Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase
Article first published online: 21 JAN 2002
The Plant Journal
Volume 29, Issue 2, pages 119–129, January 2002
How to Cite
Athwal, G. S. and Huber, S. C. (2002), Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase. The Plant Journal, 29: 119–129. doi: 10.1046/j.0960-7412.2001.01200.x
- Issue published online: 21 JAN 2002
- Article first published online: 21 JAN 2002
- Received 13 July 2001; revised 25 September 2001; accepted 10 October 2001.
- 14-3-3 proteins;
- nitrate reductase;
- cation-binding site;
- site-directed mutagenesis;
- truncated proteins
Binding of 14-3-3 proteins to nitrate reductase phosphorylated on Ser543 (phospho-NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14-3-3-dependent inactivation of phospho-NR is known to require millimolar concentrations of a divalent cation such as Mg2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine4+ and spermine3+, can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine4+ > spermidine3+ >>> cadavarine2+ ≈ putrescine2+ ≈ agmatine2+ ≈ N1-acetylspermidine2+, indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14ω, which encodes the Arabidopsis 14-3-3 isoform ω, indicated that loop 8 (residues 208–219) is the likely cation-binding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation-independent inhibition of phospho-NR activity, whereas the D219A mutant was fully Mg2+-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg2+ dependence of phospho-NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14-3-3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14-3-3s could be involved in their regulation of plant growth and development.