A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis.
The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.
Hwang
I
I
Department of Cell Biology and Molecular Genetics, and Maryland Agricultural Experiment Station, University of Maryland, College Park, MD 20742, USA.
Sze
H
H
Harper
J F
JF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017136:Ion Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010940:Plant Proteins / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D000252:Calcium-Transporting ATPases; D002147:Calmodulin; D010768:Phosphoserine; D010940:Plant Proteins; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases