Biochemical characterization of calcineurin B-like-interacting protein kinase in Vicia guard cells.
Protein phosphorylation is crucial in the signaling of guard cells in response to various factors, including light, ABA and CO2, and calcium plays a central role in these signaling processes. Despite extensive studies on the functional role of Ca(2+)-regulated protein kinases in plants, relatively little is known about the biochemical properties of the kinases in guard cells. To investigate this, we isolated the VfCIPK1 [Vicia faba calcineurin B-like calcium-binding protein (CBL)-interacting protein kinase 1] cDNA from guard cells of Vicia faba L., which encodes a Ca(2+)-regulated protein kinase that belongs to the SnRK3 subgroup, and characterized VfCIPK1 at the biochemical level. VfCIPK1 genes were expressed in guard cells and roots, but not in mesophyll cells. The VfCIPK1 protein was localized on the outer membrane of mitochondria in guard cells and interacted with VfCBL1. The immunoprecipitation experiments indicated that VfCIPK1 interacted with VfCBL1 in vivo. The recombinant VfCIPK1 phosphorylated myelin basic protein as a substrate and the activity was increased by VfCBL1, and this activity showed a maximum in the absence of Ca2+ and decreased by an elevation of the Ca2+ concentration. A pull-down assay and the measurement of surface plasmon resonance indicated that the interaction between VfCIPK1 and VfCBL1 was decreased by Ca2+. These results suggest that VfCIPK1 may be negatively regulated by cytosolic Ca2+ through VfCBL1 and may be related to mitochondrial functions in guard cells. This is the first report that shows the inhibitory effect of Ca2+ on CIPK activity in the presence of CBL.
Tominaga
Misumi
M
Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, 812-8581 Japan.
Harada
Akiko
A
Kinoshita
Toshinori
T
Shimazaki
Ken-Ichiro
K
D000595:Amino Acid Sequence; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D051336:Mitochondrial Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D031307:Vicia faba / Q000235:genetics* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D017346:Protein Serine-Threonine Kinases; D019703:Calcineurin; D002118:Calcium