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

Phosphorylation of many protein substrates by the protein kinase casei n kinase 2 (CK2) is stimulated severalfold in the presence of polyamin es such as spermine. Previous experiments have shown that CK2 is a pol yamine binding protein and that the regulatory beta subunit is require d for this binding activity. To delineate the spermine binding site of CK2, we have applied a photoaffinity labeling method using a tritiate d photoactivable analog of spermine, [H-3]sperminediazonium. The photo affinity labeled beta subunit was cleaved with cyanogen bromide, and t wo labeled peptides were separated by high performance liquid chromato graphy. The major one was the peptide T(72)EQAAEM(78) and the minor on e 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 th e 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 al pha subunit residue covalently bound to the probe. The photoaffinity l abeling method described here enabled the first elucidation, by direct microsequencing, of a polyamine binding site on CK2 for which we prop ose a provisional structural model. These observations suggest a possi ble mechanism for CK2 activation by polyamines at the molecular level.