N‐myristoylation and S‐acylation are common modifications of Ca²⁺‐regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel

Abstract

N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

Properties and functions of calcium‐dependent protein kinases and their relatives inArabidopsis thaliana

“…Moreover, the relative importance of Tyr-vs Ser/ Thr-phosphorylation remains to be evaluated because CPK Tyrphosphorylation has only been observed in vitro, which may represent marginal activity in vivo. Prediction of N-myristoylation has been confirmed in vitro for all predicted CPKs (Saito et al, 2018). *N-myristoylation and/or palmitoylation observed in vivo (Hemsley et al, 2013;Majeran et al, 2018).…”

“… Prediction of N‐myristoylation has been confirmed in vitro for all predicted CPKs (Saito et al ., ). *N‐myristoylation and/or palmitoylation observed in vivo (Hemsley et al ., ; Majeran et al ., ). …”

“…Indeed, all CRKs and 28 CPKs are predicted to be myristoylated at glycine2, a co‐translational modification involved in membrane targeting (Boudsocq & Sheen, ). In agreement, all of them were myristoylated in vitro on the N‐terminal peptide (Saito et al ., ), which was confirmed in vivo by MS analysis for AtCRK3/4/5/8 and 13 CPKs (Majeran et al ., ; Tables ). To maintain membrane association, proteins undergo reversible palmitoylation on a close cysteine or harbor a basic domain in the VNTD (Chehab et al ., ) which can be counteracted by phosphorylation.…”

Fine‐tuning of RBOHF activity is achieved by differential phosphorylation and Ca2+ binding

“…This obstacle can be exemplified using the guard cell model system. In these cells, it has been established that OST1 and CIPK11 contribute to SLAC1 ion channel regulation, and our work establishes RBOHF as an additional target of these kinases (Geiger et al ., ; Saito et al ., ). It would be challenging to distinguish the physiological contribution of each of these components to the observed phenotypes of the respective mutants.…”

Genetic variation in ZmTIP1 contributes to root hair elongation and drought tolerance in maize

“…Unlike transmembrane proteins, attachment of peripheral proteins to membranes can be brought about through lipid modifications, including N ‐myristoylation and/or S ‐acylation (Greaves and Chamberlain, ). Some evidence has been provided, indicating that N‐terminal protein myristoylation at glycine and/or S ‐acylation of an adjacent cysteine is essential for protein PM targeting in plants (Gutermuth et al , ; Hemsley et al , ; Liu, et al , ; Saito et al , ). In the present research, although the protein affinity purification was carried out in a heterogeneous plant system, we successfully identified ZmCPK9 as a substrate protein for ZmTIP1 in maize, suggesting that a conserved mechanism may exist in different plant species.…”