DIRECT IDENTIFICATION OF A POLYAMINE BINDING DOMAIN ON THE REGULATORY SUBUNIT OF THE PROTEIN-KINASE CASEIN KINASE-2 BY PHOTOAFFINITY-LABELING

Phosphorylation of many protein substrates by the protein kinase casein kinase 2 (CK2) is stimulated severalfold in the presence of polyamines such as spermine. Previous experiments have shown that CK2 is a polyamine binding protein and that the regulatory beta subunit is required for this binding activity. To delineate the spermine binding site of CK2, we have applied a photoaffinity labeling method using a tritiated photoactivable analog of spermine, [H-3]sperminediazonium. The photoaffinity labeled beta subunit was cleaved with cyanogen bromide, and two labeled peptides were separated by high performance liquid chromatography. The major one was the peptide T(72)EQAAEM(78) and the minor one was a 22-amino acid peptide comprising residues Ile(98) to Met(119). Thr(72) and His(108) were identified as the labeled amino acids of the Thr(72)-Met(78) and Ile(98) Met(119) peptides, respectively. In the same manner, we succeeded in determining the residue Leu(220) as an alpha subunit residue covalently bound to the probe. The photoaffinity labeling method described here enabled the first elucidation, by direct microsequencing, of a polyamine binding site on CK2 for which we propose a provisional structural model. These observations suggest a possible mechanism for CK2 activation by polyamines at the molecular level.