A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H⁺‐ATPase Binds 14‐3‐3 Proteins and Inhibits Fusicoccin‐Induced Activation of the H⁺‐ATPase

Abstract

A putative consensus domain for binding of 14‐3‐3 proteins to the plasma membrane (PM) H+‐ATPase was identified in the highly‐conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14‐3‐3 protein, ii) inhibited fusicoccin‐induced stimulation of the PM H+‐ATPase activity both in PM isolated from germinating radish (Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H+‐ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non‐phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H+‐ATPase is a 14‐3‐3 binding site which might cooperate with the C‐terminal domain of the'enzyme in generating a stable association between the H+‐ATPase and 14‐3‐3 protein.

14-3-3 Protein-Activated and Autoinhibited Forms of Plasma Membrane H+-ATPase

“…With regard to this it should be noted that the loss of the penultimate amino acid of H ϩ -ATPase, the phosphothreonine, is sufficient to lose the 14-3-3 binding to the regulatory domain (30,31). In addition, it would be interesting to define the biological significance of the finding of an interaction of 14-3-3 proteins with H ϩ -ATPase at a site other than its C-terminal domain (32). Like in animals (1) in plants too the 14-3-3 proteins act as multifunctional regulatory proteins, in the regulation of the metabolism, of the transcription and of the organellar protein trafficking (2,33).…”

Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H+-ATPase

“…However, 14-3-3 proteins also bind to the H ϩ -ATPase independently of FC (Fullone et al, 1998), and this FC-independent binding is phosphorylation dependent and occurs in a truncated H ϩ -ATPase lacking the C-terminal domain (Jahn et al, 1998). Recently, Marra et al (2000) identified a sequence of the PM H ϩ -ATPase highly conserved in different isoforms of different plants and localized in the cytosolic stretch connecting transmembrane segments 8 and 9, which mimics the known 14-3-3 binding motif (Muslin et al, 1996;Yaffe et al, 1997). A phosphopeptide corresponding to such a sequence bound a 14-3-3 protein and inhibited FC binding and FCinduced activation of the PM H ϩ -ATPase; all of these effects were dependent on the phosphorylation of a Ser residue.…”

The Potassium Channel KAT1 Is Activated by Plant and Animal 14-3-3 Proteins

“…When provided in large excess, it presumably titrates the endogenous 14-3-3 (see the use of this peptide in Refs. 15,20,39). The injection of this peptide strongly decreased the level of I KAT1 in oocytes (70% Ϯ 6 at Ϫ150 mV, n ϭ 4); in control experiments (data not shown) heat-inactivated Raf 621p decreased the current at Ϫ150 mV of 38% Ϯ 4 (n ϭ 4).…”