Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28)

Abstract

Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.

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

“…However, discrepant reports suggest that CaM regulation may depend on co‐factors, substrate and CaM/CML isoforms (Du et al ., ; Liu et al ., ). AtCPK3/10/28/30 and to a lesser extent AtCPK16 among 13 tested CPKs were also able to bind CaM/CML in vitro (Popescu et al ., ; Bender et al ., ). A detailed analysis of AtCPK28 mapped the CaM binding site in the JD, resulting in kinase inhibition in vitro (Bender et al ., ).…”

“…In a large‐scale analysis of 759 protein kinases, tyrosine autophosphorylation has been detected on AtCRK2/3/8 while no significant signal over the background could be observed for 17 tested CDPKs (Nemoto et al ., ). Indeed, very few CPKs could autophosphorylate on Tyr in vitro , including AtCPK28 which is closer to CRKs than to other CDPK subgroups, suggesting that CRKs and subgroup IV CDPKs could share this specific ability (Oh et al ., ; Swatek et al ., ; Nemoto et al ., ; Bender et al ., ; van Kleeff et al ., ). Nevertheless, the molecular basis of such activity remains elusive as none of the established tyrosine kinase consensus motifs is present in CDPKs or CRKs (Rudrabhatla et al ., ).…”

“…AtCPK3/10/28/30 and to a lesser extent AtCPK16 among 13 tested CPKs were also able to bind CaM/CML in vitro (Popescu et al ., ; Bender et al ., ). A detailed analysis of AtCPK28 mapped the CaM binding site in the JD, resulting in kinase inhibition in vitro (Bender et al ., ). Although the molecular mechanism of such regulation still needs to be clarified and investigated in vivo , CaM/CML could specifically regulate some CRKs and CPKs.…”

Calcium Signalling in Plant Biotic Interactions

“…In line with this hypothesis, in vitro assays determined that CPK28 is indeed Ca 2+ responsive [ 137 ]. Interestingly, CPK28 is also able to bind Ca 2+ /CaM, however, this interaction negatively affects its kinase activity in vitro [ 138 ]. Ca 2+ therefore seems to have a dual role in regulating CPK28 activity, thereby, adding another layer of complexity that has yet to be resolved.…”

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

“…Briefly, LC-MS/MS analysis was performed with a Orbitrap Fusion Trihybrid mass spectrometer (Thermo Scientific) and a nanoflow-HPLC system (Dionex Ultimate3000, Thermo Scientific) described previously [54]. The peptide identification was performed by searching the in-house N.…”