Activation of Plant Plasma Membrane H+-ATPase by 14-3-3 Proteins Is Negatively Controlled by Two Phosphorylation Sites within the H+-ATPase C-terminal Region

Abstract

The proton pump ATPase (H ؉ -ATPase) of the plant plasma membrane is regulated by an autoinhibitory C-terminal domain, which can be displaced by phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins. We performed a mass spectrometric analysis of PMA2 (plasma membrane H ؉ -ATPase isoform 2) isolated from Nicotiana tabacum suspension cells and identified two new phosphorylated residues in the enzyme 14-3-3 protein binding site: Thr 931 and Ser 938 . When PMA2 was expressed in Saccharomyces cerevisiae, mutagenesis of each of these two residues into Asp prevented growth of a yeast strain devoid of its own H ؉ -ATPases. When the Asp mutations were individually introduced in a constitutively activated mutant of PMA2 (E14D), they still allowed yeast growth but at a reduced rate. Purification of His-tagged PMA2 showed that the T931D or S938D mutation prevented 14-3-3 protein binding, although the penultimate Thr 955 was still phosphorylated, indicating that Thr 955 phosphorylation is not sufficient for full enzyme activation. Expression of PMA2 in an N. tabacum cell line also showed an absence of 14-3-3 protein binding resulting from the T931D or S938D mutation. Together, the data show that activation of H ؉ -ATPase by the binding of 14-3-3 proteins is negatively controlled by phosphorylation of two residues in the H ؉ -ATPase 14-3-3 protein binding site. The data also show that phosphorylation of the penultimate Thr and 14-3-3 binding each contribute in part to H ؉ -ATPase activation.Plasma membrane H ϩ

A Phosphorylation in the C-terminal Auto-inhibitory Domain of the Plant Plasma Membrane H+-ATPase Activates the Enzyme with No Requirement for Regulatory 14-3-3 Proteins

“…Western blotting of the purified PMA2 isoforms (Fig. 6) showed that PMA2-Ser938Asp had a lower level of phosphorylated Thr-955 and less bound 14-3-3 proteins than the wild type, as described previously (14). When compared with unmodified PMA2, the mutation of Thr-889 into alanine or aspartate did not modify FIGURE 5.…”

“…However, whether 14-3-3 protein binding was required remains unanswered. Recently, it was shown that the PMA2-Ser938Asp (PMA2-S938D) mutation prevents 14-3-3 protein binding and fails to sustain yeast growth and that, in this mutant, Thr-955 phosphorylation is decreased, but not abolished (14). If we hypothesize that activation of H ϩ -ATPase by phosphorylation of Thr-889 requires Thr-955 phosphorylation, but not the binding of 14-3-3 proteins, the double mutant PMA2-Thr889Asp-Ser938Asp (PMA2-T889D-S938D) should be devoid of bound 14-3-3 proteins but still sustain yeast growth.…”

“…The mutants PMA2-Glu14Asp (24), PMA2-Thr955Ala (9), PMA2-Ser938Asp (14), and PMA2-⌬52 (12) have been described previously. PMA2-⌬Cter, coding for PMA2 lacking the last 102 amino acids, was obtained by introducing a stop codon at position 855 between the ApaI and BglII restriction sites of 2p(PMA1)6hispma2 by triple PCR, and then the modified fragment was used to replace the corresponding ApaI/BglII fragment of 2p(PMA1)6hispma2.…”

“…Membrane Isolation-All buffers were supplemented with 1 mM phenylmethylsulfonyl fluoride and the protease inhibitors leupeptin, aprotinin, antipain, pepstatin, and chymostatin (2 g/ml each). Yeast and plant microsomal and plasma membrane fractions were prepared as described previously (14). Microsomal membranes from N. tabacum BY2 cells at day 3 were prepared with a homogenization buffer supplemented with 1 M fusicoccin to stabilize the H ϩ -ATPase 14-3-3 complex during subcellular fractionation (11).…”

“…Mass spectrometric analysis of the purified His-tagged plasma membrane H ϩ -ATPase isoform 2 (PMA2) from Nicotiana plumbaginifolia expressed in Nicotiana tabacum BY2 cells identified two phosphorylation sites, Thr-931 and Ser-938 (14). On mutation of these residues to aspartate, mimicking phosphorylation, the enzyme no longer binds 14-3-3 proteins and consequently cannot be activated, suggesting that both phosphorylation events negatively regulate the enzyme by interfering with 14-3-3 protein binding.…”