title: Properties of phosphorylated thymidylate synthase.
abstract: Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Tomasz
authors: T
authors: Frączyk
authors: Rzeszów University of Technology, Faculty of Chemistry, Rzeszów, Poland.
authors: Tomasz
authors: T
authors: Ruman
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Piotr
authors: P
authors: Wilk
authors: Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors: Paweł
authors: P
authors: Palmowski
authors: Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors: Adelina
authors: A
authors: Rogowska-Wrzesinska
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Joanna
authors: J
authors: Cieśla
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Zbigniew
authors: Z
authors: Zieliński
authors: Rzeszów University of Technology, Faculty of Chemistry, Rzeszów, Poland.
authors: Joanna
authors: J
authors: Nizioł
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Adam
authors: A
authors: Jarmuła
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Piotr
authors: P
authors: Maj
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Barbara
authors: B
authors: Gołos
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Patrycja
authors: P
authors: Wińska
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
authors: Sylwia
authors: S
authors: Ostafil
authors: Warsaw University of Technology, Faculty of Chemistry, Institute of Biotechnology, Warsaw, Poland.
authors: Elżbieta
authors: E
authors: Wałajtys-Rode
authors: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors: David
authors: D
authors: Shugar
authors: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland. Electronic address: rode@nencki.gov.pl.
authors: Wojciech
authors: W
authors: Rode
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D051379:Mice; D010766:Phosphorylation; D011817:Rabbits; D013940:Thymidylate Synthase / Q000378:metabolism*
chemical_list: D013940:Thymidylate Synthase
keywords: Mass spectrometry (MS); Molecular dynamics; Nuclear magnetic resonance (NMR); Post‐translational modification (PTM); Protein phosphorylation; Thymidylate synthase