Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.
The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth.
Svennelid
F
F
Department of Plant Biochemistry, Lund University, P.O. Box 117, SE-221 00 Lund, Sweden.
Olsson
A
A
Piotrowski
M
M
Rosenquist
M
M
Ottman
C
C
Larsson
C
C
Oecking
C
C
Sommarin
M
M
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006027:Glycosides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D006027:Glycosides; D009183:Mycotoxins; D010446:Peptide Fragments; C007808:fusicoccin; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D006180:Proton-Translocating ATPases