Skip to main content
ARS Home » Midwest Area » Urbana, Illinois » Global Change and Photosynthesis Research » Research » Publications at this Location » Publication #204325

Title: Polycations Globally Enhance Binding of 14-3-3 omega to Target Proteins in Spinach Leaves

item Huber, Steven

Submitted to: Plant Cell Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/6/2006
Publication Date: 6/1/2006
Citation: Shen, W., Huber, S.C. 2006. Polycations Globally Enhance Binding of 14-3-3 omega to Target Proteins in Spinach Leaves. Plant Cell Physiology. 47:764-771.

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 by specifically binding to certain proteins (often called client proteins) that are modified by phosphorylation, and thus interact broadly with cellular proteins. In plants, the 14-3-3 proteins regulate the activities of important enzymes involved in primary carbon and nitrogen metabolism. It is known that the ability of 14-3-3 proteins to bind to phosphorylated nitrate reductase may be regulated by divalent cations, such as magnesium, and also organic cations, such as polyamines. However, it is not know whether cations enhance the binding of 14-3-3 proteins to proteins in addition to nitrate reductase, and whether polyamines might promote specific interactions with a distinct subset of cellular client proteins. In the present study we addressed these questions using a Far-Western overlay technique and demonstrate that polycations generally enhance binding of 14-3-3 (isoform omega) to cellular target proteins. Importantly, magnesium and the polyamine, spermine, promoted binding of 14-3-3s to clients to a similar extent. However, the extent of stimulation varied with the client protein, suggesting that changes in cellular cation concentrations may be an important factor influencing the function of 14-3-3 proteins in vivo.

Technical Abstract: The binding of 14-3-3' to phosphorylated NR (pNR) is stimulated by cations such as Mg2+ or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, Far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinacia oleracea) leaves were performed using digoxygenin (DIG)-labeled Arabidopsis 14-3-3'. When separated by SDS-PAGE, approximately 25 proteins greater than 35 kDa could be resolved that interacted with DIG-labeled 14-3-3' in the absence of added cations. The presence of 5 mM Mg2+ or 0.5 mM spermine enhanced binding to most of the target proteins to a maximum of about a doubling of the observed binding. In most cases, the binding was dependent on phosphorylation of the target protein, whereas that was not necessarily the case for binding to target proteins that were unaffected by polycations. The extent of stimulation varied among the target proteins but there was no indication that the nature of the cation activator (e.g., Mg2+ versus spermine4+) altered the specificity for target proteins. In addition, binding of DIG-labeled 14-3-3' to some, but not all, target proteins was reduced by 5 mM 5'-AMP. Interestingly, light-dark treatment of spinach leaves affected the subsequent binding of DIG-labeled 14-3-3' in the overlay assay to only a few of the target proteins, one of which was identified as NADH:nitrate reductase. Overall, the results suggest that the binding of 14-3-3s to targets in addition to pNR may also be regulated by polycations and 5'-AMP.