Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain

Biochemistry. 1994 Jun 14;33(23):7267-77. doi: 10.1021/bi00189a031.


CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology
  • Base Sequence
  • Calmodulin / metabolism*
  • DNA Mutational Analysis
  • DNA, Complementary
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Peptides / pharmacology
  • Protein Kinase Inhibitors*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Sequence Deletion
  • Sequence Homology, Amino Acid


  • Calmodulin
  • DNA, Complementary
  • Peptides
  • Protein Kinase Inhibitors
  • Protein Kinases
  • calcium-dependent protein kinase