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Substrate binding is a prerequisite for stabilisation of mouse thymidine kinase in proliferating fibroblasts.
- Source :
-
Journal of molecular biology [J Mol Biol] 2000 Jul 14; Vol. 300 (3), pp. 493-502. - Publication Year :
- 2000
-
Abstract
- Thymidine kinase (TK) expression in mammalian cells is strictly growth regulated, with high levels of the enzyme present in proliferating cells and low levels in resting cells. We have shown that mouse TK expressed from a constitutive promoter is still subject to this regulation. The drastic decline in TK enzyme levels in resting cells is largely due to a pronounced reduction in the half-life of the protein. Deletion of the 30 C-terminal amino acid residues from TK abrogates growth regulation, rendering the enzyme very stable. Moreover, the substrate thymidine was sufficient to stabilise the labile TK protein in quiescent cells. Here, we report that the ability of TK to bind substrates is essential for both growth-dependent regulation and stabilisation by the substrate. By mutation or elimination of the binding sites for either of the two substrates, ATP and thymidine, we expressed TK proteins lacking enzymatic activity which abolished growth-regulated expression in both cases. Mutant TK proteins impaired in substrate binding were subject to rapid degradation in exponentially growing cells and thymidine was no longer sufficient to inhibit this rapid decay. A C-terminal truncation known to stabilise the TK wild-type protein in resting cells did not affect the rapid turnover of enzymatically inactive TK proteins. Proteasome inhibitors also failed to stabilise these substrate-binding mutants. By cross-linking experiments, we show that TK proteins with mutated substrate-binding sites exist only as monomers, whereas active TK enzyme forms dimers and tetramers. Our data indicate that, In addition to the C terminus intact substrate-binding sites are required for growth-dependent regulation of TK protein stability.<br /> (Copyright 2000 Academic Press.)
- Subjects :
- Adenosine Triphosphate metabolism
Amino Acid Sequence
Animals
Binding Sites
Catalysis
Cell Line
Conserved Sequence genetics
Cross-Linking Reagents metabolism
Cysteine Endopeptidases metabolism
Dimerization
Enzyme Stability
Fibroblasts cytology
Fibroblasts enzymology
Fibroblasts metabolism
Glutaral metabolism
Half-Life
Mice
Molecular Sequence Data
Multienzyme Complexes antagonists & inhibitors
Multienzyme Complexes metabolism
Mutation genetics
Proteasome Endopeptidase Complex
Protein Binding
Protein Structure, Quaternary
Protein Structure, Tertiary
Sequence Alignment
Thymidine Kinase chemistry
Thymidine Kinase genetics
Transfection
Cell Division
Thymidine metabolism
Thymidine Kinase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0022-2836
- Volume :
- 300
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 10884346
- Full Text :
- https://doi.org/10.1006/jmbi.2000.3876