Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition ofArabidopsis thalianaCa2+-dependent Protein Kinase 28 (CPK28).
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. Usingin situautophosphorylated 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.
From the Department of Plant Biology, University of Illinois, Urbana, Illinois 61801, kwbender@illinois.edu.
Kyle W
0000-0002-1805-8097
KW
Bender
the Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695.
R Kevin
RK
Blackburn
The Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom, and.
Jacqueline
J
Monaghan
The Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom, and.
Paul
P
Derbyshire
The Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom, and.
Frank L H
FL
Menke
The Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom, and.
Cyril
C
Zipfel
the Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695.
Michael B
MB
Goshe
From the Department of Plant Biology, University of Illinois, Urbana, Illinois 61801, rez@illinois.edu.
Raymond E
RE
Zielinski
From the Department of Plant Biology, University of Illinois, Urbana, Illinois 61801.|the United States Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801.
Steven C
SC
Huber
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects*; D007700:Kinetics; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D029681:Arabidopsis Proteins; D002118:Calcium; D002147:Calmodulin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases
Arabidopsis thaliana; Ca2+-dependent protein kinase; autophosphorylation; calcium; calmodulin (CaM); protein-protein interaction