Journal Article

Polycations Globally Enhance Binding of 14-3-3ω to Target Proteins in Spinach Leaves

Wei Shen and Steven C. Huber

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 6, pages 764-771
Published in print June 2006 | ISSN: 0032-0781
Published online June 2006 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcj050
Polycations Globally Enhance Binding of 14-3-3ω to Target Proteins in Spinach Leaves

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The binding of 14-3-3ω to phosphorylated nitrate reductase (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 digoxigenin (DIG)-labeled Arabidopsis 14-3-3ω. When separated by SDS–PAGE, approximately 25 proteins of >35 kDa could be resolved that interacted with DIG-labeled 14-3-3ω 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+ vs. spermine4+) altered the specificity for target proteins. In addition, binding of DIG-labeled 14-3-3ω 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 14-3-3ω 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.

Keywords: Divalent cations; Far-Western overlay; Nitrate reductase; 14-3-3 protein; Spermine; Spinacia oleracea; DIG, digoxigenin; MALDI-TOF, matrix-assisted desorption ionization-time of flight; NR, nitrate reductase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride

Journal Article.  5834 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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