The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end.
14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine) in the extreme C-terminal end of the H+-ATPase interacts with the binding cleft of 14-3-3 protein (Wurtele, M., Jelich-Ottmann, C., Wittinghofer, A., and Oecking, C. (2003) EMBO J. 22, 987-994). We report binding of 14-3-3 protein to a nonphosphorylated peptide representing the 34 C-terminal residues of the Arabidopsis plasma membrane H+-ATPase isoform 2 (AHA2). Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3. Among these, Thr-924 is important for interaction with 14-3-3 protein even when Thr-947 is phosphorylated. We suggest that the role of phosphorylation, which is accentuated by fusicoccin, is to stabilize protein-protein interaction between 14-3-3 protein and several residues of the H+-ATPase C-terminal domain.
Fuglsang
Anja T
AT
Department of Plant Biology, Plant Physiology and Anatomy Laboratory, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Borch
Jonas
J
Bych
Katrine
K
Jahn
Thomas P
TP
Roepstorff
Peter
P
Palmgren
Michael G
MG
D048948:14-3-3 Proteins; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000378:metabolism*
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D010446:Peptide Fragments; D014446:Tyrosine 3-Monooxygenase; D006180:Proton-Translocating ATPases; C479903:AHA2 protein, Arabidopsis