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.
Highlights
Intracellular release of Ca2ϩ ions belongs to the earliest events in signal perception in eukaryotes [1,2,3]
All our repeated attempts to overexpress full-length CIPK1 protein in either transiently or stably transformed tobacco BY-2 cell lines, transiently transformed Arabidopsis protoplasts, or stably transformed Arabidopsis plants failed due to toxicity problems or alternatively due to gene silencing that we observed after stable transformation
We performed a detailed biochemical characterization of recombinant CIPK1, CIPK23, and CIPK24 to determine their Km and Vmax values in phosphorylation analyses using synthetic substrates, Syntide-2 and ALARA peptide. These experiments confirmed that the recombinant CIPK1, CIPK23, and CIPK24 proteins harbor substrate phosphorylation activity and revealed that at least toward these artificial substrates CIPK24 exhibits significantly higher Km and Vmax values than CIPK1 and CIPK23 (Table 1 and supplemental Fig. S3)
Summary
Intracellular release of Ca2ϩ ions belongs to the earliest events in signal perception in eukaryotes [1,2,3]. Even stronger hyperactivation of kinase activity was achieved by deleting the inhibitory C-terminal regions of CIPK24/SOS2, including the NAF domain [24, 31, 39] Because these artificial kinases do not interact with CBLs, investigations of these recombinant proteins may be error-prone and cannot address CBL-dependent aspects of kinase regulation that bring about Ca2ϩ dependence and cellular targeting of CBL-CIPK complexes. In vitro analyses revealed strong Mn2ϩ dependence of the autophosphorylation activity of all CIPKs investigated Using these recombinant proteins, we identified phosphorylation of a conserved single Ser residue within the C terminus of CBLs by their interacting CIPKs as a general feature of CBL-CIPK complexes. These data suggest that CIPK-mediated phosphorylation of CBL proteins likely results in conformational changes of their flexible C terminus that enhance activity of CBL-CIPK complexes toward their target proteins
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