction cascades. Experiments in animal systems have emphasized the role(s) of 14-3-3s as binding partners of kinases involved in signal transduction pathways. Some of the examples of 14-3-3/kinase interactions include Raf, Bcl, and protein kinase C. Although plant kinases have not widely emerged as binding partners with 14-3-3, interaction with the plant calcium-dependent protein kinase (CDPK) does indicate that plant signal transduction pathways are likely to be similarly regulated by 14-3-3s. Experiments in plant systems have emphasized role(s) of 14-3-3s as binding partners of key metabolic enzymes, enzymes that are regulated by phosphorylation as a terminus of a signal transduction pathway. Some of the examples of 14-3-3/enzyme interactions include NR, sucrose phosphate synthase, glutamate synthases, and even the plasma membrane ATPase. Whereas fewer animal metabolic enzymes have emerged as being regulated by 14-3-3s, the tyrosine and tryptophan hydroxyases of the mammalian brain are activated by phosphorylation and binding by 14-3-3s, indicating that other animal metabolic enzymes are likely to be similarly regulated by 14-3-3s. Divalent cations (Me2+) are indicated by purple circles. ATP, adenosine triphosphate.

Hence, the regulation of metabolic enzymes appeared to represent different or additional roles for 14-3-3s in plants (5). Indeed, the presence of 14-3-3s in plants had already been well documented, as had the strong evolutionary conservation of 14-3-3s from animals to fungi and plants (5). Early assumptions were made that plant and animal 14-3-3s would share common functions because of the large degree of sequence conservation. However, the discovery of additional functions for plant 14-3-3 proteins has led to research taking a different track in plants as compared to animals, with the regulation of metabolic enzymes receiving more attention than signal transduction cascades.

Plant and animal 14-3-3 proteins recognize a well-conserved binding motif, Arg-X-X-pSer/pThr-X-Pro, where X is any amino acid and Ser or Thr is phosphorylated. Indeed, the addition of the phosphorylated 14-3-3 target peptide of Raf from animals was sufficient to compete off 14-3-3s from NR and reactivate the enzyme (4). Unlike the animal systems studied to date, however, plants require divalent cations for efficient 14-3-3/NR binding under physiological conditions. The early finding of structural changes within the 14-3-3s upon incubation with millimolar levels of Mg²⁺ suggests that binding competence results from structural changes in the 14-3-3 COOH-terminus (6). Proteolytic access to Lys²⁴⁷ in the loop between helices 8 and 9 of the 14-3-3 structure demonstrated the divalent cation-dependent repositioning of the COOH-terminus in a manner analogous to closing a flap over the open end of the substrate binding channel (6). This model is strengthened by supporting evidence correlating divalent metal binding and reduced surface hydrophobicity in 14-3-3s (7).

The prototypical method of 14-3-3/NR-type regulation has now been identified in several vastly different plant metabolic processes. Perhaps the most studied is the regulation of the plant plasma membrane H⁺-ATPase (adenosine triphosphatase) proton pump that is involved in maintenance of turgor pressure and is the target of the wilt-inducing fungal toxin fusicoccin (FC). Phosphorylation and 14-3-3 binding activate the H⁺-ATPase proton pump. However, the H⁺-ATPase proton pump/14-3-3 complex constitutes an FC receptor, and the binding of FC to the proton pump maintains the proton pump as an active enzyme to the point of decreasing turgor pressure in the guard cells, causing the leaves to wilt (8-10). Because the binding of 14-3-3 to the H⁺-ATPase proton pump can occur without FC, it is thought that binding of 14-3-3 under normal biological conditions serves to regulate the gating process of the pump in an environmentally responsive manner (1, 11).

Other examples of this same basic two-step process for plant metabolic regulation have recently been identified in such enzymes as sucrose phosphate synthase and glutamate synthetases (12-14). In addition, plant extracts that were "panned" for potential phosphorylated 14-3-3 binding partners revealed several other metabolic enzymes that are potentially regulated by interacting with 14-3-3 proteins (14). Given this seeming propensity for the involvement of 14-3-3s in the regulation of metabolic enzymes, we still cannot necessarily conclude that there is a fundamental difference in how animals and plants use 14-3-3s. More likely this apparent difference in 14-3-3 targets simply reflects the relative emphases and opportunities in the plant and animal experimental systems. Indeed, the original discovery of 14-3-3s as relatively abundant brain proteins may well stem from the requirement of 14-3-3s for activation of the metabolic enzymes tyrosine hydroxylase and tryptophan hydroxylase for neurotransmitter production. Additionally, in plants the involvement of 14-3-3s in more "animal-like" signaling cascade events is suggested by their association with calcium-dependent protein kinases (15). Thus, it is most likely that both plants and animals use 14-3-3s as regulatory partners in kinase signaling cascades, and also as regulatory partners at signaling pathway endpoints where the signal message is passed to a metabolic enzyme.

These issues of functional similarities and differences are confounded by the fact that 14-3-3 proteins in both plants and animals can dimerize with many other 14-3-3 isoforms. The large family size of 14-3-3s begs the question of isoform-specific interactions: Do all 14-3-3 isoforms perform the same function? Or does the high degree of divergence in the NH₂- and COOH-terminal regions define specific interactions and, therefore, specialized functions? The ability to substitute plant, yeast, and animal 14-3-3s in various systems suggests that all 14-3-3s do have similar capabilities and/or similar core functions [e.g., (16)]. Indeed, all 14-3-3s apparently can bind certain phosphorylated peptide sequences, and the high degree of sequence identity of all known 14-3-3 isoforms [as high as 60% (5)] suggests that this is not unexpected. However, although the conservation of the core phosphoprotein-binding domain supports the notion of conserved function, the extreme divergence of the NH₂- and COOH-termini of 14-3-3s argues that target specificity is possible (1). For example, in the case of NR, inactivation by 14-3-3 binding reveals differences among isoforms, which strongly supports the idea of specificity of isoform function (4).

The counterargument would state that the various isoforms have arisen simply to ensure that 14-3-3 activity is present in all cells, and that there is no specificity of biochemical function. Certainly functional overlap exists among the known individual 14-3-3 isoforms, and various 14-3-3 isoforms are differentially expressed in specific tissues. However, this argument is difficult to support because of the variation in activity and binding of different isoforms to NR, for example. In addition, many 14-3-3/target complexes appear to contain only certain subsets of all the 14-3-3s present in cells.

A corollary to this argument suggests that some specific 14-3-3 isoforms are destined for certain intracellular compartments where they ensure delivery of the same fundamental biochemical activity to those arenas. The recent demonstration of a subset of 14-3-3s inside a chloroplast supports this notion (17). The chloroplast is arguably an important metabolic organelle in plants because of the channeling of photosynthetic carbon assimilates; hence, the chloroplast 14-3-3s are potentially involved in two of the most important metabolic functions in plants: nitrogen fixation and carbon assimilation. In addition, recent work from our lab (unpublished results) has shown that specific 14-3-3 isoforms are also associated with and localized in plant mitochondria. Subsets of 14-3-3s have also been found in the nuclei of both animal and plant cells. Thus, specific isoforms seem to have certain restricted habitats within which they perform their function, but it remains to be demonstrated whether they are directed to those sites by intrinsic signals or if they arrive there because of isoform-specific interaction with client proteins that are themselves targeted.

Given the presence of plant 14-3-3s in the cytoplasm, nuclei (18, 19), chloroplasts (17), and most likely mitochondria, their potential for involvement in kinase cascades and metabolic processes looms ever larger. The expression pattern of 14-3-3s, their isoform-specific organellar presence, and their sequence terminal specificity suggests that the size and diversity of the 14-3-3 families is a reflection of their omnipotent roles in coregulating important plant processes whose regulation is primed by specific kinase cascades.

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© 2000 American Association for the Advancement of Science