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Title: Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28)

Author
item BENDER, KYLE - University Of Illinois
item BLACKBURN, R KEVIN - North Carolina State University
item MONAGHAN, JACQUELINE - Sainsbury Laboratory
item ZIPFEL, CYRIL - Sainsbury Laboratory
item GOSHE, MICHAEL - North Carolina State University
item ZIELINSKI, RAYMOND - University Of Illinois
item Huber, Steven

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2017
Publication Date: 3/10/2017
Citation: Bender, K.W., Blackburn, R., Monaghan, J., Zipfel, C., Goshe, M.B., Zielinski, R.E., Huber, S.C. 2017. Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28). Journal of Biological Chemistry. 292:3988-4002.

Interpretive Summary: Signals from the environment often affect the intracellular concentration of calcium, which is then monitored by various calcium-binding proteins that function either as sensor relays or sensor responders. Sensor relays bind calcium and then interact with other proteins, while sensor responders are themselves calcium-regulated enzymes. Plants contain a unique family of sensor responders that are calcium-dependent protein kinases (CPKs) that are activated by calcium binding and link changes in calcium with alterations in protein phosphorylation. While regulation of CPKs by calcium has been well studied, the potential contribution of other factors such as autophosphorylation of the CPK and possible interaction with sensor relay proteins has not been explored. We demonstrate here that one member of the Arabidopsis CPK family, CPK28, autophosphorylates on numerous serine, threonine and tyrosine residues, and secondly that CPK28 interacts in a high-affinity manner with the sensor relay protein, calmodulin. Interestingly, the autophosphorylation status of CPK28 reduces the interaction with calcium-bound calmodulin, and eliminates the dependence of kinase activity on added calcium. The latter result indicates that when autophosphorylated, CPK28 directly binds calcium with increased affinity such that basal levels of calcium are sufficient to maintain full activity. This suggests a molecular mechanism to allow the protein kinase to ‘remember’ prior calcium-signaling events. Overall, the results uncover new complexities in calcium signaling in plants.

Technical Abstract: Plant calcium (Ca2+) dependent protein kinases (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) and represent the primary Ca2+-dependent protein kinase activities in plant systems. While regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation to control of CPK activity is less well understood. Furthermore, whether Calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases (CaMKs) outside of the plant lineage, remains an open question. We screened a subset of plant CPKs for CaM-binding and demonstrate 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 and overlapping the known site of intramolecular interaction between the J domain and CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites and compared activity of phosphorylated and dephosphorylated CPK28 towards a peptide substrate. Similar to metazoan CaMKs, activity of autophosphorylated CPK28 was essentially Ca2+-independent. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Our analyses uncover previously unrecognized complexities in the regulation of CPK activity by Ca2+, autophosphorylation, and CaM-binding.