A single Arabidopsis GF14 isoform possesses biochemical characteristics of diverse 14-3-3 homologues

Abstract

Arabidopsis cDNA clones of GF14 proteins originally were isolated on the basis of their association with the G-box DNA/protein complex by a monoclonal antibody screening approach. GF14 proteins are homologous to the 14-3-3 family of mammalian proteins. Here we demonstrate that recombinant GF14 omega, one member of the Arabidopsis GF14 protein family, is a dimeric protein that possesses many of the attributes of diverse mammalian 14-3-3 homologues. GF14 omega activates rat brain tryptophan hydroxylase and protein kinase C in a manner similar to the bovine 14-3-3 protein. It also activates exoenzyme S of Pseudomonas aeruginosa as does bovine brain factor activating exoenzyme S (FAS), which is itself a member of 14-3-3 proteins. In addition, GF14 omega binds calcium, as does the human 14-3-3 homologue reported to be a phospholipase A2. These results indicate that a single isoform of this plant protein family can have multiple functions and that individual GF14 isoforms may have multiple roles in mediating signal transductions in plants. However, GF14 omega does not regulate growth in an in vivo test for functional similarity to the yeast 14-3-3 homologue, BMH1. Thus, while a single plant GF14 isoform can exhibit many of the biochemical attributes of diverse mammalian 14-3-3 homologues, open questions remain regarding the physiological functions of GF14/14-3-3 proteins.

Four Arabidopsis thaliana 14‐3‐3 protein isoforms can complement the lethal yeast bmh1bmh2 double disruption

“…On a Western blot probed with a monoclonal antiserum raised against the GF14-14 protein no bands are visible in extracts from strains expressing the BMH1 or BMH2 gene. This confirms our previous observations that this antiserum does not detect the yeast 14-3-3 proteins [45]. Clear bands are visible after expression of the GF14-14, GF14-4 and GF14-5 isoforms.…”

Summary

“…This observation implies that 14-3-3 proteins from plants, animals and fungi have fundamentally similar properties. Lack of functional specificity of plant 14-3-3 isoforms has also been demonstrated in other systems (van Heusden et al, 1996;Lu et al, 1994a). Therefore, the ability to bind fusicoccin seems to be determined by the presence of both the plant H ϩ -ATPase and a 14-3-3 protein and is not dependent on any specific 14-3-3 protein isoform.…”

Binding of 14-3-3 Protein to the Plasma Membrane H+-ATPase AHA2 Involves the Three C-terminal Residues Tyr946-Thr-Val and Requires Phosphorylation of Thr947

“…The AHA2 cDNA encoding the C-terminal regulatory domain (residues Leu 851 -Val 948 ) was amplified by PCR as described (18) using full-length AHA2 contained within plasmid pMP-136 as a template. The plasmid derived from pAS1-CYH (28), pAS1-CYH1/14-3-3 GF14-, contained a fusion between the GAL4 DNA binding domain and Arabidopsis GF14-14-3-3 protein (29). Yeast cells were transformed with both fusion plasmids.…”

“…Regions of AHA2, fused in frame to the GAL4 DNA binding domain vector pACT2, were cotransformed into yeast with an Arabidopsis 14-3-3 gene (GF14-; Ref. 29) contained in the GAL4 transcriptional activator domain vector pAS1-CYH. The two-hybrid constructs employed are indicated in Fig.…”