title: Phosphorylation of calcineurin B-like (CBL) calcium sensor proteins by their CBL-interacting protein kinases (CIPKs) is required for full activity of CBL-CIPK complexes toward their target proteins.
abstract: Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K(+) channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.
authors.affiliation: Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 4, 48149 Münster, Germany.
authors.forename: Kenji
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Christian
authors.initials: C
authors.lastname: Eckert
authors.forename: Uta
authors.initials: U
authors.lastname: Anschütz
authors.forename: Martin
authors.initials: M
authors.lastname: Scholz
authors.forename: Katrin
authors.initials: K
authors.lastname: Held
authors.forename: Rainer
authors.initials: R
authors.lastname: Waadt
authors.forename: Antonella
authors.initials: A
authors.lastname: Reyer
authors.forename: Michael
authors.initials: M
authors.lastname: Hippler
authors.forename: Dirk
authors.initials: D
authors.lastname: Becker
authors.forename: Jörg
authors.initials: J
authors.lastname: Kudla
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014908:Triticum / Q000737:chemistry / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C417306:CBL protein, Arabidopsis; D002135:Calcium-Binding Proteins; D046912:Multiprotein Complexes; D015221:Potassium Channels; C074500:AKT1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases