Skip to main content
ARS Home » Research » Publications at this Location » Publication #156148
Title: THE C-TERMINAL TAIL OF ARABIDOPSIS 14-3 FUNCTIONS AS AN AUTOINHIBITOR AND MAY CONTAIN A TENTH A-HELIX

Author
item SHEN, WEI - UNIV OF ILLINOIS
item CLARK, CLAY - NC STATE UNIV
item Huber, Steven

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2003
Publication Date: 5/15/2003
Citation: Shen, W., Clark, C., Huber, S.C. 2003. The c-terminal tail of arabidopsis 14-3 functions as an autoinhibitor and may contain a tenth a-helix. Plant Journal. 34:473-484.

Interpretive Summary: The '14-3-3 proteins' were originally discovered as abundant brain proteins but are now known to be ubiquitous among plants and animals. These proteins function as binding proteins, and in plants, are known to be involved in transcription factor complexes (that control gene expression) and as components that regulate the activities of important plant enzymes involved in primary carbon and nitrogen metabolism (e.g., nitrate assimilation). It is known that the ability of 14-3-3 proteins to bind to their target proteins may be regulated by divalent cations, such as magnesium, which bind to the 14-3-3 proteins. However, the mechanism underlying the cation effect is not known. The results obtained in the present study indicate that the C-terminal tail of the 14-3-3 protein functions as an "autoinhibitor," that blocks binding to nitrate reductase when magnesium is not present. Removal of the C-terminal tail allows for binding of the truncated 14-3-3 protein to nitrate reductase, and inhibition of nitrate reductase enzymatic activity. These results increase our understanding of the molecular mechanisms that control 14-3-3 binding, and also provide unique experimental tools to use in future experiments to study the function of 14-3-3 proteins in vivo using transgenic plants.

Technical Abstract: The eukaryotic regulatory protein 14-3-3 is involved in many important plant cellular processes including regulation of nitrate assimilation, through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations and some polyamines interact with the loop 8 region of 14-3-3 proteins and allow them to bind and inhibit phosphorylated NR in vitro (Athwal and Huber, 2002, Plant J., 29, 119-129). The role of the highly variant C-terminal regions of 14-3-3 isoforms in regulation by polycations is not clear. In this study we did structural analyses on the C-terminal tail of the Arabidopsis 14-3-3 isoform and evaluated its contributions to inhibition of pNR. Nested C-terminal truncations of the recombinant 14-3-3 protein revealed that removal of the C-terminal tail renders the protein partially Mg2+-independent in both pNR binding and inhibition of activity, suggesting that the C-terminus functions as an autoinhibitor. The C-terminus of 14-3-3 appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys-247. C-terminal truncation of 14-3-3 at Thr-255 increased its interaction with antibodies to the C-terminus of 14-3-3 in non-denaturing conditions but not in denaturing conditions, suggesting that the C-terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism analysis of a C-terminal peptide, from Trp-234 to Lys-249, revealed the C-terminal tail might contain a tenth -helix, in agreement with the in silico predictions. The function of the putative tenth -helix is not clear, because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg2+. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14-3-3 protein is restricted by the C-terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.