Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain.
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.
Harper
J F
JF
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Huang
J F
JF
Lloyd
S J
SJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism*; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002147:Calmodulin; D018076:DNA, Complementary; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases