Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains.
A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.
Ramachandiran
S
S
Department of Horticulture, Washington State University, Pullman 99164-6414, USA.
Takezawa
D
D
Wang
W
W
Poovaiah
B W
BW
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive / Q000235:genetics / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry*; D010944:Plants / Q000201:enzymology / Q000235:genetics; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D010940:Plant Proteins; C045140:visinin; D017871:Calcium-Calmodulin-Dependent Protein Kinases