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

Biochim Biophys Acta. 1999 Sep 14;1434(1):6-17. doi: 10.1016/s0167-4838(99)00166-1.

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 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Calcium / pharmacology*
  • Cloning, Molecular
  • Daucus carota / enzymology*
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Escherichia coli
  • Molecular Sequence Data
  • Phospholipids / pharmacology*
  • Phosphorylation
  • Protein Kinase Inhibitors
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Phospholipids
  • Protein Kinase Inhibitors
  • Recombinant Proteins
  • Protein Kinases
  • calcium-dependent protein kinase
  • Calcium