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.