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Properties of Phosphorylated Thymidylate Synthase

Frączyk, Tomasz and Ruman, Tomasz and Wilk, Piotr and Palmowski, Paweł and Rogowska-Wrzesinska, Adelina and Cieśla, Joanna and Zieliński, Zbigniew and Nizioł, Joanna and Jarmuła, Adam and Maj, Piotr and Gołos, Barbara and Wińska, Patrycja and Ostafil, Sylwia and Wałajtys-Rode, Elżbieta and Shugar, David and Rode, Wojciech (2015) Properties of Phosphorylated Thymidylate Synthase. Biochimica et Biophysica Acta, 1854 . pp. 1922-1934. ISSN 1570-9639

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Official URL: http://www.sciencedirect.com/science/article/pii/S...


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 E. 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 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.

Item Type:Article
Subjects:Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Divisions:Department of Biophysics
ID Code:1077
Deposited By: Tomasz Frączyk
Deposited On:05 Jan 2016 13:36
Last Modified:08 Mar 2018 15:33

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