Calcium and phospholipid activation of a recombinant calcium-dependent protein kinase (DcCPK1) from carrot (Daucus carota L.).

Abstract 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 μM) 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.