Calcium and phospholipid activation of a recombinant calcium-dependent protein kinase (DcCPK1) from carrot (Daucus carota L.).
A calmodulin-like domain protein kinase (DcCPK1, previously designated CDPK431) cloned from carrot (Daucus carota L.) was expressed at high levels in Escherichia coli and partially purified. Ca(2+)-induced gel mobility shift and (45)Ca(2+) ligand binding assays confirmed that recombinant DcCPK1 binds Ca(2+) through its calmodulin-like domain and undergoes a significant conformational change. Ca(2+) activated the kinase activity of recombinant DcCPK1 (K(0.5)=1.7 microM) up to 20-fold. Ca(2+) combined with certain lipids, including phosphatidic acid, phosphatidylserine and phosphatidylinositol, but not diolein or lysophosphatidylcholine, provided even greater Ca(2+)-dependent protein kinase activity. DcCPK1 phosphorylated casein and histone III-S, and a variety of peptide substrates containing a hydrophobic and a basic residue situated P-5 and P-3 amino acids N-terminal to a Ser or Thr residue. The calmodulin and protein kinase inhibitors, W-7 and staurosporine, inhibited CDPK activity. The similarities between DcCPK1 and mammalian protein kinase C (PKC) in substrate specificity, sensitivity to inhibitors, and activation by Ca(2+) and phospholipid suggest that various CDPK isoforms may be responsible for some PKC-like activities in plant cells.
Farmer
P K
PK
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA.
Choi
J H
JH
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D003001:Cloning, Molecular; D018552:Daucus carota / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D010743:Phospholipids; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases