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
Authors: Uta Ansch√ºtz
Antonella Reyer
J√∂rg Kudla
Katrin Held
Martin Scholz
Michael Hippler
Kenji Hashimoto
Christian Eckert
Rainer Waadt
Dirk Becker
Journal: Journal of Biological Chemistry (2012)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Calcineurin B-like proteins; CBL-CIPK complexes; in vitro transcription/translation protocol; phosphorylation; CIPK NAF domain
Affiliations: Institut f√ºr Biologie und Biotechnologie der Pflanzen, Universit√§t M√ºnster, Schlossplatz 4, 48149 M√ºnster, Germany.

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. 

Retracted: No

PubPeer comments:
 Figure 1, it seems there is a duplicated panel here (red boxes), but detected proteins are supposed to be different (ellipses). Could the authors please check?  , 
 Thank you very much for bringing this duplication to our attention. Regretfully, this mistake happened during the assembly of the final figure version. This resulted in a duplication of the data from the CIPK1 autophosphorylation panel that were overlaid onto the data representing CIPK24 autophosphorylation data in the lower panel. The autophosphorylation experiments depicted in Figure 1 of this publication have been performed in triplicate. We have now replaced the erroneous CIPK24 panel with the original data from December 2010, which we intended to publish. The correct figure is now provided. , 
 #2 Thank you very much for your reply, and for going to correct this mistake. ,