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.


Abstract:
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 proteinprotein 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 photocrosslink 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.

Influence of pAzF incorporation into CK2β on holoenzyme formation Capillary electrophoresis analysis of CK2 activity
The phosphorylation of a substrate peptide (SP) by CK2α alone and in addition of CKβ or CK2β pAzF was analyzed at 37°C.

SP SP P
Incorporation of pAzF into CK2β keeps its function to stabilize CK2α unaffected CKβ pAzF was incubated with CK2α and irradiated with UV light of 365 nm. The αβ-photo-crosslink (*) was analysed by SDS-PAGE with Coomassie staining and by Western Blot with a primary antibody against CK2α.

Results and Discussion
The interaction of CK2α and CK2β pAzF was covalently captured by photocrosslinking

Photo-crosslinking of CK2β pAzF and CK2α
Photocrosslinking of CK2β pAzF and CK2α in presence of bovine serum albumin (BSA) as a non-binding partner of CK2β CKβ pAzF was incubated with CK2α and a two fold higher concentration of BSA. The proteins were irradiated with UV light of 365 nm and separated by SDS-PAGE. (*) CK2αβ-photo-crosslink; (**) CK2 ββ-photocrosslink Photo-crosslinking reaction is not influenced by background proteins like BSA

Results and Discussion
Specificity of photo-crosslinking reaction in presence of non-interaction partners ** ** Crosslinking of CK2β and CK2α with disuccinimidyl suberate (DSS) CKβ and CK2α were incubated with different concentrations of the homo-bifunctional NHS-ester DSS that crosslinks primary amino groups. Crosslinks were analysed by SDS-PAGE.

Non-site-directed crosslinking method in comparison
One αβ-photo-crosslink with CK2β pAzF +CK2α -Multiple crosslinks with DSS+CK2β+CK2α  The unnatural amino acid p-azidophenylalanine (pAzF) was incorporated into the regulatory CK2 subunit CK2β  This mutant CK2β pAzF was still able to increase the activity of CK2α  CK2β pAzF was successfully photo-crosslinked with CK2α  It could be shown, that the photo-crosslinking reaction is not influenced by background proteins like bovine serum albumin  Compared to another crosslinking method using DSS, photo-crosslinking with incorporated pAzF offers the advantage of a site directed reaction with only one crosslinking product per CK2β pAzF protein