Characterization of the mechanism of regulation of Ca2+/ calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase.
Ca2+/calmodulin-dependent protein kinase I (CaMKI) is maintained in an autoinhibited state by the interaction of a COOH-terminal helix-loop-helix (Ile286-Met316) regulatory domain with the catalytic core. Activation of the enzyme by calmodulin (CaM) also allows CaMKI to be phosphorylated and activated by a second enzyme, CaMK kinase (CaMKK). To more thoroughly characterize the regulation of CaMKI by CaM and its interrelationship with phosphorylation by CaMKK, we have carried out a detailed structure-function analysis using recombinant wild-type (WT) and mutant forms of CaMKI and CaMKK. CaMKI-WT, in the absence of CaM, or CaMKI-299 and CaMKI-298 were autoinhibited and could not be phosphorylated by CaMKK-433 (a truncated constitutively active form of CaMKK). Removal of Phe298 (CaMK-297) generated a constitutively active form of CaMKI that was also phosphorylated by CaMKK-433. CaMKI-WT was essentially inactive in the absence of CaM (K0.5 for activation by CaM approximately 30 nM). Mutation of Ile294 and Phe298 to alanine (CaMKI-2A) resulted in measurable basal enzyme activity. Additional mutation of Ile286 and Val290 to alanine (CaMKI-4A) increased this basal activity. Mutation of Trp303 (CaMKI-W303S) resulted in a large increase in the K0.5 for CaM ( approximately 100 microM), supporting a role for this residue as an initial target for CaM. Mutation of Phe307 (CaMKI-F307A) resulted in increased basal enzyme activity, supporting a role for this residue in autoinhibition of CaMKI. Together these studies demonstrate the critical role of specific amino acids in the autoinhibition of CaMKI and also in its activation by CaM and phosphorylation by CaMKK.
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats
D002147:Calmodulin; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517599:Camk1 protein, rat; C517686:Camk4 protein, rat; C517682:Pnck protein, rat