Purification and characterization of Ca2+/calmodulin-dependent protein kinase IV kinase from rat brain.
Calmodulin-dependent protein kinase IV (CaM-kinase IV) kinase was recently discovered in the rat brain by its activity to activate the inactive recombinant CaM-kinase IV expressed in Escherichia coli [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170]. In the present study, CaM-kinase IV kinase was purified approximately 2,000-fold from rat cerebral cortex by purification procedures including calmodulin affinity chromatography, and its properties were examined. The highly purified CaM-kinase IV kinase gave one major protein band corresponding to a molecular weight of about 66,000 upon SDS-PAGE. The purified CaM-kinase IV kinase phosphorylated and concomitantly activated CaM-kinase IV purified from rat brain as well as the recombinant kinase expressed in Escherichia coli in a Ca2+/calmodulin-dependent manner. The phosphorylation of CaM-kinase IV by CaM-kinase IV kinase occurred on only serine residue(s). Among a number of proteins, including several known to be phosphorylated by the various protein kinases tested, CaM-kinase IV was the best substrate for CaM-kinase IV kinase. Since syntide-2, a synthetic peptide known to be a good peptide substrate for calmodulin-dependent protein kinase II (CaM-kinase II), was a fairly good substrate for CaM-kinase IV kinase, some kinetic properties of CaM-kinase IV kinase were examined using syntide-2 as a substrate. The Km value for the peptide substrate in the presence of Ca2+/calmodulin was almost two orders of magnitude lower than that in its absence, although the Vmax value was almost the same in the presence and absence of Ca2+/calmodulin.
Okuno
S
S
Department of Biochemistry, Asahikawa Medical College, Hokkaido.
Kitani
T
T
Fujisawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012685:Sepharose / Q000031:analogs & derivatives
D002147:Calmodulin; C029483:Blue-Sepharose; D012685:Sepharose; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat