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

Fraczyk, TomaszNencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland (författare)

Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2alpha and CK2alpha' characterized by similar K(m) values, 4.6microM and 4.2microM, respectively, but different efficiencies, the apparent turnover number with CK2alpha 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 CK2beta, 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 CK2alpha. MALDI-TOF MS analysis led to identification of phosphorylated Ser(124) in human TS, within a sequence LGFS(124)TREEGD, 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 CK2alpha, resulting in incorporation of 0.4mol of phosphate per mol of dimeric TS, human TS exhibits unaltered K(m) values for dUMP and N(5,10)-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser(124) phosphorylation on its catalytic efficiency.

Kubiński, KonradDepartment of Molecular Biology, John Paul II Catholic University of Lublin, 102 Krasnicka Av., 20-718 Lublin, Poland (författare)
Masłyk, MaciejDepartment of Molecular Biology, John Paul II Catholic University of Lublin, 102 Krasnicka Av., 20-718 Lublin, Poland (författare)
Cieśla, JoannaNencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland (författare)
Hellman, UlfUppsala universitet,Ludwiginstitutet för cancerforskning(Swepub:uu)ulfhm (författare)
Shugar, DavidInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego St., 02-106 Warszawa, Poland (författare)
Rode, WojciechNencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland (författare)
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, PolandDepartment of Molecular Biology, John Paul II Catholic University of Lublin, 102 Krasnicka Av., 20-718 Lublin, Poland (creator_code:org_t)

Av författaren/redakt...: Fraczyk, Tomasz; Kubiński, Konrad; Masłyk, Maciej; Cieśla, Joanna; Hellman, Ulf; Shugar, David; visa fler...; Rode, Wojciech; visa färre...

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