title: Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase.
abstract: The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(-) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.
authors.affiliation: Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA Connecticut Healthcare System, New Haven, CT 06520, USA.
authors.forename: Xiukun
authors.initials: X
authors.lastname: Lin
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Frank
authors.initials: F
authors.lastname: Maley
authors.forename: Edward
authors.initials: E
authors.lastname: Chu
mesh_terms: D000409:Alanine / Q000235:genetics; D045744:Cell Line, Tumor; D003545:Cysteine / Q000235:genetics* / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D008156:Luciferases; D013940:Thymidylate Synthase; D003545:Cysteine; D000409:Alanine