The regulatory region of calcium/calmodulin-dependent protein kinase I contains closely associated autoinhibitory and calmodulin-binding domains.
The mechanism for the regulation of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was investigated using a series of COOH-terminal truncated mutants. These mutants were expressed in bacteria as fusion proteins with glutathione S-transferase and purified by affinity chromatography using glutathione Sepharose 4B. A mutant (residues 1-332) showed complete Ca2+/CaM-dependent activity. Truncation mutants (residues 1-321, 1-314, and 1-309) exhibited decreasing affinities for Ca2+/CaM and also exhibited decreasing Ca2+/CaM-dependent activities. Truncation mutants (residues 1-305 or 1-299) were unable to bind Ca2+/CaM and were inactive. In contrast, truncation mutants (residues 1-293 or 1-277) were constitutively active at a slightly higher level (2-fold) than fully active CaM kinase I. These results indicate the location of the Ca2+/CaM-binding domain on CaM kinase I (residues 294-321) and predict the existence of an autoinhibitory domain near, or overlapping, the Ca2+/CaM-binding domain. These conclusions were supported by studies which showed that a synthetic peptide (CaM kinase I (294-321)) corresponding to residues 294-321 of CaM kinase I inhibited the fully active kinase in a manner that was competitive with Ca2+/CaM and also inhibited the constitutively active mutant (residues 1-293) in a manner that was competitive with Syntide-2, a peptide substrate, (Ki = 1.2 microM) but was non-competitive with ATP. Thus, these results suggest that CaM kinase I is regulated through an intrasteric mechanism common to other members of the family of Ca2+/CaM-dependent protein kinases.
Yokokura
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H
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Picciotto
M R
MR
Nairn
A C
AC
Hidaka
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002147:Calmodulin; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517599:Camk1 protein, rat; C517682:Pnck protein, rat; D009219:Myosin-Light-Chain Kinase