Autophosphorylation of creatine kinase: characterization and identification of a specifically phosphorylated peptide.
We report that several different chicken and rabbit creatine kinase (CK)1 isoenzymes showed an incorporation of 32P when incubated with [gamma-32P]ATP in an autophosphorylation assay. This modification was was shown to be of covalent nature and resulted from an intramolecular phosphorylation reaction that was not dependent on the CK enzymatic activity. By limited proteolysis and sequence analysis of the resulting peptides, the autophosphorylation sites of chicken brain-type CK could be localized within the primary sequence of the enzyme to a 4.5 kDa peptide, spanning a region that is very likely an essential part of the active site of creatine kinase. Homologous peptides were found to be autophosphorylated in chicken muscle-type CK and a mitochondrial CK isoform. Phosphopeptide as well as mutant enzyme analysis provided evidence that threonine-282(2), threonine-289 and serine-285 are involved in the autophosphorylation of CK. Thr-282 and Ser-285 are located close to the reactive cysteine-283. Thr-289 is located within a conserved glycine-rich region highly homologous to the glycine-rich loop of protein kinases, which is known to be important for ATP binding. Thus, it seems likely that the described region constitutes an essential part of the active site of CK.
Hemmer
W
W
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0654, USA.
Furter-Graves
E M
EM
Frank
G
G
Wallimann
T
T
Furter
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011817:Rabbits
D007527:Isoenzymes; D010455:Peptides; D003402:Creatine Kinase