Abstract
This study describes the identification of over 150 target proteins of the five 14-3-3 isoforms in 7-d-old barley (Hordeum vulgare) cv Himalaya seedlings using yeast two-hybrid screens complemented with 14-3-3 protein affinity purification and tandem mass spectrometry. Independent experiments for a subset of genes confirmed the yeast two-hybrid interactions, demonstrating a low false positive identification rate. These combined approaches resulted in the identification of more than 150 putative targets; 15% were previously reported to be 14-3-3 interactors, including, for example, Serpin, RF2A, WPK4 kinase, P-type proton-translocating adenosine triphosphatase, EF1A, glutamine synthetase, and invertases. The affinity purification resulted in 30 interactors, of which 44% function in metabolism, while the yeast two-hybrid screens identified 132 different proteins, with 35% of the proteins involved in signal transduction. A number of proteins have a well-described function in hormonal signaling, such as the auxin transport protein PIN1 and NPH3 and components of the brassinosteroid pathway, such as the receptor kinase BAK1 (OsPERK1) and BRI1-kinase domain-interacting protein 129. However, 14-3-3 interactions with these signal mediators have not been confirmed in the affinity purification. Confirmations of the 14-3-3 interaction with the three ABF-like transcription factors are shown using far western analysis. Also, a REPRESSION OF SHOOT GROWTH ortholog named RF2A was identified; these transcription factors play important roles in the abscisic acid and gibberellin pathways, respectively. We speculate that 14-3-3 proteins have a role in cross talk between these hormonal pathways. The specificity and complementary nature of both the affinity purification and the yeast two-hybrid approaches is discussed.
Highlights
Phosphorylation dependent protein-protein interactions play crucial roles in the execution of various biological functions (Mukherji, 2005)
Later it was found that 14-3-3 proteins are present in all eukaryotes. 14-3-3 proteins belong to a conserved protein family consisting of 7 isoforms in human, 2 isoforms in yeast, 15 isoforms in Arabidopsis, and 8 isoforms in rice
In this study we show that the yeast two-hybrid screen is a good tool to identify putative 14-3-3 targets
Summary
Phosphorylation dependent protein-protein interactions play crucial roles in the execution of various biological functions (Mukherji, 2005). Most widespread amongst proteins that interact with phosphoserine or phosphothreonine motifs are members of the 14-3-3 family (Van Heusden, 2005). Co-crystallization of the 14-3-3 dimer together with a 14-3-3 interacting peptide derived from the kinase c-Raf-1, showed that the clamp shape structure has two binding grooves that both can bind a peptide (Yaffe et al, 1997). It was shown that the 14-3-3 binding sites contain a conserved amino acid composition that belongs either to the mode I (R/K-S-X-pS/T-X-P), mode II (R/K-X-Y/F-X-pS/T-X-P) or mode III (pS/pT(X1-2)-COOH) type (Muslin et al, 1996; Yaffe et al, 1997; Ganguly et al, 2005). Binding to the target protein depends on the phosphorylation of the serine or threonine residues inside the binding site in most, but not all cases
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