AUTOPHOSPHORYLATION OF CREATINE-KINASE - CHARACTERIZATION AND IDENTIFICATION OF A SPECIFICALLY PHOSPHORYLATED PEPTIDE

We report that several different chicken and rabbit creatine kinase (C K)(1) isoenzymes showed an incorporation of P-32 when incubated with [ gamma-P-32]ATP in an autophosphorylation assay. This modification was shown to be of covalent nature and resulted from an intramolecular pho sphorylation reaction that was not dependent on the CK enzymatic activ ity. By limited proteolysis and sequence analysis of the resulting pep tides, the autophosphorylation sites of chicken brain-type CK could be localized within the primary sequence of the enzyme to a 4.5 kDa pept ide, spanning a region that is very likely an essential part of the ac tive site of creatine kinase. Homologous peptides were found to be aut ophosphorylated in chicken muscle-type CK and a mitochondrial CK isofo rm. Phosphopeptide as well as mutant enzyme analysis provided evidence that threonine-282(2), threonine-289 and serine-285 are involved in t he autophosphorylation of CK. Thr-282 and Ser-285 are located close to the reactive cysteine-283. Thr-289 is located within a conserved glyc ine-rich region highly homologous to the glycine-rich loop of protein kinases, which is known to be important for ATP binding. Thus, it seem s likely that the described region constitutes an essential part of th e active site of CK.