Maize plasma membrane aquaporins belonging to the PIP1 and PIP2 subgroups are in vivo phosphorylated.
Aquaporins are channel proteins that facilitate transmembrane water movement. In this study, we showed that plasma membrane intrinsic proteins (PIPs) from maize shoots are in vitro and in vivo phosphorylated on serine residues by a calcium-dependent kinase associated with the membrane fraction. Mass spectrometry identified phosphorylated peptides corresponding to the C-terminal region of (i) ZmPIP2;1, ZmPIP2;2 and/or ZmPIP2;7; (ii) ZmPIP2;3 and/or ZmPIP2;4; (iii) ZmPIP2;6; together with (iv) a phosphorylated peptide located in the N-terminal region of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and/or ZmPIP1;4. The role of phosphorylation in the water channel activity of wild-type and mutant ZmPIP2;1 was studied in Xenopus laevis oocytes. Activation of endogenous protein kinase A increased the osmotic water permeability coefficient of ZmPIP2;1-expressing oocytes, suggesting that phosphorylation activates its channel activity. Mutation of S126 or S203, putative phosphorylated serine residues conserved in all plant PIPs, to alanine decreased ZmPIP2;1 activity by 30-50%, without affecting its targeting to the plasma membrane. Mutation of S285, which is phosphorylated in planta, to alanine or glutamate did not affect the water channel activity. These results indicate that, in oocytes, S126 and S203 play an important role in ZmPIP2;1 activity and that phosphorylation of S285 is not required for its activity.
Van Wilder
Valérie
V
Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 5-15, B-1348 Louvain-la-Neuve, Belgium.
Miecielica
Urszula
U
Degand
Hervé
H
Derua
Rita
R
Waelkens
Etienne
E
Chaumont
François
F
D000818:Animals; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D009865:Oocytes / Q000378:metabolism; D009995:Osmosis; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018520:Plant Shoots / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D020346:Aquaporins; D017868:Cyclic AMP-Dependent Protein Kinases; D018076:DNA, Complementary; D018744:DNA, Plant; D049452:Green Fluorescent Proteins; D010940:Plant Proteins; D011494:Protein Kinases; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases