Phosphorylation of thymidylate synthase from various sources by human protein kinase CK2 and its catalytic subunits

Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2α and CK2α′ characterized by similar Km values, 4.6 μM and 4.2 μM, respectively, but different efficiencies, the apparent turnover number with CK2α being 10-fold higher. With both catalytic subunits, phosphorylation of human TS, like calmodulin and BID, was strongly inhibited in the presence of the regulatory subunit CK2β, the holoenzyme being activated by polylysine. Phosphorylation of recombinant human, rat, mouse and Trichinella spiralis TSs proteins was compared, with the human enzyme being apparently a much better substrate than the others. Following hydrolysis and TLC, phosphoserine was detected in human and rat, and phosphotyrosine in T. spiralis, TS, used as substrates for CK2α. MALDI-TOF MS analysis led to identification of phosphorylated Ser124 in human TS, within a sequence LGFS124TREEGD, atypical for a CK2 substrate recognition site. The phosphorylation site is located in a region considered important for the catalytic mechanism or regulation of human TS, corresponding to the loop 107–128. Following phosphorylation by CK2α, resulting in incorporation of 0.4 mol of phosphate per mol of dimeric TS, human TS exhibits unaltered Km values for dUMP and N5,10-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser124 phosphorylation on its catalytic efficiency.

Thymidylate synthase (TS) was a substrate for human CK2 catalytic subunits. MALDI-TOF MS analysis of sulfonated protein digest led to identification of phosphorylated Ser124 in human TS, within a sequence LGFS124TREEGD.Figure optionsDownload as PowerPoint slide