ABSTRACT
Interaction of 14-3-3 proteins with their targets depends not only on the phosphorylation status of the target but also on that of 14-3-3 (Fu et al., 2000). In this work we demonstrated that the maize 14-3-3 isoform GF14-6 is a substrate of the tyrosine kinase insulin growth factor receptor 1. By means of site-directed mutants of GF14-6, we identified Tyr-137 as the specific tyrosine residue phosphorylated by the insulin growth factor receptor 1. Phosphorylation of GF14-6 on Tyr-137 lowered its affinity for a peptide mimicking the 14-3-3 binding site of the plant plasma membrane H+-ATPase. Moreover, phosphorylation in planta of 14-3-3 tyrosine residues, resulting from incubation with the tyrosine phosphatase inhibitor, phenylarsine oxide, decreased their association to the H+-ATPase.
Subject(s)
Plant Proteins/chemistry , Plant Proteins/metabolism , Proton-Translocating ATPases/metabolism , Tyrosine 3-Monooxygenase/chemistry , Tyrosine 3-Monooxygenase/metabolism , Tyrosine/chemistry , 14-3-3 Proteins , Arabidopsis/metabolism , Arsenicals/pharmacology , Base Sequence , Cell Membrane/enzymology , DNA, Plant/genetics , Enzyme Inhibitors/pharmacology , Mutagenesis, Site-Directed , Phosphorylation , Plant Proteins/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Receptor, IGF Type 1/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tyrosine 3-Monooxygenase/genetics , Zea mays/genetics , Zea mays/metabolismABSTRACT
To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H(+)-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H(+)-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H(+)-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H(+)-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H(+)-ATPase.
