Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II.
Regulation of Ca(2+)/calmodulin-dependent protein kinase II is likely based on an auto-inhibitory mechanism in which a segment of the kinase occupies the catalytic site in the absence of calmodulin. We analyze potential auto-inhibitory associations by employing charge reversal and hydrophobic-to-charged residue mutagenesis. We identify interacting amino acid pairs by using double mutants to test which modification in the catalytic domain complements a given change in the auto-inhibitory domain. Our studies identify the core pseudosubstrate sequence (residues 297-300) but reveal that distinct sequences centered about the autophosphorylation site at Thr-286 are involved in the critical auto-inhibitory interactions. Individual changes in any of the residues Arg-274, His-282, Arg-283, Lys-291, Arg-297, Phe-293, and Asn-294 in the auto-inhibitory domain or their interacting partners in the catalytic domain produces an enhanced affinity for calmodulin or generates a constitutively active enzyme. A structural model of Ca(2+)/calmodulin-dependent protein kinase II that incorporates these interactions shows that Thr-286 is oriented inwardly into a hydrophobic channel. The model explains why calmodulin must bind to the auto-inhibitory domain in order for Thr-286 in that domain to be phosphorylated and why introduction of phospho-Thr-286 produces the important Ca(2+)-independent state of the enzyme.
Yang
E
E
Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125, USA.
Schulman
H
H
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D013379:Substrate Specificity
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases