Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Clinical Science (2002) 30, (411–415) (Printed in Great Britain)

Abstract

PDF

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H -ATPase

J. Borch*†, K. Bych*, P. Roepstorff†, M. G. Palmgren* and A. T. Fuglsang*¹

*Department of Plant Biology, Plant Physiology and Anatomy Laboratory, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871-Frederiksberg C, Copenhagen, Denmark, and †Department of Biochemistry and Molecular Biology, Odense University Campus, University of Southern Denmark, Campusvej 55, DK 5230 Odense M, Denmark

Key words: binding motif, fusicoccin.

Abbreviations used: FC, fusicoccin; SPR, surface plasmon resonance; pRaf, phosphorylated Raf peptide; AHA2, Arabidopsis thaliana H⁺-ATPase isoform 2; PMA2, Nicotiana plumbaginifolia H⁺-ATPase; ExoS, exoenzyme S.

¹To whom correspondence should be addressed (e-mail atf@kvl.dk).

Abstract

14-3-3 proteins interact with a novel phosphothreonine motif (Y⁹⁴⁶pTV) at the extreme C-terminal end of the plant plasma membrane H⁺-ATPase molecule. Phosphorylation- independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H ⁺-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H⁺-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H⁺-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H⁺-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.

Introduction

The mechanism by which 14-3-3 proteins bind to their targets has been studied extensively (for recent reviews, see [ 1–6]). In most cases, targets of 14-3-3 proteins contain an interacting epitope involving a phosphoserine [ 7, 8]. A common 14-3-3-binding motif of RSXpSXP (where pS is phosphoserine) interacts in the crystal structure with a conserved amphipathic groove in the 14-3-3 protein. However, epitopes distinct from the phosphoserine motif have been examined [ 9].

14-3-3 proteins bind to several important plant enzymes. Among them is the nitrate reductase involved in the assimilation of nitrate [ 10, 11], the F₀F₁-ATP synthase [ 12] and the plasma membrane H⁺-ATPase important for generating the electrochemical gradient across the plasma membrane (for a review, see [ 13]). The binding of 14-3-3 proteins to these enzymes can result in either activation or inactivation, dependent on the target enzyme. In the case of plasma membrane H⁺-ATPase, activation of proton pumping is the result of 14-3-3 binding. This observation has generated considerable interest since most of the secondary transporters in plant cells depend upon the activity of the H⁺-ATPase [ 14].

A novel binding motif

By expressing the plant H⁺-ATPase in yeast, it has been possible to precisely identify the site of interaction between the 14-3-3 protein and the C-terminus of the H⁺-ATPase by the use of site-directed mutagenesis [ 9, 15, 16]. This has demonstrated that the 14-3-3-binding motif is located in the extreme C-terminal end of the H⁺-ATPase protein and consists of Y⁹⁴⁶TV as the last three amino acids. The motif YTV is a novel binding motif sharing no homology with other identified 14-3-3-binding motifs [ 7, 8]. This observation was surprising since the groove in 14-3-3 proteins that accommodates phosphorylated peptides is extremely well conserved [ 2, 17]. Database searches reveal only one other protein, besides the plant plasma membrane H⁺-ATPases, that contains a YTV motif in the extreme C-terminal end of the protein, namely a plant flavonoid 3,5 hydroxylase (gi: 1785488), which so far has not been identified as a 14-3-3 target. However, the very fact that new binding sites not resembling other 14-3-3-binding motifs can be found indicates that the number of 14-3-3-binding proteins in plants and animals might be much higher than previously thought.

Co-crystallization of 14-3-3 and its interacting peptides has demonstrated that a proline residue after the phosphorylated motif is important in order for the peptide to exit the binding groove [ 18]. Since the YTV motif is located at the extreme end of the protein, the H⁺-ATPase polypeptide apparently just enters the binding groove, but does not exit.

Phosphorylation-dependent 14-3-3 binding

In order for the 14-3-3 protein to bind to the H⁺-ATPase, phosphorylation of Thr⁹⁴⁷ (YpTV) is a prerequisite. This penultimate threonine in the plant plasma membrane H⁺ -ATPase is phosphorylated in vivo [ 19] and, importantly, increased phosphorylation in vivo occurs as a response to the exposure of plant material to blue light, a treatment known to activate the plasma membrane H⁺ -ATPase [ 20]. Whereas most other 14-3-3 targets only bind 14-3-3 protein following phosphorylation of a serine residue, few other examples are known where binding depends on phosphorylation of a threonine residue [ 21].

Phosphorylation-independent 14-3-3 binding

Fusicoccin (FC) is a fungal toxin that has been used widely to study regulation of the plant plasma membrane H⁺-ATPase. It has been known for a long time that FC activates the H⁺ -ATPase [ 22]. When FC is added to the 14-3-3–H⁺-ATPase complex in vitro the interaction is stabilized and becomes almost irreversible. When 14-3-3 protein is allowed to interact with a peptide derived from the C-terminal end of plant H⁺-ATPase, addition of FC causes the dissociation constant (K_D) of the complex to shift by an order of magnitude, as demonstrated by the use of surface plasmon resonance (SPR) spectroscopy (see Table 1) [ 9].

Table 1 Binding affinities (dissociation constant, K_D, values) and binding sequences for various peptides and proteins to 14-3-3 protein

The K_D values were determined by SPR spectroscopy (except for R18). pS, phosphoserine; pT, phosphothreonine.

Protein	Binding site (consensus)	KD (nM)	Reference
Raf-1 kinase	RSTpSTP (mode 1)	122	[ 8]
Peptide (artificial ligand)	RLSHpSLP (mode 2)	56	[ 7]
Peptide (artificial ligand)	RLYHpSLP (mode 2)	37	[ 7]
ExoS (non-phosphorylated)	DLASKPERSGEVQEQDVRLRMRGLDLA*	7	[ 27, 28]
R18 (artificial ligand)	PHCVPRDLSWLDLEANMCLP	80	[ 25]
AHA2	VKLVGLDIETPSYpTV*	88	[ 9]
AHA2+addition of FC	VKLVGLDIETPSYpTV*	7	[ 9]

* The peptide represents the extreme C-terminus of the protein.

Interestingly, FC not only stabilizes the phosphorylated complex, but, in addition, overcomes the requirement for phosphorylation of Thr⁹⁴⁷ in order for 14-3-3 to bind. This has been shown in 14-3-3-overlay assays using non-phosphorylated fusion proteins involving the H⁺-ATPase C-terminal hydrophilic domain (100 residues) [9,23,24]. However, even in the presence of FC, peptides representing the last 16 amino acids of the C-terminus of an Arabidopsis plasma membrane H⁺-ATPase (AHA2) can only bind 14-3-3 protein when Thr ⁹⁴⁷ is phosphorylated [ 9]. This indicates that specific residues or secondary structures located further upstream in the C-terminus of the H⁺-ATPase must be important for the non-phosphorylated FC-induced 14- 3-3 binding.

A range of C-terminal/GST-fusion proteins were derived from Nicotiana plumbaginifolia H⁺-ATPase (PMA2), and tested in 14-3-3 overlay assays in the presence of FC. This study revealed that the last 37 amino acid residues are sufficient for phosphorylation-independent 14-3-3 binding [ 24].

In this study, peptides corresponding to the last 34 amino acids of AHA2 have been synthesized. Threonine or phosphothreonine residues were introduced at the penultimate position. These peptides have been used for SPR protein–protein interaction studies ( Figure 1). Both the non-phosphorylated and the phosphorylated peptides bound 14-3-3 in an FC-stimulated manner ( Figure 1). The rates of 14-3-3 binding to the two H⁺-ATPase-derived peptides were different. The phosphorylated peptide exhibited the highest rate of association with 14-3-3 protein. Notably, the unphosphorylated H⁺-ATPase peptide bound 14-3-3 very weakly, even in the absence of FC ( Figure 1A). The addition of FC increased the binding of 14-3-3 significantly ( Figure 1B). This implies that preformed low-affinity complexes between 14-3-3 protein and AHA2 create the basis for FC-dependent high-affinity complexes and this seems to be true for phosphorylated as well as unphosphorylated AHA2. The additional residues in the longer peptides (compared with the 16-residue peptides studied previously) do not resemble any previously defined 14-3- 3-binding motifs.

Figure 2 summarizes some of the different H⁺-ATPase peptides used in the identification of the 14-3-3-binding site and the role of phosphorylation.

A number of cases have been described in the literature where 14-3-3 proteins interact with a target in a phosphorylation-independent manner. Screening of a random peptide phage library revealed several non-phosphorylated peptides that bind 14-3-3 [ 25]. Some of these peptides resemble the prototype phosphoserine motif but without phosphorylation of the serine residue. The most frequently occurring peptide, R18, contains a WLDLE motif and was shown to compete with phosphorylated Raf peptide (pRaf) in order to bind 14-3-3 [ 25]. Furthermore, co-crystallization shows that R18 binds in the amphipathic groove [ 26].

Exoenzyme S (ExoS) from Pseudomonas aeruginosa binds 14-3-3 in a non-phosphorylated form [ 27, 28]. Interestingly, ExoS contains a motif homologous to the R18 peptide, F²⁴⁵GADAE, that may mediate an interaction similar to the one between 14-3-3 and R18, as suggested by Zhang et al. [ 29]. However, the site of interaction with the 14-3-3 protein was later located to the last 27 amino acid residues in the C-terminal of ExoS not containing this motif [ 28]. Mutational analysis of 14-3-3 proteins has demonstrated that ExoS selectively employs residues in the amphipathic binding groove in order to bind. Unlike phosphorylated ligands, ExoS does not require hydrophobic residues in the binding groove, but ExoS still uses the same charged residues as pRaf as contact sites [ 29–31].

Recently an additional non-phosphorylated 14-3-3-binding epitope, the guanine nucleotide exchange factor p190RhoGEF, was identified [ 32]. The binding site was narrowed down to I¹³⁷⁰QAIQNL and it was suggested that the polar and non-polar residues in the binding site may be instrumental in binding to the charged and uncharged surfaces in the amphipathic groove.

Table 1 summarizes some of the identified 14-3-3 binding peptides and their binding affinity to 14-3-3 proteins. Notably non-phosphorylated and phosphorylated peptides bind with similar affinity. It remains to be tested whether FC has any effect on protein–protein interactions involving 14-3-3 protein other than the H⁺-ATPase–14-3-3 complex.

Structural role of FC in the 14-3-3–H⁺-ATPase complex

In a search for the plant FC receptor, 14-3-3 proteins were identified as FC-binding proteins [ 33–35]. However, it was soon realized that 14-3-3 protein alone is not able to bind FC, indicating the presence of a second component in the FC binding complex. After a direct interaction between the C-terminus of the H⁺-ATPase and the 14-3-3 proteins had been demonstrated [ 13, 36, 37], it was found that the FC receptor is a complex between the C-terminal domain of H⁺-ATPase and 14-3-3 protein [ 38, 39].

What is the function of FC in the 14-3-3–H⁺-ATPase complex? Several possibilities exist. FC is a strongly hydrophobic compound carrying no negative charges and therefore does not mimic phosphorylation by directly introducing a local negative charge. Alternatively, FC may change the conformation of the H⁺-ATPase polypeptide and by doing so in some way compensate for phosphorylation of the penultimate threonine residue. Another possibility is that FC binds to the interface between both of the two protein components in the complex. This might explain why the simultaneous presence of 14-3-3 protein and H⁺-ATPase is required for FC binding.

Crystallization of the 14-3-3–H⁺-ATPase complex in the presence and absence of FC will be of importance to fully understand the binding mechanism in this unusual 14-3-3-binding motif and the role of the FC molecule in the stabilization of the complex.

References

1 Tzivion, G., Shen, Y. H. and Zhu, J. (2001) Oncogene 20, 6331–6338
Medline   1st Citation

2 Fu, H. A., Subramanian, R. R. and Masters, S. C. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 617–647
Medline   1st Citation  2nd

3 Finnie, C., Borch, J. and Collinge, D. B. (1999) Plant Mol. Biol. 40, 545–554
Medline   1st Citation

4 Roberts, M. R. (2000) Curr. Opin. Plant Biol. 3, 400–405
Medline   1st Citation

5 Yaffe, M. B. and Elia, A. E. H. (2001) Curr. Opin. Cell Biol. 13, 131–138
Medline   1st Citation

6 Yaffe, M. B. and Smerdon, S. J. (2001) Structure 9, R33–R38
Medline   1st Citation

7 Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J. and Cantley, L. C. (1997) Cell 91, 961–971
Medline   1st Citation  2nd  3rd  4th

8 Muslin, A. J., Tanner, J. W., Allen, P. M. and Shaw, A. S. (1996) Cell 84, 889–897
Medline   1st Citation  2nd  3rd

9 Fuglsang, A. T., Visconti, S., Drumm, K., Jahn, T., Stensballe, A., Mattei, B., Jensen, O. N., Aducci, P. and Palmgren, M. G. (1999) J. Biol. Chem. 274, 36774–36780
Medline   1st Citation  2nd  3rd  4th  5th  6th  7th  8th  9th

10 Bachmann, M., Huber, J. L., Athwal, G. S., Wu, K., Ferl, R. J. and Huber, S. C. (1996) FEBS Lett. 398, 26–30
Medline   1st Citation

11 Moorhead, G., Douglas, P., Morrice, N., Scarabel, M., Aitken, A. and MacKintosh, C. (1996) Curr. Biol. 6, 1104–1113
Medline   1st Citation

12 Bunney, T. D., van Walraven, H. S. and de Boer, A. H. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4249–4254
Medline   1st Citation

13 Jahn, T., Fuglsang, A. T., Olsson, A., Bruntrup, I. M., Collinge, D. B., Volkmann, D., Sommarin, M., Palmgren, M. G. and Larsson, C. (1997) Plant Cell 9, 1805–1814
Medline   1st Citation  2nd

14 Palmgren, M. G. (2001) Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 817–845
Medline   1st Citation

15 Svennelid, F., Olsson, A., Piotrowski, M., Rosenquist, M., Ottman, C., Larsson, C., Oecking, C. and Sommarin, M. (1999) Plant Cell 11, 2379–2391
Medline   1st Citation

16 Maudoux, O., Batoko, H., Oecking, C., Gevaert, K., Vandekerckhove, J., Boutry, M. and Morsomme, P. (2000) J. Biol. Chem. 275, 17762–17770
Medline   1st Citation

17 Xiao, B., Smerdon, S. J., Jones, D. H., Dodson, G. G., Soneji, Y., Aitken, A. and Gamblin, S. J. (1995) Nature (London) 376, 188–191
Medline   1st Citation

18 Rittinger, K., Budman, J., Xu, J. A., Volinia, S., Cantley, L. C., Smerdon, S. J., Gamblin, S. J. and Yaffe, M. B. (1999) Mol. Cell 4, 153–166
Medline   1st Citation

19 Olsson, A., Svennelid, F., Ek, B., Sommarin, M. and Larsson, C. (1998) Plant Physiol. 118, 551–555
Medline   1st Citation

20 Kinoshita, T. and Shimazaki, K. (1999) EMBO J. 18, 5548–5558
Medline   1st Citation

21 Obsil, T., Ghirlando, R., Klein, D. C., Ganguly, S. and Dyda, F. (2001) Cell 105, 257–267
Medline   1st Citation

22 Marrè, E. (1979) Annu. Rev. Plant Physiol. 30, 273–288
1st Citation

23 Fullone, M. R., Visconti, S., Marra, M., Fogliano, V. and Aducci, P. (1998) J. Biol. Chem. 273, 7698–7702
Medline   1st Citation

24 Jelich-Ottmann, C., Weiler, E. W. and Oecking, C. (2001) J. Biol. Chem. 276, 39852–39857
Medline   1st Citation  2nd  3rd

25 Wang, B., Yang, H., Liu, Y. C., Jelinek, T., Zhang, L., Ruoslahti, E. and Fu, H. (1999) Biochemistry 38, 12499–12504
Medline   1st Citation  2nd  3rd

26 Petosa, C., Masters, S. C., Bankston, L. A., Pohl, J., Wang, B. C., Fu, H. I. and Liddington, R. C. (1998) J. Biol. Chem. 273, 16305–16310
Medline   1st Citation

27 Masters, S. C., Pederson, K. J., Zhang, L. X., Barbieri, J. T. and Fu, H. A. (1999) Biochemistry 38, 5216–5221
Medline   1st Citation  2nd

28 Henriksson, M. L., Troller, U. and Hallberg, B. (2000) Biochem. J. 349, 697–701
Medline   1st Citation  2nd  3rd

29 Zhang, L. X., Wang, H. N., Masters, S. C., Wang, B. C., Barbieri, J. T. and Fu, H. A. (1999) Biochemistry 38, 12159–12164
Medline   1st Citation  2nd

30 Zhang, L., Wang, H., Liu, D., Liddington, R. and Fu, H. (1997) J. Biol. Chem. 272, 13717–13724
Medline   1st Citation

31 Wang, H., Zhang, L., Liddington, R. and Fu, H. (1998) J. Biol. Chem. 273, 16297–16304
Medline   1st Citation

32 Zhai, J., Lin, H., Shamim, M., Schlaepfer, W. and Canete-Soler, R. (2001) J. Biol. Chem. 276, 41318–41324
Medline   1st Citation

33 Oecking, C., Eckerskorn, C. and Weiler, E. W. (1994) FEBS Lett. 352, 163–166
Medline   1st Citation

34 Korthout, H. and Deboer, A. H. (1994) Plant Cell 6, 1681–1692
Medline   1st Citation

35 Aducci, P., Ballio, A., Fogliano, V., Fullone, M. R., Marra, M. and Proietti, N. (1993) Eur. J. Biochem. 214, 339–345
Medline   1st Citation

36 Olivari, C., Meanti, C., De Michelis, M. I. and Rasi-Caldogno, F. (1998) Plant Physiol. 116, 529–537
Medline   1st Citation

37 Oecking, C., Piotrowski, M., Hagemeier, J. and Hagemann, K. (1997) Plant J. 12, 441–453
1st Citation

38 Baunsgaard, L., Fuglsang, A. T., Jahn, T., Korthout, H. A., de Boer, A. H. and Palmgren, M. G. (1998) Plant J. 13, 661–671
Medline   1st Citation

39 DeMichelis, M. I., RasiCaldogno, F., Pugliarello, M. C. and Olivari, C. (1996) Plant Physiol. 110, 957–964
1st Citation

Received 20 March 2002

Copyright 2002 Biochemical Society