Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM -binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. GEl band shift assays and densitometry experiments with intact CAM kinase I and the CAM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that confirmational changes CaM on binding CaM kinase I orCaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of Ca, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of MET small mutants of CaM showed that the Trp-containing N-terminal lobe of CaM was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicated that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase (AU)

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cystosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose CI 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca2+/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase VI (AU)