Determination of CK2 Specificity and Substrates by Proteome-Derived Peptide Libraries

Understanding
the specificity of kinases enables prediction of
their substrates and uncovering kinase functions in signaling pathways.
Traditionally synthesized peptide libraries are used to determine
the kinase specificity. In this study, a proteomics-based method was
developed to determine the specificity of kinase by taking the advantages
of proteome-derived peptide libraries and quantitative proteomics.
Proteome-derived peptide libraries were constructed by digesting proteins
in total cell lysate followed with dephosphorylation of the resulting
peptides. After incubating the peptide libraries with/without CK2
for in vitro kinase assay, stable isotopic labeling based quantitative
phosphoproteomics was applied to distinguish the in vitro phosphosites
generated by CK2. By using the above approach, 404 CK2 in vitro phosphosites
were identified by 1D LC–MS/MS. Those sites allowed the statistic
determination of the CK2 specificity. In addition to the easy construction
of the proteome-derived peptide library, another significant advantage
of this method over the method with synthesized peptide libraries
is that the identified phosphosites could be directly mapped to proteins
for the screening of putative kinase substrates. It was found that
the confidence for substrate identification could be significantly
improved by comparing the in vitro CK2 sites with the in vivo sites
identified by phosphoproteomics analysis of the same cell lines. By
applying this integrated strategy, 138 phosphosites from 105 putative
CK2 substrates of high confidence were determined.