The Arabidopsis GORK K+-channel is phosphorylated by calcium-dependent protein kinase 21 (CPK21), which in turn is activated by 14-3-3 proteins

Abstract

Potassium (K+) is a vital ion for many processes in the plant and fine-tuned ion channels control the K+-fluxes across the plasma membrane. GORK is an outward-rectifying K+-channel with important functions in stomatal closure and in root K+-homeostasis. In this study, post-translational modification of the Arabidopsis GORK ion channel and its regulation by 14-3-3 proteins was investigated. To investigate the possible interaction between GORK and 14-3-3s an in vivo pull-down from an Arabidopsis protein extract with recombinant GORK C-terminus (GORK-C) indeed identified endogenous 14-3-3s (LAMBDA, CHI, NU) as binding partners in a phosphorylation dependent manner. However, a direct interaction between 14-3-3's and GORK-C could not be demonstrated. Since the pull-down of 14-3-3s was phosphorylation dependent, we determined GORK-C as substrate for CPK21 phosphorylation and identified three CPK21 phospho-sites in the GORK protein (T344, S518 and S649). Moreover, interaction of 14-3-3 to CPK21 strongly stimulates its kinase activity; an effect that can result in increased GORK phosphorylation and change in activity. Using the non-invasive vibrating probe technique, we measured the predominantly GORK mediated salt induced K+-efflux from wild-type, gork, cpk21, aha2 and 14-3-3 mutant roots. The mutants cpk21 and aha2 did not show statistical significant differences compared to WT. However, two (out of six) 14-3-3 isoforms, CHI and PHI, have a clear function in the salt induced K+-efflux. In conclusion, our results show that GORK can be phosphorylated by CPK21 and suggest that 14-3-3 proteins control GORK activity through binding with and activation of CPK21.