Linked Life Data

10884346

Source:http://linkedlifedata.com/resource/pubmed/id/10884346

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2000-8-10
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0022-2836
pubmed:author
pubmed:copyrightInfo
Copyright 2000 Academic Press.
pubmed:issnType
Print
pubmed:day
14
pubmed:volume
300
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
493-502
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:10884346-Adenosine Triphosphate, pubmed-meshheading:10884346-Amino Acid Sequence, pubmed-meshheading:10884346-Animals, pubmed-meshheading:10884346-Binding Sites, pubmed-meshheading:10884346-Catalysis, pubmed-meshheading:10884346-Cell Division, pubmed-meshheading:10884346-Cell Line, pubmed-meshheading:10884346-Conserved Sequence, pubmed-meshheading:10884346-Cross-Linking Reagents, pubmed-meshheading:10884346-Cysteine Endopeptidases, pubmed-meshheading:10884346-Dimerization, pubmed-meshheading:10884346-Enzyme Stability, pubmed-meshheading:10884346-Fibroblasts, pubmed-meshheading:10884346-Glutaral, pubmed-meshheading:10884346-Half-Life, pubmed-meshheading:10884346-Mice, pubmed-meshheading:10884346-Molecular Sequence Data, pubmed-meshheading:10884346-Multienzyme Complexes, pubmed-meshheading:10884346-Mutation, pubmed-meshheading:10884346-Proteasome Endopeptidase Complex, pubmed-meshheading:10884346-Protein Binding, pubmed-meshheading:10884346-Protein Structure, Quaternary, pubmed-meshheading:10884346-Protein Structure, Tertiary, pubmed-meshheading:10884346-Sequence Alignment, pubmed-meshheading:10884346-Thymidine, pubmed-meshheading:10884346-Thymidine Kinase, pubmed-meshheading:10884346-Transfection
pubmed:year
2000
pubmed:articleTitle
Substrate binding is a prerequisite for stabilisation of mouse thymidine kinase in proliferating fibroblasts.
pubmed:affiliation
Institute of Molecular Biology, University of Vienna, Austria.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't