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 β subunit is required for this binding acti...

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Bibliographic Details
Published in:The Journal of biological chemistry 1995-07, Vol.270 (29), p.17400-17406
Main Authors: Leroy, Didier, Schmid, Nathalie, Behr, Jean-Paul, Filhol, Odile, Pares, Serge, Garin, Jérome, Bourgarit, Jean-Jacques, Chambaz, Edmond M., Cochet, Claude
Format: Article
Language:English
Subjects:
Affinity Labels - metabolism
Amino Acid Sequence
Animals
Binding Sites
Casein Kinase II
Molecular Sequence Data
Protein Conformation
Protein-Serine-Threonine Kinases - chemistry
Protein-Serine-Threonine Kinases - metabolism
Spermine - metabolism
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Summary: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 β 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, [3H]sperminediazonium. The photoaffinity labeled β subunit was cleaved with cyanogen bromide, and two labeled peptides were separated by high performance liquid chromatography. The major one was the peptide T72EQAAEM78 and the minor one was a 22-amino acid peptide comprising residues Ile98 to Met119. Thr72 and His108 were identified as the labeled amino acids of the Thr72-Met78 and Ile98-Met119 peptides, respectively. In the same manner, we succeeded in determining the residue Leu220 as an α 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.270.29.17400