Photo-crosslinking of human protein kinase regulatory subunit CK2β for the identification of CK2 binding partners

Human protein kinase CK2 is a heterotetrameric Ser/Thr kinase, consisting of two catalytic (CK2α/α’) and two regulatory (CK2β) subunits. CK2 plays a key role in several physiological and pathological processes. Moreover in cancer cells it was shown that CK2 is upregulated [1]. Although the number of more than 300 substrates is still increasing, the regulation of CK2 remains unclear [2]. It is assumed that several protein-protein interactions are involved in the regulation of CK2. Thereby CK2β modulates the substrate specificity of CK2 and also functions as a docking platform for regulators and substrates. This study aims for the identification of binding partners by photo-crosslinking coupled with mass spectrometry. Therefore the unnatural amino acid p‑azidophenylalanine (pAzF) is incorporated into CK2β [3].

Here we report the establishment of the photo-crosslinking procedure with purified CK2β‑pAzF with its strongest binding partner CK2α as a proof of principle. The photo-crosslinking product of CK2β-pAzF and CK2α was detected by SDS-PAGE analysis and immunostaining. Furthermore it was shown, that the photo-crosslink reaction is specific for interaction partners and is not affected by other proteins. The site directed photo-crosslinking reaction was compared to the common used homo-bifunctional NHS-ester disuccinimidyl suberate (DSS) that crosslinks primary amino groups.

References:

[1] Tawfic, S. et al.: Histol Histopathol. 2001, 16:573-582.

[2] Meggio, F.and Pinna, L.A.: FASEB J. 2003, 17:349-368.

[3] Chin, J.W. et al.: J. Am. Chem. Soc. 2002, 124, 9026-9027.